Charakterisierung peroxisomaler und Lipid-Droplet ... · PLC Phospholipase C PLD Phospholipase D...

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Charakterisierung peroxisomaler und Lipid-Droplet assoziierter Proteine der Hefe Saccharomyces cerevisiae Dissertation zur Erlangung des Grades eines Doktors der Philosophie der Fakultät für Biologie und Biotechnologie der Ruhr-Universität Bochum Internationalen Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum Abteilung für Systembiochemie vorgelegt von Dipl.-Biol. & Biochem. Mykhaylo O. Debelyy aus Dnepropetrovsk, Ukraine Bochum Juli, 2011

Transcript of Charakterisierung peroxisomaler und Lipid-Droplet ... · PLC Phospholipase C PLD Phospholipase D...

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Charakterisierung peroxisomaler und Lipid-Droplet

assoziierter Proteine der Hefe Saccharomyces cerevisiae

Dissertation zur Erlangung des Grades eines Doktors der Philosophie

der Fakultät für Biologie und Biotechnologie der Ruhr-Universität Bochum

Internationalen Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum

Abteilung für Systembiochemie

vorgelegt von Dipl.-Biol. & Biochem. Mykhaylo O. Debelyy

aus Dnepropetrovsk, Ukraine

Bochum Juli, 2011

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Zusammenfassung

In der vorliegenden Arbeit wurden Peroxisomen und Lipid-Droplet assoziierte Proteine der Hefe S. cerevisiae untersucht. Lpx1p und Ldh1p sind putative Hydrolasen und/oder Lipasen von Peroxisomen beziehungsweise Lipid-Droplets; Pex1p und Pex6p sind peroxisomale AAA Proteine und Ubp15p stellt ein deubiquitinilierendes Enzym dar.

Es konnte gezeigt werden, dass Lpx1p in Peroxisomen lokalisiert ist, wo hingegen Ldh1p überwiegend zu Lipid-Droplets dirigiert wird. Lpx1p wie auch Ldh1p besitzen das für Lipasen der a/ß-Hydrolase-Familie typische Sequenzmotiv GXSXG. Beide Proteine tragen ein putatives, peroxisomales Typ 1 targeting Signal (PTS1) und weisen untereinander zwei homologe Bereiche auf. Während gezeigt werden konnte, das es sich bei Lpx1p um ein peroxisomales Enzym handelt, wurde anhand subzellulärer Lokalisationsstudien für Ldh1p eine überwiegende Lokalisation an Lipid-Droplets dargelegt. Für Lpx1p konnte ferner gezeigt werden, das dessen Lokalisation vom PTS1-Rezeptor Pex5p abhängt. Ferner konnte für Lpx1p ein Huckepack-Transport in die Peroxisomen gezeigt werden. Dem gegenüber ist der Transport von Ldh1p zu den Lipid-Droplets unabhängig von dessen PTS1.

Für Lpx1p und Ldh1p konnte in vitro mittels rekombinanter Proteine eine

Triacylglycerol-Lipase wie auch–hydrolase Aktivität belegt werden. Es konnte gezeigt werden, dass Lpx1p nicht für das Vorhandensein funktioneller Peroxisomen benötigt wird, ein Umstand der eher auf eine metabolische als auf eine Biogenesefunktion des Proteins hinweist. Ldh1p ist hingegen notwendig für die Aufrechterhaltung normaler Konzentrationen an nicht-polaren und polaren Lipiden in den Lipid-Droplets. Ein Charakteristikum der Δldh1-Mutante ist das Auftreten übergroßer Lipid-Droplets sowie einer übermäßigen Akkumulation nicht-polarer Lipide und Phospholipiden nach Wachstum auf Medium mit Ölsäure als alleinige Kohlenstoffquelle. Basierend auf den Daten wird eine Funktion von Ldh1p in der Aufrechterhaltung der Lipid-Homeostase in der Hefe durch die Regulation des Spiegels an Phospholipiden wie auch nicht-polaren Lipiden diskutiert.

Der peroxisomale Matrix Proteinimport wird durch zyklisierende Rezeptoren

ermöglicht, die zwischen dem Zytosol und der peroxisomalen Membran pendeln. Die Ubiquitinilierung des Rezeptors dient dabei als dessen Exportsignal. Ein entscheidender Schritt innerhalb dieses Zyklus ist die ATP-abhängige Ablösung des Rezeptors von der peroxisomalen Membran. Dieser Schritt wird durch die peroxisomalen AAA ATPasen Pex1p und Pex6p bewerkstelligt. In der vorliegenden Arbeit konnte gezeigt werden, dass der AAA-Komplex sowohl die Pex5p-Dislokaseaktivität wie auch eine deubiquitinilierende Aktivität beinhaltet. Im Einklang mit dieser Beobachtung konnte Ubp15p, eine Ubiquitin-Hydrolase, als neuer Bestandteil des AAA-Komplexes identifiziert werden. Ubp15p ist partiell peroxisomal lokalisiert und in der Lage Ubiquitinreste vom modifizierten PTS1-Rezeptor Pex5p abzuspalten. Des Weiteren weisen Ubp15p-defiziente Zellen einen stress-induzierten PTS1-Importdefekt auf. Diese Ergebnisse führen zu dem Modell nachdem die Entfernung des Ubiquitins von Pex5p ein spezifisches Ereignis darstellt welches ein wesendlicher Schritt im Rezeptor- Recycling darstellt.

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ABKÜRZUNGSVERZEICHNIS

AAA ATPases Associated with various cellular Activities ATP Adenosine triphosphate BPC 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-

undecanoyl)-sn-glycero-3-phosphocholine (bis-BODIPY-FL C11-PC) Cdc48p Cell division cycle 48 protein CRL Candida rugosa lipase DGR 1,2-O-dilauryl-rac-glycero-3-glutaric acid (6-methyl resorufin) ester DPG 1,2-dioleoyl-3-(pyren-1-yl) decanoyl-rac-glycerol DUB Deubiquitinating enzyme ERAD Endoplasmic-Reticulum-Associated protein Degradation

GFP Green Fluorescence Protein E1 Ubiquitin-activating enzymes E2 Ubiquitin-conjugating enzymes E3 Ubiquitin ligases E. coli Escherichia coli min Minutes ml Millilitre NPL Non-polar lipids NSF N-ethylmaleimide sensitive factor (fusion protein) NTD N-terminal domain pex peroxisome assembly PC Phosphatidylcholine PE Phosphatidylethanolamine PL Polar lipids PLA Phospholipase A PLC Phospholipase C PLD Phospholipase D PMPs Peroxisome Membrane Proteins PNB p-Nitrophenyl butyrate PNS post nuclear supernatant ProtA Protein A PTS Peroxisomal Targeting Signal RING really interesting new gene SRH Second region of homology TEV Tobacco Etch virus Ub Ubiquitin Ubc Ubiquitin-conjugating enzyme VCP Valosin-containing protein

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Characterization of peroxisome- and lipid droplet-related proteins of Saccharomyces cerevisiae

Dissertation to obtain the degree Doctor Philosophiae (Doctor of Philosophy, PhD)

at the Faculty of Biology and Biotechnology Ruhr-University Bochum

International Graduate School of Biosciences Ruhr-University Bochum

Department of Systems Biochemistry

submitted by Dipl.-Biol. & Biochem. Mykhaylo O. Debelyy

from Dnepropetrovsk, Ukraine

Bochum July, 2011

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ERKLÄRUNG

Hiermit erkläre ich, dass ich die Arbeit selbständig verfasst und bei keiner anderen Fakultät

eingereicht und dass ich keine anderen als die angegeben Hilfsmittel verwendet habe. Es

handelt sich bei der heute von mir eingereichten Dissertation um sechs in Wort und Bild

völlig übereinstimmende Exemplare.

Weiterhin erkläre ich, dass digitale Abbildung nur die originalen Daten enthalten und in

keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde.

Bochum, den

_______________________________________

(Unterschrift)

Vorsitzender der Prüfungskommission: Prof. Dr. Franz Narberhaus (Fakultät für Biologie, RUB) 1. Gutachter: Prof. Dr. Ralf Erdmann (Medizinische Fakultät, RUB) 2. Gutachter: Prof. Dr. Ulrich Kück (Fakultät für Biologie, RUB) 3. Gutachter: PD Dr. Mathias Lübben (Fakultät für Biologie, RUB)

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INDEX__________________________________________________________________ 3

INDEX 3 CHAPTER 1. INTRODUCTION 4 ABSTRACT 5 1.1 Biology of peroxisomes 5

1.1.1 Structure and function of peroxisomes 5 1.1.2 Biogenesis of peroxisomes 7 1.1.3 Posttranslational modifications of peroxins 12 1.1.4 Peroxisomal AAA-ATPase peroxins 14

1.2 Biology of lipid droplets 19 1.2.1 Structure and function of lipid droplets 19 1.2.2 Biogenesis of lipid droplets 21 1.2.3 Interactions between peroxisome and lipid droplets 22

1.3 Objectives 24 CHAPTER 2. ORIGINAL WORKS 25 2.1 Biology of peroxisomes 25

2.1.1 Lpx1p is a peroxisomal lipase required for normal peroxisomes morphology 25

2.1.2 The AAA peroxins Pex1p and Pex6p function as dislocases for the ubiquitinated peroxisomal import receptor Pex5p 36

2.1.3 Ubp15p, an ubiquitin hydrolase associated with the peroxisomal export machinery 42

2.2 Biology of lipid droplets 65 2.2.1 The putative Saccharomyces cerevisiae hydrolase Ldh1p is

localized to lipid droplets 65 2.2.2 Involvement of the Saccharomyces cerevisiae hydrolase Ldh1p

in lipid homeostasis 71

CHAPTER 3. DISCUSSION 77 3.1 Novel hydrolases of S. cerevisiae 77 3.2 Ubp15p, a novel compound of AAA-complex 83 CHAPTER 4. REFERENCES 90 CHAPTER 5. MISCELLANEOUS 104 5.1 Publications 104 5.2 Personal contribution to the papers 105 5.3 Conferences 106 5.4 Curriculum Vitae 107 5.5 Acknowledgement 108 5.6 Global scientific outlook for human race 109

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ABSTRACT

The peroxisomal and lipid droplets related proteins of yeast S. cerevisiae were characterized in this work. Lpx1p and Ldh1p are putative hydrolases and/or lipases of peroxisome and lipid droplets respectively; Pex1p and Pex6p are peroxisomal AAA ATPases; and Ubp15p is a deubiquitinating enzyme.

It was shown that Lpx1p is present in the peroxisome but Ldh1p is

predominantly localized to lipid droplets. Lpx1p as well as Ldh1p comprises the typical GXSXG-type lipase motif of members of the α/β-hydrolase family. Both proteins carry a putative peroxisomal targeting signal type-1 (PTS1) and can be aligned with two regions of homology. While Lpx1p was shown to be a peroxisomal enzyme, subcellular localization studies revealed that Ldh1p is predominantly localized to lipid droplets. It was shown that Lpx1p import is dependent on the PTS1 receptor Pex5p. Moreover, it was shown that Lpx1p is piggyback-transported into peroxisomes. But it was demonstrated that targeting of Ldh1p to lipid droplets occurs independently of the PTS1 receptor Pex5p.

Triacylglycerol lipase as well as hydrolase activities were shown for both

recombinant proteins Lpx1p and Ldh1p in vitro. It was shown that the Lpx1p protein is not required for wild-type-like steady-state function of peroxisomes, which might be indicative of a metabolic rather than a biogenetic role. It was clearly shown that peroxisomes in Δlpx1 mutants have an aberrant morphology characterized by intraperoxisomal vesicles or invaginations. It was shown that Ldh1p is not required for the function and biogenesis of peroxisomes. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of the Δldh1 strain is the appearance of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and nonpolar lipid levels.

It is known that the peroxisomal matrix protein import is facilitated by cycling

receptors shuttling between the cytosol and the peroxisomal membrane. One crucial step in this cycle is the ATP-dependent release of the receptors from the peroxisomal membrane. This step is facilitated by the peroxisomal AAA ATPases Pex1p and Pex6p with ubiquitination of the receptor being the main signal for its export. It was shown in this work that the AAA-complex contains Pex5p dislocase as well as deubiquitinating activity. Ubp15p, an ubiquitin hydrolase, was identified as novel constituent of the complex. Ubp15p partially localizes to peroxisomes and is capable to cleave off ubiquitin-moieties from the PTS1-receptor Pex5p. Furthermore, Ubp15p-deficient cells are characterized by a stress related PTS1-import defect. The results merge to a picture in which removal of ubiquitin of the PTS1-receptor Pex5p is a specific event and might represent a vital step in receptor recycling.

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CHAPTER 1. INTRODUCTION

1.1 Biology of peroxisomes

1.1.1 Structure and function of peroxisomes

Peroxisomes or microbodies are a class of structurally and functionally related ubiquitous

eukaryotic organelles that are involved in lipid and antioxidant metabolism (179). Originally

these structures were described as cellular organelles in 1966 by C. de Duve and P. Baudhuin

(33) after they had been first mentioned in a PhD thesis of J. Rhodin a more than a decade

earlier (177). Generally, peroxisomes are spherical organelles with diameter from 0.1 to 1 µm

envelop by a single phospholipid bilayer membrane (218) (Fig. 1.1.1.1).

Fig. 1.1.1.1 Induction of peroxisomes in yeast S. cerevisiae by oleic acid. Localization and morphology of peroxisome. (A) Red staining: labeling of peroxisome by DsRed-PTS2; Green staining: partial labeling of peroxisome by GFP-Ubp15p; (B) Electron microscopy image of wild-type; (C) Electron microscopy image of peroxisome free mutant (Δpex19); C, cytosol; ER, endoplasmic reticulum; L, lipid droplets; M, mitochondria; N, nucleus; P, peroxisome.

The peroxisome-family includes peroxisomes, glyoxysomes of plants and fungi, glycosomes

of trypanosomes, and Woronin-bodies of filamentous fungi (11, 138, 179) (Fig.1.1.1.2). The

unique variability in function of peroxisomes is displayed by an electron-dense proteinaceous

organellar matrix that contains no DNA (179), but is extremely variable in their enzyme

content, adjusted to metabolic functions according to the cellular needs.

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Fig. 1.1.1.2 Induction of peroxisomes in yeast species and filamentous fungi. Peroxisomes can have highly variable sizes and shapes. Furthermore, they can be present in clusters but can also be dispersed throughout the cytoplasm. A) Aspergillus tamarii cell grown on oleate showing peroxisomes. In addition, Woronin bodies are present near the septum (arrow). Many lipid bodies are present. B) Hansenula polymorpha cell from a methanol-limited chemostat. More than 80% of the cell is filled with cuboid-shaped peroxisomes. C) Saccharomyces cerevisiae cell grown on oleate showing clustered peroxisomes. D) Penicillium chrysogenum hyphae producing the fluorescent peroxisomal protein green fluorescent protein-Ser-Lys-Leu-COOH (GFP-SKL). Cells were grown in penicillin-producing medium and treated with Mitotracker Orange to stain the mitochondria. The bar represents 1 mm, unless indicated otherwise. L, lipid body; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. Taken with modifications from (107).

The peroxisomal matrix harbours at least 50 different enzymes that are linked to

diverse biochemical pathways (96). The β-oxidation of fatty acids and the detoxification of

hydrogen peroxide are regarded as the central function of peroxisomes (191, 219). But there is

one exception: the Woronin-bodies, function of which is only the plugging of the septal pores

in case of hyphal injury. While the ß-oxidation in fungi and plants exclusively take place in

peroxisomes (179, 191, 219), in mammalian cells only the very long chain fatty acids are

oxidized in peroxisomes (97, 121). Moreover, peroxisomes are involved in the synthesis of

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plasmalogens (which contribute more than 80% of the phospholipid content of the white

matter in the brain), cholesterol and bile acids (17, 76, 118, 119) as well as the oxidation of

alcohols, metabolism of prostaglandins, catabolism of purines and polyamines, the main

reaction of photorespiration in plant leafs and final steps of penicillin biosynthesis in some

filamentous fungi (80, 153, 215, 216, 218). They are the source of signalling molecules such

as jasmonates in plants (36, 160, 225) or lipid-derived ligands for PPARs

(peroxisomeproliferator-activated receptors) in humans (51).

The existence of severe inherited diseases in human caused by malfunctions in

peroxins, encoded by PEX genes, stimulates intensive research interest to the field of

peroxisome biogenesis. So far 34 peroxins were discovered to be involved in different stages

of peroxisome biogenesis (217). Human peroxisomal disorders can be categorized as either

single-enzyme disorders or peroxisomal biogenetic defects (229). Single-enzyme disorders,

such as for example Refsum disease is caused by a defect of phytanoyl-CoA hydroxylase,

whereas X-linked adrenoleukodystrophy is caused by a defect in a peroxisomal ATP-

transporter. In contrast, biogenesis defects are mostly caused by mutations in the PEX genes

(211). Peroxisomal disorders are associated with morphological peroxisomal defects such as

inclusions or invaginations (56, 151).

1.1 Biology of peroxisomes

1.1.2 Biogenesis of peroxisomes

As peroxisomes do not contain genetic material, their protein content is determined by

the import of nuclear encoded proteins (26). Peroxisomes can multiply by division (152) or de

novo by budding from the endoplasmic reticulum (84, 123). Without exception, peroxisomal

matrix proteins are synthesized on free ribosomes and are subsequently imported in a post-

translational manner (136, 188). Like the sorting of proteins to other cellular compartments,

protein targeting to peroxisomes depends on signal sequences. Peroxisomal import of most

matrix proteins depends on the conserved PTS1 (peroxisomal targeting signal type 1) receptor

Pex5p, which recognizes the PTS1 localized at the very C-terminus of the cargo proteins

(170, 212). The three-amino-acid signal SKL (serine–lysine–leucine) was the first PTS1 to be

discovered, and is in many cases sufficient for directing a protein to peroxisomes. Based on

mutagenesis experiments, amino acid permutations and sequence comparisons between

different species, the PTS1 generally fits the consensus (S/A/C)-(K/R/H)-(L/M). A second

peroxisomal targeting signal (PTS2) (188) is present in considerably fewer peroxisomal

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proteins. PTS2 is usually located within the first 20 amino acids of the protein, and has been

defined as (RK)-(LVIQ)-XX-(LVIHQ)-(LSGAK)-X-(HQ)-(LAF) (166). PTS2-bearing

proteins are recognized by the cytosolic conserved receptor Pex7p (188).

Based on the concept of cycling receptors (38, 144), the matrix protein import can be

divided into four steps: 1) receptor-cargo recognition in the cytosol, 2) docking at the

peroxisomal membrane, 3) cargo-translocation and release, and 4) receptor release from the

membrane and recycling. After the cargo recognition by their cognate receptor in the cytosol

(72), in yeast the second step in receptor cycle is facilitated by Pex14p together with Pex13p

and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this

cycle with both soluble import receptors Pex5p and Pex7p (176) (Fig. 1.1.2.1).

Fig. 1.1.2.1 The receptor cycle. According to the model of the cycling receptor, the peroxisomal protein import conceptually can be divided in five steps: (I) cargo recognition in the cytosol and (II) docking of the receptor–cargo complexes to the peroxisomal membrane. (III) Cargo-translocation into the peroxisomal matrix. (IV) Disassembly of the receptor–cargo complex and (V) export of the receptor back to the cytosol. PTS1-containing proteins are recognized by the soluble import receptor Pex5p in the cytosol. Proteins harbouring the PTS2 are recognized by Pex7p and the cofactors Pex18p and Pex21p in S. cerevisiae, the orthologous Pex20p in other fungi or Pex5L in plants and mammals. After this step, the receptor–cargo complex targets to and associates with the peroxisomal membrane via the docking complex consisting of Pex14p, Pex13p and Pex17p. The transport of PTS1-proteins across the membrane is facilitated by formation of a pore mainly consisting of Pex14p and Pex5p. Pex8p connects the RING-complex to the docking complex. The three ubiquitin ligases Pex2p, Pex10p and Pex12p form the RING-complex and together with ubiquitin conjugating enzymes like Pex4p are responsible for receptor ubiquitination. In the last step of the cycle, the receptor Pex5p is exported back to the cytosol by the two AAA-peroxins Pex1p and Pex6p and is enabled for the next round of import. Taken from (179).

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It was demonstrated by using the yeast two-hybrid system and pull-down assays that yeast S.

cerevisiae Pex5p directly interacts with two separate regions of Pex14p, amino acid residues

1-58 and 235-308. The latter binding site at the C-terminus of Pex14p overlaps with a binding

site of Pex7p at amino acid residues 235-325 (158). The functional assessment of these two

binding sites of Pex14p with the PTS-receptors indicates that they have distinct roles.

Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in

Δpex14 cells expressing the Pex14-(59-341)-p fragment; however, it did not lead to a pex

phenotype. In contrast, truncation of the C-terminal 106 amino acids of Pex14p completely

blocked this process (158). It was proposed that the C-terminus of Pex14p contains the actual

docking site and that the N-terminus could be involved in a Pex5p-Pex14p association inside

the peroxisomal membrane (158).

The molecular mechanism of how the cargo proteins traverse the peroxisomal

membrane remains unclear. However, recent reports demonstrated the transient formation of a

dynamic pore which is adapted to the size of the cargo and could facilitate the translocation of

at least 9 nm particles (222).

The final step in the receptor cycle is the release of the receptor back to the cytsosol to

facilitate a new round of import. With respect to the PTS1-receptor Pex5p, recent reports

demonstrated that its dislocation from the peroxisomal membrane to the cytosol at the end of

the receptor cycle is ATP-dependent and catalyzed by the AAA-peroxins Pex1p and Pex6p

(150, 172). With this respect in accordance to the export-driven import model it is believed,

that the export of receptor delivers the energy for cargo-translocation (184).

Pex4p-catalysed mono-ubiquitination of Pex5p direct the receptor for recycling,

thereby enabling further rounds of matrix protein import, whereas Ubc4p-catalysed

polyubiquitination targets Pex5p to proteasomal degradation (44, 106, 171, 172).

The import of peroxisomal membrane proteins (PMPs) is differing from the import

machinery of peroxisomal matrix proteins (43, 68). This is in agreement with the fact that

most pex-mutants are distinguished by an affected import of matrix proteins but not affected

import of PMPs. In these mutants, the PMPs are imported in peroxisomal remnants, so called

ghosts (25, 183, 188). Only several mutants were characterized by the complete absence of

detectable peroxisomal membrane ghosts. Functional complementation of these mutants led

to the identification of Pex3p, Pex19p and in some organisms Pex16p which are involved in

the biogenesis of the peroxisomal membrane (10, 41, 60, 67, 88, 146, 179, 200) (Fig. 1.1.2.2).

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Fig. 1.1.2.2 Topogenesis of peroxisomal membrane proteins. Two routes are proposed for the targeting of peroxisomal membrane proteins (PMPs). Class I proteins are directly imported into existing peroxisomes. Class II proteins are first targeted to ER where they concentrate in pre-peroxisomal vesicles which then are targeted to existing peroxisomes or function as an origin for de novo formation of peroxisomes. Currently, it is controversially discussed whether class I PMPs are also targeted to the ER and whether class II PMPs are also targeted to existing peroxisomes. Taken from (179).

Various roles have been suggested for Pex19p. Initially, due to its capacity to interact

with the majority of the peroxisomal membrane proteins (PMPs), and according, to its

multiple localization at the peroxisomal membrane and in the cytosol, Pex19p is considered to

be a soluble import receptor for newly synthesized PMPs (100, 185). As a consequence,

Pex19p binds PMPs in the cytosol and delivers them to the peroxisomal membrane by

docking to its membrane anchored binding partner Pex3p (67, 179) (Fig. 1.1.2.3).

Subsequent, Pex19p is additionally assumed to behave as a PMP-specific chaperone.

Correspondingly, Pex19p bear the capacity to adhere and sustain PMP by the development of

a soluble complex and in this manner anticipating conglomeration of the PMP (105, 192).

Also, Pex19p has possibility to function as an insertion factor during PMP import (54, 198) or

act as an assembly/disassembly factor for peroxisomal membrane complexes at the

peroxisomal membrane (52, 179). Recently, it was shown that Pex19p is required for the

transport of Pex3p from the endoplasmic reticulum (ER) to the peroxisomal membrane (84).

Pex3p is an integral membrane protein at the peroxisomal membrane with a topology distinct

all over species (60, 75, 93, 179, 199).

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Fig. 1.1.2.4 Pex19p-dependent import of PMPs. Class I peroxisomal membrane proteins (PMPs) harbour a peroxisomal membrane protein targeting signal (mPTS) which is recognized in the cytosol by the import receptor and/or PMP-specific chaperone Pex19p, a farnesylated, mostly cytosolic protein with a small portion of the protein found associated with the peroxisomal membrane. In the next step, the cargo-loaded Pex19p docks to the peroxisomal membrane via association with its docking factor Pex3p. Then the PMP is inserted into the membrane in an unknown manner but presumably with assistance of Pex19p, Pex3p and, in some organisms, Pex16p. The requirement of ATP for this process is not clear. Finally, Pex19p shuttles back to the cytosol where it might start a new round of import. Taken from (179). In Saccharomyces cerevisiae, Pex3p bear an N-terminal transmembrane region and a large C-

terminal domain to be turned toward the cytosolic side of the peroxisome (86, 179). Pex3p

performs a pivotal function in the import of PMPs at which point it assists as a docking factor

at the peroxisomal membrane and acts as binding partner for Pex19p-PMP-complexes

meanwhile import of the PMPs (49, 53, 147). Pex3p additionally acts as a crucial factor in the

de novo development of peroxisomes as it is considered to be the initiating step for this

peroxisome assembling action.

PMP insertion into the peroxisomal membrane in some organism required assistance

of Pex16p. It was demonstrated that Pex16p is an integral membrane protein which is mainly

found in higher eukaryotes (129, 179, 200) and in the yeast Y. lipolytica (41). Although the

mammalian Pex16p is an integral membrane protein with the C- as well as the N-terminus

facing the cytosol (87), the yeast Pex16p is a membrane associated protein facing the

peroxisomal lumen (41). It was shown that Pex16p execute distant activities in peroxisome

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biogenesis. The mammalian Pex16p is required for the topogenesis of membrane proteins and

acts in the very early stages of peroxisome biogenesis while the yeast Pex16p is a negative

regulator of peroxisomal fission (41, 108, 179).

1.1 Biology of peroxisomes

1.1.3 Posttranslational modifications of peroxins

The theory of cycling receptors Pex5p and Pex7p imply consecutive interaction of the

receptors to distinct proteins or protein complexes at the peroxisome (116, 158). The

regulation of such interactions are implementing by reversible posttranslational modification

such as phosphorylation and/or ubiquitination (116, 171).

Actually, the membrane proteins Pex14p and Pex15p were shown to be

phosphorylated (42, 99, 114). Nevertheless, the physiological functions of phosphorylation in

peroxisomal matrix protein import are unexplored (116).

Recently, two peroxins have been shown to be ubiquitinated. For example, Pex18p, a

protein involved in the PTS2 pathway, is constitutively degraded in an ubiquitin-dependent

manner (174). Considering such observation two independent research groups demonstrated

polyubiquitination of Pex5p in cells deficient in constituents of the AAA or Pex4p-Pex22p

complexes (106, 171). Besides, it was shown for Pex5p that polyubiquitination leads to the

proteasomal degradation (171). It was demonstrated that ubiquitination of proteins requires

the consecutive activity of at least three types of enzymes: a ubiquitin-activating enzyme (E1),

a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) (167, 226) (Fig. 1.1.3.1).

At the terminating stage of the ubiquitination cascade an isopeptide bond amongst

ubiquitin and the lysine residue of the substrate is arranged. This reaction is catalyzed by the

E2 enzyme, usually in association with the E3 ligase. The length of the ubiquitin chain

conjugated to a protein substrate is carrying out a considerably meaningful function.

Polyubiquitinated proteins (the minimal chain length is four ubiquitin moieties) are

normally distinguished from other non-ubiquitinated proteins and degraded by the proteasome

(213). In contrast, monoubiquitination, an attachment of a single ubiquitin moiety, regulates

cellular processes such as endocytosis, sorting into multivesicular bodies and virus budding in

a proteasome-independent way (81).

It was shown that Pex5p is a monoubiquitinated and stable protein in wild-type cells.

Pex5p monoubiquitination occurs at the peroxisome and is blocked in cells deficient of

operative docking or RING finger complexes, evoking concept that Pex5p

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monoubiquitination is a late event in peroxisomal matrix protein import (116, 171). It was

shown that polyubiquitination of Pex5p protein is not a prerequisite for functional

peroxisomal protein import in S. cerevisiae (169).

Fig. 1.1.3.1 The ubiquitylation pathway. Free ubiquitin (Ub) is activated in an ATP-dependent manner with the formation of a thiol-ester linkage between E1 and the carboxyl terminus of ubiquitin. Ubiquitin is transferred to one of a number of different E2s. E2s associate with E3s, which might or might not have substrate already bound. For HECT domain E3s, ubiquitin is next transferred to the active-site cysteine of the HECT domain followed by transfer to substrate (S) (as shown) or to a substrate-bound multi-ubiquitin chain. For RING E3s, current evidence indicates that ubiquitin might be transferred directly from the E2 to the substrate. Taken with modifications from (226).

It was shown that Pex5p is a monoubiquitinated and stable protein in wild-type cells.

Pex5p monoubiquitination occurs at the peroxisome and is blocked in cells deficient of

operative docking or RING finger complexes, evoking concept that Pex5p

monoubiquitination is a late event in peroxisomal matrix protein import (116, 171). It was

shown that polyubiquitination of Pex5p protein is not a prerequisite for functional

peroxisomal protein import in S. cerevisiae (169). Moreover, it was shown that

polyubiquitinated forms of Pex5p concentrate in definite pex mutants in an Ubc4p-dependent

fashion (116), an observation that is in agreement with previous reports (106, 171). Despite,

monoubiquitination of Pex5p in wild-type cells is not controlled by Ubc4p, and peroxisome

biogenesis is not disturbed in cells deficient of Ubc4p (116). Besides, it was demonstrated the

polyubiquitination of Pex5p is part of a quality control system that direct membrane-

accumulated Pex5p for proteasomal degradation (44, 106, 172).

The protein Pex19p acts as a receptor and chaperone of peroxisomal membrane

proteins (PMPs) (190). The conserved CaaX box peroxin Pex19p is known to be modified by

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farnesylation (67, 104, 146). It was recently shown that the complete pool of Pex19p is

processed by farnesyltransferase in vivo and that this modification is independent of

peroxisome induction or the Pex19p membrane anchor Pex3p. Moreover, it was demonstrated

that genomic mutations of PEX19, which blocks farnesylation are critical for correct matrix

protein import into peroxisomes. It was shown that mutants defective in Pex19p farnesylation

are characterized by a significantly reduced steady-state concentration of prominent

peroxisomal membrane proteins Pex11p and Ant1p as well as constitutive compounds of the

peroxisomal import machinery such as RING peroxins (180).

1.1 Biology of peroxisomes

1.1.4 Peroxisomal AAA-peroxins

The highly diverse and adaptive character of peroxisomes is accomplished by modulation of

their enzyme content, which is mediated by dynamically operating protein-import

machineries. The import of matrix proteins into the peroxisomal lumen has been described as

the ATP-consuming step. It was shown that the peroxisomal AAA-ATPase (ATPase

Associated with various cellular Activities) proteins Pex1p and Pex6p are mechano-enzymes

and core components of a complex which dislocates the cycling import PTS1-receptor Pex5p

from the peroxisomal membrane back to the cytosol. Such release of Pex5p has been regarded

as the final step of the peroxisomal protein import cascade. The AAA-mediated process is

regulated by the ubiquitination status of the receptor Pex5p. Pex4p-catalysed mono-

ubiquitination of Pex5p primes the receptor for recycling, thereby enabling further rounds of

matrix protein import, whereas Ubc4p-catalysed polyubiquitination targets Pex5p to

proteasomal degradation (168).

AAA-proteins are characterized by a typical modular architecture as they contain an

N-terminal non-ATPase domain which is followed by at least one conserved AAA domain.

Each AAA-cassette usually contains an ATP-binding site (Walker A) and an ATP-hydrolysis

site (Walker B) along with other motifs, such as the SRH (46) (Fig 1.1.4.1).

Pex1p and Pex6p are type II AAA-proteins, which are characterized by two AAA

domains. In both AAA peroxins, the second AAA domain is more conserved than the first

one. Interaction and subsequent oligomerization of Pex1p and Pex6p is believed to be

initiated in the cytosol and involves their first less conserved AAA domains (D1) (19, 204).

Although neither binding nor hydrolysis of ATP at D1 seems to be essential for

functionality in both yeast and humans, the interaction of human Pex1p and Pex6p is

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stimulated by binding of ATP to D1 of human Pex1p and Pex6p (48, 204). Furthermore, ATP

binding, but not hydrolysis, at the second AAA cassette (D2) of Pex1p is required for the

Pex1p–Pex6p interaction in both systems (19, 204).

Fig 1.1.4.1 Molecular organization of the AAA complex in S. cerevisiae. The AAA peroxins Pex1p and Pex6p are composed of an NTD, a non-conserved AAA domain (D1) and a conserved AAA domain (D2). The AAA domains contain ATP-binding sites (A) and, with the exception of D1 of Pex6p, also ATP-hydrolysis sites (B). Pex1p and Pex6p form a heteromeric complex, and oligomerization requires the presence of the D1 domains and is stimulated by ATP binding to Pex1p D2. Recruitment of the AAA complex to peroxisomes occurs via binding of Pex6p NTD to Pex15p and requires ATP binding at Pex6p D1, while detachment from Pex15p needs ATP binding and hydrolysis at Pex6p D2. The peroxisomal AAA complex dynamically associates with the functional matrix protein-import machinery (importomer) and Pex4p (Ubc10p) is supposed to be required for the disconnection of the AAA complex from the importomer. Taken with modifications from (168). Pex1p and Pex6p are believed to form heterohexameric structures in the cytosol and at the

peroxisomal membrane (47, 178, 203, 204). However, it is not clear whether formation of a

heteromeric assembly of the AAA peroxins is a prerequisite for their function, as one

population of Pex1p does not co-localize with Pex6p in mammalian cells (204, 228).

Although the formation of hexameric structures is common to AAA proteins, the

formation of heterohexamers has been found in few other cases, such as the m-AAA (matrix

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AAA) complex, consisting of Yta10p and Yta12p, which is active at the matrix site of the

inner mitochondrial membrane, (7) or the six different Rpt ATPases from the 19S proteasome

(62).

The recruitment of AAA-complexes to peroxisomes is mediated by the tail-anchored

peroxisomal membrane proteins Pex15p in S. cerevisiae or its functional orthologue Pex26p

in human cells via binding of the N-terminal domain of Pex6p, stimulated by ATP binding to

the Walker A motif of Pex6p D1 (20, 145). In contrast, the Walker A and B motifs of Pex6p

D2 are required for an efficient detachment from Pex15p/Pex26p (20, 57, 204). Although

Pex15p and Pex26p have been described as adaptor proteins for the N-terminal part of Pex6p,

no adaptor has yet been identified for Pex1p (Fig. 1.1.4.2).

Fig. 1.1.4.2 Schematic representation of interaction between Pex15p and Pex6p. The N-terminus of Pex15p interacts with the N-terminal part of Pex6p, an interaction which is stimulated by ATP-binding to the first AAA domain (A1) of Pex6p. On the other side hydrolysis of ATP by the second AAA domain of Pex6p (B2) stimulates release of Pex6p from Pex15p. Taken from (20).

The NTD (N-terminal domain) of murine Pex1p represents the only available crystal

structure of the AAA peroxins (193). The NTD folds into two structurally independent

globular subdomains (N- and C-lobe), which comprise an N-terminal double-ψ fold and a C-

terminal β-barrel, separated by a shallow groove. Similar grooves were found in the adaptor-

binding sites within the NTDs of VCP, NSF and VAT (VCP-like ATPase from

Thermoplasma), suggesting functional similarity (193).

The Pex15p-anchored AAA complex itself is part of an even larger protein complex at

the peroxisomal membrane, the peroxisomal matrix protein import machinery called the

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importomer (178). To conclude, at least in S. cerevisiae, the Pex1p-bound nucleotides seem to

influence the Pex1p–Pex6p interaction, while the different nucleotide states of Pex6p regulate

the dynamic Pex6p–Pex15p/Pex26p association. The non-conserved domains are responsible

for oligomerization, while the conserved domains exhibit the main ATPase activity.

Import of folded proteins into peroxisomes occurs in a post-translational manner and

depends on ATP. The soluble PTS1 receptor Pex5p is the major signal-recognition factor of

proteins destined for the peroxisomal matrix. The receptor cycle of Pex5p involves cargo

recognition in the cytosol, docking of the receptor–cargo complex to the peroxisomal

membrane, translocation of the receptor–cargo complex to the luminal side of the membrane,

followed by release of the cargo into the matrix and retrotranslocation of the receptor back to

the cytosol (44).

Permeabilized cell systems of human fibroblasts provided the first evidence that Pex5p

accumulated reversibly at the peroxisomal membrane under ATP-modulated conditions (38).

Detailed in vitro studies revealed that the binding and translocation of Pex5p itself is ATP-

independent while the export of Pex5p back to the cytosol requires ATP (69). The identity of

the corresponding ATPase remained a matter of debate until in vitro systems in S. cerevisiae

(172) and human fibroblast cells (150) identified Pex1p and Pex6p as the motor proteins of

Pex5p export. Their function in this process requires the presence of their membrane-anchor

proteins, Pex15p or Pex26p.

The in vitro reconstitution of the complete Pex5p cycle revealed that ATP binding and

hydrolysis at both Pex1p D2 and Pex6p D2 is needed for receptor dislocation (172).

Interestingly, the Walker B motif of Pex1p D2 seems to have no function in formation or

targeting of the AAA complexes (19, 204) and thus may be exclusively required for handling

of the substrate. The binding and consumption of ATP is believed to induce conformational

changes within the AAA peroxins that generate the driving force to pull the receptor out of

the membrane by a mechanism possibly similar to the one of Cdc48p (p97/VCP) in ERAD

The mechanism of substrate recognition by the AAA peroxins is not understood.

Although Pex5p and the AAA-proteins form a complex at the peroxisomal membrane (150,

172, 178), no direct interaction of the PTS1 receptor with either Pex1p or Pex6p has been

reported. This interaction seems to be regulated or mediated by a third factor, which could

represent an unknown adaptor protein of the AAA-peroxins or post-translational modification

of the substrate. It is well known that both processes play a central role in the function of

Cdc48p (p97/VCP) (98, 227), which is the closest evolutionary relative of Pex1p and Pex6p

(58, 186). As a consequence, the question has to be addressed of how the AAA-peroxins can

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distinguish Pex5p forms destined for dislocation from cargo-loaded Pex5p species destined

for cargo translocation.

A possible solution may arise from the crystal structure of Pex1p NTD, which displays

similarities to the corresponding adaptor-binding domains of other AAA proteins (193). Data

from p97 and Ufd1 have identified a double-ψ β-barrel fold as a ubiquitin-binding domain

with binding sites for both mono- and poly-ubiquitin (163). Most interestingly, the PTS

receptors Pex5p, Pex18p and Pex20p have been demonstrated to be ubiquitinated (106, 128,

171, 174).

The PTS1 receptor Pex5p of S. cerevisiae is monoubiquitinated in wild-type cells

(116), whereas it has been shown to be polyubiquitinated in mutants of the proteasome or

cells affected in the AAA and Pex4p–Pex22p complexes of the peroxisomal protein-import

machinery (106, 171). Polyubiquitination of Pex5p, requiring the ubiquitin conjugating

enzymes Ubc4p and the partly redundant Ubc5p and Ubc1p, takes place exclusively at the

peroxisomal membrane and marks the receptor for proteasomal degradation as part of a

quality-control system (106, 116, 171). Alternatively, Pex5p is the specific molecular target

for mono-ubiquitination by Pex4p (Ubc10p) (169, 232), which is essential for peroxisomal

biogenesis (231) and is anchored via Pex22p to the peroxisomal membrane (113).

The functional role of ubiquitination in the dislocation process has been elucidated by

in vitro export assays, revealing that mono-ubiquitination of Pex5p constitute the export

signal under physiological conditions, whereas polyubiquitination seems to provide an export

signal for the release of dysfunctional PTS1 receptors from the membrane and proteasomal

degradation as part of the quality-control pathway (169).

The direct mechanistic influence of this modification on the export reaction remains to

be investigated. The AAA peroxins may interact directly or indirectly via putative adaptors

with the ubiquitin tag on Pex5p. Alternatively, the attachment of ubiquitin may induce local

conformational changes within Pex5p to expose hidden binding sites. This mode of

interaction is also discussed for Cdc48p (p97/VCP), which binds ubiquitin via adaptor

complexes such as Ufd1/Npl4 and via its N-terminal domain. This domain is capable of

recognizing ubiquitin chains and also non-modified segments of its substrates (208, 236).

Notably, the AAA complex displays significantly increased association with the importomer

in PEX4-deficient cells, indicating that the ATPase cycles of Pex1p and Pex6p are coupled to

the mono-ubiquitination-dependent receptor cycle of Pex5p (178).

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1.2 Biology of lipid droplets

1.2.1 Structure and function of lipid droplets

Lipid droplets (LDs) are remarkable dynamic subcellular organelles of globular shape with a

size range from 20 to 100 µm, depending on the cell type (37, 50, 73, 201). LDs are depots of

neutral lipids with a complex biology that exist in virtually any kind of cell, ranging from

bacteria to yeasts, plants, and higher mammals (15, 55, 73). In many cells, LDs occupy a

considerable portion of the cell volume and weight (221). As the major intracellular storage

organelles, LDs were first described in the works of R. Altmann and E. B. Wilson in the 19th

century (2, 233) (Fig. 1.2.1.1).

Fig. 1.2.1.1 Lipid droplets in yeast S. cerevisiae. Localization and morphology of lipid droplets in wild-type yeast strain BY4742. (A) Erg6p-RFP labeled lipid droplets; (B) Oil Red O-stained lipid droplets; (C) Electron microscopy image of oleic acid induced yeast cell; C, cytosol; ER, endoplasmic reticulum; L, lipid droplets; M, mitochondria; N, nucleus; P, peroxisome.

In contrast to the vesicular organelles, which have the aqueous content enclosed by a

phospholipid bilayer membrane (50, 55), mature LDs have a unique physical structure: they

have a neutral lipid core consisting of triacylglycerols (TG) and sterol esters (SE) surrounded

by a phospholipid monolayer (15, 132, 239) that contains numerous peripheral or embedded

proteins (143, 207) (Fig. 1.2.1.2).

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Fig. 1.2.1.2 Lipid droplets composition. Taken with modifications from (73).

TG, as well as SE, play a crucial role for the cell: TG is the main energy store, and

both TG and SE are depots of membrane lipid components (221). LDs can tightly regulate the

level of intracellular free cholesterol by hydrolyzing sterol ester (143). The LD core also

contains other endogenous neutral lipids, like monoacylglycerol, diacylglycerol, free

cholesterol, and retinol ester, and xenobiotic hydrophobic compounds, such as polycyclic

aromatic hydrocarbons (73, 94, 195, 205, 207). A number of proteins are specifically targeted

to the LD surface (95), where they can regulate LD dynamics and the turnover of stored lipids

(132). Lipid-metabolizing enzymes, including hydrolases and lipases, are the major classes of

LD enzymes (37). LDs play crucial roles in cellular energy homeostasis and lipid metabolism

(221). LDs can provide a rapidly mobilized lipid source for many important biological

processes. Neutral lipids may be mobilized for the generation of energy by β-oxidation (191,

219) or for the synthesis of membrane lipids and signalling molecules (37). It has been shown

that all cell types have the ability to generate LDs in response to elevated fatty acid levels and

to subsequently metabolize and disperse these LDs when conditions are reversed (143),

thereby providing an emergency energy pool for cell survival (15). Due to their unique

architecture, LDs can protect cells from the effects of potentially toxic lipid species, such as

unesterified lipids (117, 132) or toxic free fatty acids (15), by depositing them inside the LD’s

core. In addition to this lipid scavenging function, LDs can transiently store certain proteins,

which may be released or degraded at later time points (37, 55, 64, 230). LDs interact with

other organelles such as peroxisomes, endosomes, endoplasmatic reticulum (ER), plasma

membrane, mitochondria and caveolae (73). The obesity and type 2 diabetes mellitus are most

common lipid droplets-associated disorders caused by impairment of triacylglycerol (TAG)

metabolism (112). The key anabolic and catabolic enzymes involved in TAG metabolism are

conserved between yeast and mammals (8, 32, 175).

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1.2 Biology of lipid droplets

1.2.2 Biogenesis of lipid droplets

Biogenesis of LDs is tightly connected to the ER (15, 37, 73) (Fig. 1.2.2.1). Several

models were recently proposed for description of LDs de novo biosynthesis. In the ER-

budding model, the neutral lipids accumulate in the interspace between the bilayer leaflets of

the ER membrane that subsequently budding-out of the cytoplasm-oriented phospholipids

hemimembrane with formation of the nascent LDs (15, 73).

Fig. 1.2.2.1 Lipid droplets in adipocytes. (a) 3T3-L1 adipocytes that have been stimulated to induce lipolysis and then labelled for Rab18 (red) and neutral lipids (green). Rab18 is specifically recruited to the surface of a subset of lipid droplets (LDs). The scale bar represents 10 µm. (b) High-pressure frozen 3T3-L1 adipocytes that were processed for electron-microscopy observation after freeze substitution. Note the complexity of the membranes that wrap around and associate with LDs (such complexity represent result of interaction of lipid droplest with endoplasmic reticulum membranes). The scale bar represents 1 µm. Taken with modifications from (143).

In the ER-domain model, the LDs remain fused to the ER and are lipid-bearing

protrusions of the ER membrane, developing a specialized ER domain (73). In the bicelle

model, neutral lipids aggregate between the two leaflets of the ER membrane but, instead of

budding, nascent LDs are excised from the membrane, acquire phospholipids from both the

cytosolic and luminal leaflets (73, 173). In the vesicular-budding model, tiny immature

bilayer vesicles that remain attached to the ER membrane are exploited as a precursor for LDs

formation. Neutral lipids are supplied into the vesicle bilayer and blow up the intermembrane

volume, finally squeezing the vesicular lumen so that it becomes a tiny incorporation inside

the LDs (73, 221) (Fig. 1.2.2.2).

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Fig. 1.2.2.2 Lipid droplets biogenesis models. Taken with modification from (73).

1.2 Biology of lipid droplets

1.2.3 Interactions between peroxisome and lipid droplets

Peroxisomes are frequently shown to be tightly associated to lipid droplets (18, 28, 64, 65, 78,

159). It was clearly demonstrated by J. Goodman that lipid droplets and peroxisomes have

tight physiological interconnections. It was shown that oleic acid induced peroxisomes of

yeast S. cerevisiae are stabily associated with lipid droplets by formation of tubular-shaped

protrusions into the lipid droplets cores (18). It was demonstrated that peroxisomes can invade

lipid droplets with pexopodia and establish peroxisome – lipid droplets synapses. Such close

contacts could facilitate lipid molecules bidirectional transport across two organelles (18). For

instance, ether lipids, which are normally synthesized in peroxisomes, were shown to be

highly enriched in the lipid droplets core of several cell types (13, 18). Some of the

peroxisomes inside of lipid droplets constellations are often shown to be dumbbell-shaped,

indicating a dependence of the peroxisomal fission (24, 66) on lipid droplets close physical

association (21). It was shown in plants that glyoxysomal (peroxisomal) membrane lipids of

germinating cotton seeds have exclusively lipid droplets but not endoplasmic reticulum origin

(28). In that case triacylglycerols as well as fatty acids were shown to be directly trafficking

from lipid droplets to glyoxysomes. Such an observation can indicate about requirements of

lipid droplets in peroxisomal maturation and/or fission (64) in contrary to known fact that

endoplasmic reticulum membranes are the source of pre-peroxisomal vesicles (84). Moreover,

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it was shown in plant that cotyledons of a Δped1 strain (strains lacking peroxisomal fatty acid

β-oxidation pathway) have a substantial portion of tight physical contact of lipid droplets and

glyoxysomes, with tubular structures within the glyoxysomes that appear to be derived from

lipid droplets; possibly these formations are system of transportation of triacylglycerols for

glyoxysomal β-oxidation (78). It was demonstrated that yeast Saccharomyces cerevisiae can

form extensive long-term contacts between peroxisome and lipid droplets in case of their

culturing on medium containing oleic acid as a sole carbon source; in case of yeast culturing

on glucose medium only transient interactions were observed (18). Fungi commonly exhibit

peroxisome – lipid droplet intimae association. In yeast Yarrowia lipolytica grown in oleic

acid medium, many peroxisomes in a temperature-sensitive Δpex3 mutant strain (strain

partially deficient in pre-peroxisome budding from the ER) wrap around lipid droplets, as if

attempting to access core lipids for membrane assembly (14, 18). Furthermore, animal cells

also were shown to demonstrate an extensive association of peroxisomes with lipid droplets in

cultured COS-7 cells (18, 187).

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1.3 Objectives

The goal of this work was to study the biogenesis of peroxisomes and lipid droplets in yeast S.

cerevisiae. In the first part of the thesis, the objectives were to characterize the Lpx1p protein:

(1) prove the localization of Lpx1p in the peroxisome; (2) show the role of the peroxisomal

targeting signal type 1 (PTS1) in the targeting of Lpx1p to the peroxisome; (3) demonstrate the

dependence of Lpx1p transport to the peroxisome on the peroxisomal shuttling receptor Pex5p;

(4) prove the existence of piggyback-transport of Lpx1p into the peroxisomes; (5) express the

recombinant Lpx1p in Escherichia coli; (6) prove the existence of the hydrolase and

triacylglycerol lipase activities for Lpx1p in vitro; (7) investigate the role of Lpx1p in the

peroxisome metabolism and/or the biogenesis.

In the second part of the thesis, the objectives were to characterize the peroxisomal

AAA (ATPases Associated with diverse cellular Activities) ATPase proteins Pex1p and

Pex6p. In the third part of the thesis, the objectives were to characterize Ubp15p protein: (1)

prove the partial localization of Ubp15p in the peroxisome; (2) express the recombinant Ubp15p

in Escherichia coli; (3) prove the existence of the ubiquitin hydrolase, monoUb-Pex5p and/or

polyUb-Pex5p deubiquitinating activities for Ubp15p in vitro; (4) demonstrate the role of

Ubp15p in peroxisome metabolism and/or biogenesis (show some Ubp15p specific

phenotype); (5) show physical interaction of Ubp15p with components of peroxisomal export

machinery (Pex6p); (6) prove the existence of Ub-Pex5p dislocase and deubiquitinating

activities in the purified from yeast AAA-ATPase complex in vitro (show association of

deubiquitinating activity with dislocase activity in the endogenously expressed AAA-ATPase

complex); (7) show the role of Ubp15p in the cycle of shuttling receptor Pex5p; (8) prove the

requirement of enzymatic deubiquitination of Ub-Pex5p in vivo; (9) show steady state level of

Ub-Pex5p in wild-type and Δubp15 yeast strains.

In the forth part of the thesis, the objectives were to characterize Ldh1p protein: (1)

show the localization of Ldh1p (it was shown localization the lipid droplets); (2) show the role of

the peroxisomal targeting signal type 1 (PTS1) in the targeting of Ldh1p to the lipid droplets;

(3) demonstrate the dependence of Ldh1p transport to the lipid droplets on the peroxisomal

shuttling receptor Pex5p; (4) express the recombinant Ldh1p in Escherichia coli; (5)

investigate the existence of the hydrolase and triacylglycerol lipase activities for Ldh1p in

vitro; (6) show the role of conserved serine in the hydrolase/lipase motif GXSXG for enzyme

activity; (7) elucidate the role of Ldh1p in the lipid droplets metabolism and/or the biogenesis

(show some Ldh1p specific phenotype).

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Lpx1p is a peroxisomal lipase required for normalperoxisome morphologySven Thoms1,*, Mykhaylo O. Debelyy1, Katja Nau1,†, Helmut E. Meyer2 and Ralf Erdmann1

1 Institut fur Physiologische Chemie, Abteilung fur Systembiochemie, Ruhr-Universitat Bochum, Germany

2 Medizinisches Proteomcenter, Ruhr-Universitat Bochum, Germany

Peroxisomes are ubiquitous eukaryotic organelles that

are involved in lipid and antioxidant metabolism.

They are versatile and dynamic organelles engaged in

the b-oxidation of long and very long chain fatty

acids, in a-oxidation, bile acid and ether lipid synthe-

sis, and in amino acid and purine metabolism [1].

Peroxisomes are a source of reactive oxygen species,

and are involved in the synthesis of signalling mole-

cules in plants. Remarkably, peroxisomes are the only

site of fatty acid b-oxidation in plants and fungi.

Human peroxisomal disorders can be categorized

as either single-enzyme disorders or peroxisomal

biogenetic defects [2]. Single-enzyme disorders, for

example Refsum disease caused by a defect of

phytanoyl CoA hydroxylase, or X-linked adrenoleu-

kodystrophy caused by a defect in a peroxisomal

ATP-transporter. Biogenetic defects are mostly caused

by mutations in the peroxisomal biogenesis genes,

the PEX genes, that code for peroxins [3]. Peroxi-

somal disorders are associated with morphological

Keywords

lipase; peroxin; peroxisome; proteomics;

PTS1

Correspondence

R. Erdmann, Abteilung fur

Systembiochemie, Ruhr-Universitat

Bochum, Universitatsstr. 150,

44780 Bochum, Germany

Fax: +49 234 32 14266

Tel: +49 234 322 4943

E-mail: [email protected]

Present address

*Universitatsmedizin Gottingen, Abteilung

fur Padiatrie und Neuropadiatrie, Georg-

August-Universitat, Germany

†Forschungszentrum Karlsruhe, Institut fur

Toxikologie und Genetik, Germany

(Received 20 September 2007, revised 22

November 2007, accepted 30 November

2007)

doi:10.1111/j.1742-4658.2007.06217.x

Lpx1p (systematic name: Yor084wp) is a peroxisomal protein from Saccha-

romyces cerevisiae with a peroxisomal targeting signal type 1 (PTS1) and a

lipase motif. Using mass spectrometry, we have identified Lpx1p as present

in peroxisomes, and show that Lpx1p import is dependent on the PTS1

receptor Pex5p. We provide evidence that Lpx1p is piggyback-transported

into peroxisomes. We have expressed the Lpx1p protein in Escherichia coli,

and show that the enzyme exerts acyl hydrolase and phospholipase A activ-

ity in vitro. However, the protein is not required for wild-type-like steady-

state function of peroxisomes, which might be indicative of a metabolic

rather than a biogenetic role. Interestingly, peroxisomes in deletion mutants

of LPX1 have an aberrant morphology characterized by intraperoxisomal

vesicles or invaginations.

Abbreviations

BPC, 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-undecanoyl)-sn-glycero-3-phosphocholine (bis-BODIPY-FL C11-PC);

DGR, 1,2-O-dilauryl-rac-glycero-3-glutaric acid (6-methyl resorufin) ester; DPG, 1,2-dioleoyl-3-(pyren-1-yl)decanoyl-rac-glycerol;

PNB, p-nitrobutyrate.

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peroxisomal defects such as inclusions or invagin-

ations [4,5].

Peroxisomal import of most matrix proteins depends

on the PTS1 (peroxisomal targeting signal type 1)

receptor Pex5p, which recognizes the PTS1 localized at

the very C-terminus [6,7]. The three-amino-acid signal

SKL (serine–lysine–leucine) was the first PTS1 to be

discovered, and is in many cases sufficient for directing

a protein to peroxisomes. Most PTS1 are tripeptides of

the consensus [SAC][KRH][LM] located at the extreme

C-terminus.

A second matrix protein peroxisomal targeting sig-

nal (PTS2) is present in considerably fewer peroxi-

somal proteins. PTS2 is usually located within the first

20 amino acids of the protein, and has been defined

as [RK][LVIQ]XX[LVIHQ][LSGAK]X[HQ][LAF] [8].

PTS2-bearing proteins are recognized by the cytosolic

receptor Pex7p.

Systems biology approaches led to the identification

of Lpx1p as an oleic acid-inducible peroxisomal matrix

protein of unknown function [9,10]. The gene sequence

of LPX1 predicts a lipase motif of the GxSxG type

that is typical for a ⁄ b hydrolases [11,12]. Using mass

spectrometry, we identify Lpx1p as present in peroxi-

somes, and analyse its peroxisomal targeting. We show

that it acts as a phospholipase A, and, by electron

microscopy and morphometry, we provide the first evi-

dence for an interesting peroxisomal phenotype of the

Dlpx1 deletion mutant.

Results

Identification of Lpx1p in peroxisomes by mass

spectrometry

We identified Lpx1p (lipase 1 of peroxisomes;

EC 3.1.1.x) in a follow-up study to an exhaustive pro-

teomic characterization of peroxisomal proteins [13].

This involved purification of peroxisomes from oleic-

acid induced Saccharomyces cerevisiae, and subsequent

membrane extraction using low- and high-salt buffers.

Low-salt-extractable proteins were solubilized in SDS

buffer, and separated by RP-HPLC [14]. Proteins in

individual HPLC fractions were further separated by

SDS–PAGE, and protein bands were cut out and anal-

ysed by mass spectrometry. Lpx1p (systematic name:

Yor084wp) was extractable by low salt and identified

together with the peroxisomal aspartate aminotransfer-

ase Aat2p in HPLC fraction 7 at a molecular mass of

approximately 45 kDa (Fig. 1A) [15].

The predicted molecular mass of Lpx1p is 44 kDa.

It carries a peroxisomal targeting signal type 1, gluta-

mine–lysine–leucine (QKL) (Fig. 1B,D). The amino

acid sequence comprises the lipase motif GHSMG of

the general GxSxG type [11,16] with the central serine

being part of the catalytic triad. This lipase motif is

indicative of a ⁄ b hydrolase family members [12].

Hydrophobicity predictions [17] indicate a pronounced

hydrophobic region in the central domain, consisting

of amino acids 154–177 with the core region 164LLI-

LIEPVVI173 (Fig. 1C).

By homology searches with other prokaryotic and

eukaryotic hydrolases (not shown) using profile hidden

Markov models [18], we identified a conserved histi-

dine that is probably part of the catalytic triad of the

active site (Fig. 1B). The third member of the catalytic

triad could not be identified by sequence-based

searches.

PTS1-dependent targeting of Lpx1p

to peroxisomes

The majority of the Lpx1p in a cell homogenate was

pelleted at 25 000 g, consistent with an organellar

localization of the protein (Fig. 2A). In this experi-

ment, more of the peroxisomal soluble thiolase Fox3p

(EC 2.3.1.x) than of Lpx1p appears to be present in

the supernatant. This is probably due to partial peroxi-

some rupture during preparation, and might indicate

that Lpx1p, in contrast to Fox3p, is loosely associated

with the peroxisomal membrane.

The peroxisomal localization of Lpx1p had been

demonstrated indirectly by immuno-colabelling of a

heterozygous C-terminally Protein A-tagged version

of Lpx1p in a diploid strain [10]. Peroxisomal locali-

zation under these conditions would depend on the

presence of copies of Lpx1p that are not blocked by

a C-terminal tag, and by the interaction of Lpx1p

with itself (piggyback import). We wished to analyse

whether Lpx1p directly localized to peroxisomes, and

cloned LPX1 for expression from a yeast shuttle

plasmid using an N-terminal GFP tag. This fusion

protein was localized to peroxisomes in a Dlpx1 dele-

tion strain (Fig. 2B), indicating that Lpx1p by itself

targets to peroxisomes. Peroxisomal localization of

Lpx1p was abolished when Lpx1p was expressed with

a C-terminal tag (Fig. 2C), indicating that the

C-terminus has to be free for Pex5p-dependent

import. Peroxisomal localization was abolished in the

absence of Pex5p (Fig. 2C), and was not affected by

the absence of Pex7p (Fig. 2C), indicating that its

targeting to peroxisomes is dependent on the PTS1

pathway.

We confirmed the peroxisomal localization of Lpx1p

by subcellular fractionation. On a sucrose density gra-

dient, GFP–Lpx1p co-migrated with Fox3p (alternative

S. Thoms et al. Peroxisomal lipase Lpx1p

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name: Pot1p), with Pex11p, and with the catalase (EC

1.11.1.6) activity peak in the same density fraction at

about 1.225 gÆcm)3 (fraction 10) (Fig. 2D). The activ-

ity of the mitochondrial marker fumarase (EC 4.2.1.2)

together with the mitochondrial Mir1p showed a

clearly separate peak at a density of 1.192 gÆcm)3 in

fraction 14 (Fig. 2D).

Lpx1p was identified from low-salt-extractable mem-

branes (Fig. 1A), and the amount of Lpx1p that is not

membrane-associated or found in the non-peroxisomal

low-density fractions (Fig. 2D; fractions 19–29) is low

compared to Fox3p.

Although the QKL C-terminus of Lpx1p does not

match the PTS1 consensus [SAC][KRH][LM], a QKL

terminus is able to target a test substrate to peroxi-

somes [19]. Lpx1p is one of four S. cerevisiae proteins

that end in QKL (Fig. 1D), and is probably the only

one that is localized to peroxisomes.

Self-interaction of Lpx1p

C-terminally tagged Lpx1p localizes only to peroxi-

somes when endogenous copies of the protein are pres-

ent [10]. This suggests piggyback import of Lpx1p,

which, in turn, would rely on self-interaction of Lpx1p.

We tested this hypothesis by two-hybrid analysis of

LPX1. Neither the fusion of Lpx1p with the GAL4

binding domain nor its fusion with the activation

domain were auto-activating (Fig. 3A). The strains

expressing both fusions exhibit a strong two-hybrid

interaction signal, exceeding that of the control PEX11

with PEX19 (Fig. 3A). Because complex formation

Fig. 1. Identification of Lpx1p from Saccharomyces cerevisiae

peroxisomes by proteomics. (A) Isolation of putative peroxisomal

proteins by preparative chromatographic separation. Low salt-

extractable peroxisomal proteins were solubilized by SDS and

separated by reverse phase HPLC. Polypeptides of selected frac-

tions were separated by SDS–PAGE and visualized by Coomassie

blue staining. Only the first 13 lanes of the HPLC profile are shown

[15]. The band marked by an asterisk contains the peroxisomal

proteins Lpx1p (predicted molecular mass 44 kDa) and Aat2p (pre-

dicted molecular mass 44 kDa) in HPLC fraction 7 at a molecular

mass of approximately 45 kDa. (B) Alignment of the LPX1 gene

with a Mycoplasma genitale (Mg) gene encoding a putative ester-

ase ⁄ lipase (AAC71532) and with the putative triacylglycerol lipase

AAB96044 from Mycoplasma pneumoniae (Mp). Identical amino

acids are indicated by an asterisk and similar amino acids are indi-

cated by a colon and full point, depending on degree of similarity.

The conserved GxSxG lipase motif is shaded in grey. The lipase

motif contains the putative active-site serine. The arrowhead

indicates the probable active-site histidine, as determined from

alignments using eukaryotic esterase lipase family members (not

shown). The third member of the catalytic triad could not be identi-

fied by sequence-based analysis. (C) Hydropathy plot of Lpx1p.

A Kyte–Doolittle plot was calculated with window size of 11.

Values > 1.8 may be regarded as highly hydrophobic regions.

(D) Termini of all four QKL proteins from S. cerevisiae. Only Lpx1p

is predicted to target to peroxisomes. Positions relative to the

(putative) PTS1 are indicated. Grey boxes, lysine in position -1 and

valine in position -5 are probably required to target Lpx1p to

peroxisomes.

Peroxisomal lipase Lpx1p S. Thoms et al.

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might play a significant role in peroxisomal (piggyback)

protein import [20], we determined the size of the Lpx1p

complex by gel filtration of cell lysates of oleate-induced

cells on a Superdex 200 column. We found that the

majority of Lpx1p is not present in high-molecular-

mass complexes, but elutes at molecular masses cor-

responding to monomers, dimers and trimers (Fig 3B).

The two-hybrid interaction probably reflects the com-

plex formation. However, our identification of low-

molecular-mass complexes of Lpx1 does not exclude

the possibility that higher-molecular-mass complexes

are transiently formed during topogenesis of the protein.

Lpx1p is not required for peroxisome biogenesis

Having shown that Lpx1p is targeted to peroxisomes

by the soluble PTS1 receptor, we wished to determine

whether Lpx1p is required for the biogenesis of peroxi-

somes. We first tested the Dlpx1 knockout for growth

on oleate. However, Lpx1p is dispensable for growth

on oleate as the only carbon source (Fig. 4A). To

determine the influence of Lpx1p on peroxisome bio-

genesis in more detail, post-nuclear supernatants were

prepared from wild-type and Dlpx1 strains. The post-

nuclear supernatants were analysed by Optiprep gradient

analysis and subsequent tests of gradient fractions for

peroxisomal catalase and mitochondrial cytochrome c

oxidase (EC 1.9.3.l; Fig. 4B). None of these marker pro-

teins indicated a significant change in the abundance or

density of peroxisomes or mitochondria, suggesting

that peroxisomal and mitochondrial biogenesis remain

functional after deletion of the LPX1 gene.

Lipase activity of Lpx1p

Characteristic GxSxG motifs and similarities with

a ⁄ b hydrolases in the predicted protein sequence sug-

gest that Lpx1p is an esterase, possibly a lipase

[11,12,16]. To directly investigate Lpx1p, we expressed

the full-length protein as a fusion protein with a C-ter-

Fig. 2. Localization and PTS1-dependent targeting of Lpx1p to peroxisomes. (A) Immunological detection of GFP–Lpx1p in a sedimentation

experiment. A cell-free homogenate (T) was separated into supernatant (S) and an organelle-containing pellet fraction (P) by centrifugation at

25 000 g (30 min). Amounts corresponding to equal T content of each fraction were analysed by SDS–PAGE and western blotting with

antibodies against GFP and the peroxisomal marker protein oxoacyl CoA thiolase, Fox3p (alternative name: Pot1p). (B) Lpx1p is localized to

peroxisomes. Coexpression of PTS2-dsRed and GFP–Lpx1p in yeast cells. Cells were grown on ethanol to induce the expression of PTS2-

dsRed. (C) Import of Lpx1p into peroxisomes is dependent on Pex5p and independent of Pex7p. Lpx1p was expressed as either a C-terminal

fusion (top images) or N-terminal fusion (bottom images) with GFP. In the Dpex5 deletion mutant, Lpx1p cannot be imported into peroxi-

somes, irrespective of the position of the tag (right). Deletion of PEX7 does not influence Lpx1p targeting if the PTS1 is not blocked by GFP

(top left). GFP fusion proteins that are not targeted to peroxisomes mislocalize to the cytosol. Bar = 2 lm. (D) Sucrose density gradient anal-

ysis of GFP–LPX1-transformed yeast. A cell-free organelle sediment from oleate induced cells was analysed on a density gradient with

sucrose concentrations form 32 to 54% w ⁄ v. Individual fractions were analysed for catalase activity (peroxisomal marker) and fumarase

activity (mitochondrial marker). In addition, the presence of GFP–Lpx1p, Fox3p, Pex11p (peroxisomal membrane protein) and Mir1p (mito-

chondrial phosphate carrier) was tested by western blotting and immunodetection.

S. Thoms et al. Peroxisomal lipase Lpx1p

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minal hexahistidine tag in Escherichia coli and purified

the protein using immobilized metal-ion affinity chro-

matography (Fig. 5A). The protein was further puri-

fied by gel filtration on a Superdex 200 column

(Fig. 5B). Gel filtration indicated the propensity of

Lpx1p to oligomerize in vitro, albeit to a much lower

extent than in yeast whole-cell lysates (compare

Figs 3B and 5B).

Purified protein was used for the generation of poly-

clonal antibodies in rabbit. Antisera recognized a pro-

tein of about 43 kDa, indicating that the antiserum is

specific for Lpx1p. We used these antibodies to con-

firm that the endogenous yeast Lpx1p is induced by

oleic acid (Fig. 5A).

To analyse the enzyme activity of Lpx1p, we assayed

the E. coli-expressed protein for esterase activity, using

p-nitrophenyl butyrate (PNB) as the test substrate. PNB

can be hydrolysed by esterases, yielding free p-nitro-

phenol, which can be determined photometrically at

410 nm. Lpx1p hydrolysed the test substrate with a

KM of 6.3 lm and Vmax of 0.17 lmolÆs)1 (Table 1).

Lpx1p is strongly induced by oleic acid, regulated by

stress-associated transcription factors [21], and aligns

with human epoxide hydrolases (EC 1.14.99.x; not

shown). We found that Lpx1 hydrolysed the epoxide

hydrolase substrate [22] 4-nitrophenyl-trans-2,3-epoxy-

3-phenylpropyl carbonate (NEPC) (data not shown),

but we consider that this activity is non-specific,

because it could not be blocked by the specific epoxide

hydrolase inhibitor N,N’-dicyclohexylurea (DCU) (data

not shown).

To test for lipase activity, we used 1,2-dioleoyl-3-

(pyren-1-yl)decanoyl-rac-glycerol (DPG) as a substrate.

DPG contains the eximer-forming pyrene decanoic

acid as one of the acyl residues. Upon cleavage, the

free pyrene decanoic acid shows reduced eximer

fluorescence. Lpx1p exerts lipase activity towards

DPG of 5.6 pmolÆh)1Ælg)1 (Table 1). For comparison,

we measured the lipase activity of commercial yeast

Candida rugosa lipase towards DPG and found an

Fig. 3. Lpx1p interacts with itself. (A) Two-hybrid assay. Full-length

Lpx1p was fused to the GAL4 binding or activation domain and co-

expressed in a yeast strain with Escherichia coli b-galactosidase

under the control of a GAL4-inducible promotor. b-galactosidase

activity was measured in lysates of doubly transformed strains. No

signal was obtained when LPX1 was combined with empty

vectors. Positive control: interaction of Pex19p with Pex11p.

(B) Size-exclusion chromatography of a wild-type cell lysate of

oleate-induced cells. The lysate was fractionated by gel filtration

on a Superdex 200 column and tested by immunoblotting with

anti-Lpx1p antiserum. The molecular masses indicated were

interpolated from a calibration curve and correspond well with

monomeric, dimeric and trimeric forms of Lpx1p. The relative

distribution of the three forms was quantified using NIH Image

(National Institutes of Health, Bethesda, MD, USA). The elution vol-

ume is indicated in millilitre.

Fig. 4. Absence of pex phenotype in a Dlpx1 deletion. (A) Growth

on plates with oleate as the only carbon source. Wild-type, Dlpx1 or

Dpex1 control stains were spotted in equal cell numbers in series of

10-fold dilutions on oleate or ethanol plates. Absence of growth and

oleic acid consumption (halo formation) indicates a peroxisomal

defect. Control: growth assay on ethanol. (B) Optiprep density gradi-

ent centrifugation analysis of postnuclear supernatants prepared

from oleate-induced wild-type and Dlpx1 strains. All fractions were

analysed using catalase (peroxisome) and cytochrome c oxidase

(mitochondria) enzyme assays. The peroxisomal and mitochondrial

densities were not measurably altered by LPX1 deletion.

Peroxisomal lipase Lpx1p S. Thoms et al.

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activity of 2.0 pmolÆh)1Ælg)1 under the same assay con-

ditions (Table 1).

We sought to confirm lipase activity by testing

Lpx1p in a clinical assay for pancreatic lipase. The

assay uses the substrate 1,2-O-dilauryl-rac-glycero-

3-glutaric acid (6-methyl resorufin) ester (DGR) in a

desoxycholate-containing buffer. Lpx1p did not hydro-

lyse this substrate under the assay conditions (Table 1).

Next we tested for phospholipase C activity in a

coupled enzyme assay with phosphatidylcholine as the

substrate. In this assay, phospholipase C converts

phosphatidylcholine to phosphocholine and diacylglyc-

erol. Alkaline phosphatase hydrolyses phosphocholine

to form choline, which is then oxidized by choline

oxidase to betaine and hydrogen peroxide. The latter,

in the presence of horseradish peroxidase, reacts with

10-acetyl-3,7-dihydrophenoxazine to form fluorescent

resorufin. This assay, as well as a similar assay for

phospholipase D, gave negative results for Lpx1p

(Table 1).

Finally, we tested phospholipase A (EC 3.1.1.4)

activity using the substrate 1,2-bis-(4,4-difluoro-5,7-

dimethyl-4-bora-3a,4a-diaza-sindacene-3-undecanoyl)-

sn-glycero-3-phosphocholine (bis-BODIPY-FL C11-PC,

BPC). BPC is a glycerophosphocholine with BODIPY

dye-labeled sn-1 and sn-2 C11 acyl chains. Cleavage

reduces dye quenching and leads to a fluorecence

increase at 530 nm upon excitation at 488 nm. Lpx1p

exerts phospholipase A activity of 7.9 pmolÆh)1Ælg)1.

As a control enzyme, we used commercial porcine

pancreas lipase, which hydrolysed 195 pmolÆh)1Ælg)1.

In summary, Lpx1p shows acyl esterase, lipase and

phospholipase A activity towards PNB, DPG and

BPC, respectively.

Altered peroxisome morphology in deletion

mutants of LPX1

Lastly, we analysed electron microscopic (EM) images

of knockouts of LPX1. To our surprise, a large

number of Dlpx1 peroxisomes showed an abnormal

morphology. The peroxisomes appear vesiculated

Fig. 5. Protein expression, antibody genera-

tion and oleate induction of Lpx1p. Expres-

sion of Lpx1p in Escherichia coli. (A) Lpx1p

was expressed as a fusion protein with a

C-terminal hexahistidine tag and purified by

His-trap chromatography. The purified Lpx1p

(lane 1) was used to generate polyclonal

antibodies in rabbit that recognize the puri-

fied recombinant protein (lane 4). Endoge-

nous Lpx1p in whole yeast lysates is

recognized only after induction of peroxi-

somes and Lpx1p by oleate (lane 2 versus

lane 3). Molecular masses are shown in

kDa. (B) Second purification step: gel filtra-

tion on Superdex 200 column. The elution

profile indicates that most of the protein

behaves as a monomer, but a small propor-

tion forms dimers and trimers.

Table 1. Esterase, lipase, and phospholipase activity of Lpx1p.

Esterase activity was measured using PNB (p-nitrobutyrate) as a

substrate. KM and Vmax values were calculated using Michaelis–

Menten approximations. Lipase activity was determined using DPG

as a substrate. Activity was measured from two independent pro-

tein preparations in triplicate. Candida rugosa lipase (CRL) was used

as a positive control for lipase measurement. (Pancreas) lipase

activity assays used DGR in a coupled enzyme assay as a sub-

strate. Phospholipase C and D (PLC and PLD) activities were mea-

sured in coupled enzyme assays using phosphatidylcholine (PC).

Phospholipase A measurements used BPC (bis-BODIPY-FL C11-PC)

as a test substrate. Porcine pancreas lipase (PPL) was used as a

control.

Enzyme Substrate Activity

Activity parameters

(pmolÆh)1Ælg)1)

Lpx1p PNB Acyl esterase KM 6.3 lM;

Vmax 0.17 lmolÆs)1

Lpx1p DPG (Triacylglycerol)

lipase

5.6 ± 1.5

CRL DPG (Triacylglycerol)

lipase

2.0 ± 0.1

Lpx1p DGR (Pancreas) lipase Below detection limit

Lpx1p PC PLC Below detection limit

Lpx1p PC PLD Below detection limit

Lpx1p BPC PLA 7.9

PPL BPC PLA 195

S. Thoms et al. Peroxisomal lipase Lpx1p

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(Fig. 6B), and either contain intraperoxisomal vesicles

or their membrane is grossly invaginated. On average,

one vesiculated peroxisome is visible in every fifth

mutant cell (Fig. 6E). When the average number of

altered peroxisomes is counted, we find that every

third peroxisome shows this vesiculation phenotype

(Fig. 6D). This is a high percentage, considering the

fact that the peroxisomes were viewed in thin micro-

tome sections. In three dimensions, every single peroxi-

some might contain a vesicular membrane or

indentation that escapes notice in two-thirds of the

‘two-dimensional’ sections.

The average number of peroxisomes per cell is

insignificantly increased in Dlpx1 (2.95 versus 2.76,

Fig. 6C). Wild-type cells did not contain any vesicu-

lated peroxisome (Fig. 6A,D,E). The drastic phenotype

of Dlpx1 is reminiscent of the peroxisomal morphology

found in peroxisomal disorders.

Discussion

Lpx1p is a peroxisomal protein with an unusual

PTS1

LPX1 is one of the most strongly induced genes fol-

lowing a shift from glucose to oleate, as determined by

serial analysis of gene expression (SAGE) experiments

[9]. The oleate-induced increase in mRNA abundance

is abolished in the Dpip2 Doaf1 double deletion strain,

indicating that its induction is dependent on the tran-

scription factor pair Pip2p and Oaf1p [9]. The Lpx1p

protein itself is induced by oleic acid as determined

using a Protein A tag [10] or by use of an antibody

raised against Lpx1p (see Results).

Lpx1p does not conform to the general PTS1 con-

sensus. The other three QKL proteins in S. cerevisiae

are probably not peroxisomal (Fig. 1D): Efb1p

(systematic name: Yal003wp) is the elongation factor

EF-1b [23], Rpt4p (Yor259cp) is a mostly nuclear

19S proteasome cap AAA protein [24], and Tea1p

(Yor337wp) is a nuclear Ty1 enhancer activator [25].

However, QKL is sufficient to sponsor Pex5p binding

[19]. Why are these QKL proteins not imported into

peroxisomes? This is probably due to the upstream

sequences. Lpx1p has a lysine at position -1 (relative

to the PTS1 tripeptide) and a hydrophobic amino acid

at position -5 (Fig. 1D). These features promote Pex5p

binding and are not found in the other three QKL

proteins (Fig. 1D) [19]. Our views were confirmed

by applying a PTS1 prediction algorithm (http://

mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp)

[26], which predicted peroxisomal localization for

Lpx1p only of the four proteins listed in Fig. 1D.

Fig. 6. Peroxisome morphology phenotype of the Dlpx1 deletion.

Absence of LPX1 leads to drastic peroxisomal vesiculation or invagi-

nation. Electron microscopic images of cells from (A) wild-type and

(B) Dlpx1. All cells were grown on medium with 0.1% oleic acid. Per-

oxisomes are marked by arrowheads. Bar = 2 lm. (C) Comparison of

per cell peroxisome numbers in wild-type and Dlpx1 strains. (D) Aver-

age number of vesicles per peroxisome (wild-type, n = 94; Dlpx1,

n = 142). In Dlpx1, about every third peroxisome contains a vesicle.

(E) Percentage of cells with vesicle-containing peroxisomes. Roughly

one in five Dlpx1 cells carries peroxisomes with a vesicle or invagi-

nations (wild-type, n = 34; Dlpx1, n = 48). px, peroxisome(s).

Peroxisomal lipase Lpx1p S. Thoms et al.

510 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS

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Lipase activity and cellular function of Lpx1p

Lpx1p could be involved in various processes: (a)

detoxification and stress response, (b) lipid mobiliza-

tion, or (c) peroxisome biogenesis. As Lpx1p expres-

sion may be regulated by Yrm1p and Yrr1p [21], a

transcription factor pair that mediates pleiotropic drug

resistance effects, we speculate that Lpx1p is required

for a multidrug resistance response that did not show

a phenotype in our experiments. We could, however,

exclude epoxide hydrolase activity for Lpx1p, because

hydrolysis of the epoxide hydrolase test substrate

was not affected by a specific epoxide hydrolase

inhibitor.

We investigated the dimerization of Lpx1p in the

context of piggyback protein import into peroxisomes.

Self-interaction (dimerization) is frequently found in

regulation of the enzymatic activity of other lipases

such as C. rugosa lipase or human lipoprotein lipase

[27,28]. The putative active-site serine of Lpx1p is

located next to the region of highest hydropathy, sug-

gesting that Lpx1p is a membrane-active lipase that

contributes to metabolism or the membrane shaping of

peroxisomes.

Peroxisomes are sites of lipid metabolism. It is thus

not surprising to find a lipase associated with peroxi-

somes. Our experiments show that Lpx1p has triacyl-

glycerol lipase activity; however, activities towards the

artificial test substrates DPG and DGR were low. Our

evidence for phospholipase A activity of the enzyme,

together with the EM phenotype, suggest that Lpx1

has a more specialized role in modifying membrane

phospholipids.

Recently, a mammalian group VIB calcium-indepen-

dent phospholipase A2 (iPLA2c) was identified that

possesses a PTS1 SKL and a mitochondrial targeting

signal [29,30]. The enzyme is localized in peroxisomes

and mitochondria, and is involved, among others, in

arachinoic acid and cardiolipin metabolism [31,32].

Knockout mice of iPLA2c show mitochondrial ⁄ cardio-logical phenotypes [33]. It will be exciting to determine

whether human iPLA2c and yeast Lpx1p are function-

ally related.

We have provided evidence that peroxisomes are still

functional in the absence of LPX1. This suggests a

non-essential metabolic role for Lpx1p in peroxisome

function. The morphological defect found in electron

microscopic images of a deletion of Lpx1p

(peroxisomes containing inclusions or invaginations) is

symptomatic of a yeast peroxisomal mutant, and is

reminiscent of the phenotypes found in human peroxi-

somal disorders [4,5]. Out data suggest that Lpx1p is

required to determine the shape of peroxisomes.

Experimental procedures

Strains and expression cloning

The S. cerevisiae strains BY4742, BY4742 Dyor084w,BY4742 Dpex5, BY4742 Dpex7 and BY4742 Dpex1 were

obtained from EUROSCARF (Frankfurt, Germany). S. ce-

revisiae strain BJ1991 (Mata leu2 trp1 ura3-251 prb1-1122

pep4-3) has been described previously [34].

Genomic S. cerevisiae DNA was used as a PCR template

for PCR. For construction of pUG35-LPX1 (LPX1–GFP),

PCR-amplified YOR084w (primers 5¢-GCTCTAGAATG

GAACAGAACAGGTTCAAG-3¢ and 5¢-CGGAATTCCA

GTTTTTGTTTAGTCGTTTTAAC-3¢) was subcloned into

EcoRV-digested pBluescript SK+ (Stratagene, La Jolla,

CA, USA), and then introduced into the XbaI and EcoRI

sites of pUG35 (HJ Hegemann, Dusseldorf, Germany). For

construction of pUG36-LPX1 (GFP–LPX1), PCR-amplified

YOR084w (primers 5¢-GAGGATCCATGGAACAGAACA

GGTTCAAG-3¢ and 5¢-CGGAATTCTTACAGTTTTTGT

TTAGTCGTTTTAAC-3¢) was subcloned into EcoRV-

digested pBluescript SK+, and then cloned into the BamHI

and EcoRI sites of pUG36 (HJ Hegemann).

pET21d-LPX1 was constructed by introducing PCR-

amplified YOR084w (primers 5¢-GAATCCATGGAACAG

AACAGGTTCAA-3¢ and 5¢-CGGTACCGCGGCCGCCA

GTTTTTGTTTAGTCGTTTT-3¢) into the NcoI and NotI

sites of pET21d (EMD Chemicals, Darmstadt, Germany).

For construction of pPC86-LPX1 and pPC97-LPX1,

YOR084w was amplified using primers 5¢-CCCGGGAAT

GGAACAGAACAGGTTCAAG-3¢ and 5¢-AGATCTTTA

CAGTTTTTGTTTAGTCGTTTT-3¢, and introduced into

pGEM-T (Promega, Mannheim, Germany). The ORF was

excised using XmaI and BglII, and introduced into pPC86

and pPC97 [35]. All constructs were confirmed by DNA seq-

uencing. pPTS2-DsRed has been described previously [36].

Image acquisition

Samples were fixed with 0.5% w ⁄ v agarose on microscopic

slides. Fluorescence microscopic images were recorded on

an Axioplan2 microscope (Zeiss, Koln, Germany) equipped

with an aPlan-FLUAR 100 x ⁄ 1.45 oil objective and an

AxioCam MRm camera (Zeiss) at room temperature. If

necessary, contrast was linearly adjusted using the image

acquisition software Axiovision 4.2 (Zeiss).

Protein purification and antibody generation

Lpx1p was expressed from pET21d-LPX1 in BL21(DE3)

E. coli. Cells were harvested by centrifugation (SLA3000,

4000 g, 15 mins), and resuspended in buffer P (1.7 mm

potassium dihydrogen phosphate, 5.2 mm disodium hydro-

gen phosphate, pH 7.5, 150 mm sodium chloride) containing

S. Thoms et al. Peroxisomal lipase Lpx1p

FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS 511

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a protease inhibitor mix (8 lm antipain-dihydrochloride,

0.3 lm aprotinin, 1 lm bestatin, 10 lm chymostatin, 5 lm

leupeptin, 1.5 lm pepstatin, 1 mm benzamidin, and 1 mm

phenylmethane sulfonylfluoride) and 50 lgÆmL)1 lysozyme,

22.5 lgÆmL)1 DNAse I and 40 mm imidazole. Cells were

sonicated 20 times for 20 s each using a 250D Branson

digital sonifier (Danbury, CT, USA) with an amplitude

setting of 25%. After removal of cell debris (SS34,

27 000 g, 45 min) the supernatant was clarified by 0.22 lmfiltration (Sarstedt, Numbrecht, Germany) and loaded

on Ni-Sepharose columns (GE Healthcare, Munich,

Germany) equilibrated with buffer W (buffer P containing

300 mm sodium chloride, 1 mm dithiothreitol, 40 mm

imidazole). The column was washed in buffer W until no

further protein was eluted. Recombinant Lpx1p was eluted

by a continuous 40–500 mm imidazole gradient based on

buffer W. Peak fractions (identified by SDS–PAGE) were

pooled and concentrated using VivaSpin concentrators

(30 kDa cutoff, Sartorius, Gottingen, Germany). Lpx1p

was further purified by gel-filtration chromatography.

Protein was stored at 0 �C. For the production of poly-

clonal antibodies, gel bands corresponding to 150 lgprotein were excised and used for rabbit immunization

(Eurogentec, Seraing, Belgium).

Size-exclusion chromatography

For analysis of endogenous Lpx1p by gel filtration, 5 mL of

a glass bead lysate of oleate-induced BY4742 wild-type cells

in buffer A (buffer P, pH 7.3, 300 mm sodium chloride) with

a protease inhibitor mix were injected into a HiLoad 16 ⁄ 60Superdex 200 prepgrade column (GE Healthcare) and eluted

using buffer A at a flow rate of 1 mL)1Æmin and a fraction

size of 2 mL. Fractions were analysed by SDS–PAGE and

Western blotting. A 500 lL aliquot of the concentrated

Ni-Sepharose eluate of Lpx1p from E. coli expression was

purified in the same buffer under the same conditions. For

estimation of Lpx1p complex sizes, molecular masses were

interpolated from a calibration curve generated using

ovalbumin (45 kDa), carboanhydrase (29 kDa), trypsin

inhibitor (20.1 kDa), lactalbumin (14.2 kDa) and aprotinin

(6.5 kDa) as molecular mass standards.

Enzyme assays

Esterase activity was determined using 0.5 mm p-nitrophe-

nyl butyrate (Sigma-Aldrich, Seelze, Germany) in NaCl ⁄Pi

(pH 7.4) in a total volume of 200 lL at 37 �C. The amount

of free p-nitrophenol was determined at 410 nm in 96-well

plates. Michaelis–Menten kinetics were analysed using

GraphPad Prism4 (Graph Pad Software, San Diego, CA,

USA).

Lipase activity was determined using 0.5 mm DPG (Mar-

ker Gene Technologies, Eugene, OR, USA) in 0.1 m gly-

cine, 19 mm sodium deoxicholate, pH 9.5, in a total volume

of 200 lL at 37 �C. Hydrolysis of DPG was followed in

96-well plates at 460 nm with 360 nm excitation using

a Sirius HT fluorescence plate reader (MWG Biotech,

Ebersberg, Germany). Lipase activity towards DPG was

measured in assay setups containing 2–10 lg Lpx1p (from

two independent protein preparations), with C. rugosa

triacylglycerol lipase (Lipase AT30 Amano, 1440 UÆmg)1,

Sigma-Aldrich) as a control.

Phospholipase A activity was measured using bis-

BODIPY FL C11-PC (Molecular Probes ⁄ Invitrogen,Eugene, OR, USA) as the substrate. The assay setup con-

tained 70 lg Lpx1p in 50 lL assay buffer (50 mm Tris,

100 mm sodium chloride, 1 mm calcium carbonate, pH 8.9)

together with 50 lL substrate-loaded liposomes. Liposomes

were prepared by injecting 90 lL of an ethanolic mixture of

3.3 mm dioleyl phosphatidylcholine (Avanti Polar Lipids,

Alabaster, AL, USA), 3.3 mm dioleyl phosphatidylglycerol

(Avanti Polar Lipids) and 0.33 mm bis-BODIPY FL C11-

PC into 5 ml assay buffer. Substrate turnover was mea-

sured at 528 nm emission after 488 nm excitation. Activity

was calculated from the initial velocity. Porcine pancreas

phospholipase A2 (Fluka ⁄Sigma-Aldrich, Buchs, Swizer-

land) was used as a control.

Density gradient centrifugation

Gradient centrifugation was carried out essentially as

described previously [37]. Briefly, oleate-induced yeast cells

were converted to spheroblasts using 25 UÆg)1 Zymoly-

ase 100T (MP Biomedicals, Illkirch, France). Spheroblasts

were gently ruptured by Potter–Elvehjem homogenization,

and centrifuged at low speed (3 · 10 min at 600 g) to

generate postnuclear supernatants. These supernatants, con-

taining 5 mg protein, were loaded on a 32–54% sucrose

gradient (Fig. 2D) or an Optiprep gradient (Fig. 4B) and

centrifuged for 3 h at 19 000 g (Sorvall SV288, 19 000 rpm,

4 �C). The gradient was fractionated into about 29 frac-

tions of 1.2 mL. Fractions were analysed using enzyme

assays for oxoacyl CoA thiolase, catalase, fumarase and

cytochrome c oxidase [37].

Other methods

Mass spectrometry and high-pressure lipid chromatography

have been described previously [14,15,38,39]. Subcellular

fractionation, yeast two-hybrid assays and electron micros-

copy were carried out as described previously [37].

Acknowledgements

We thank Elisabeth Becker, Monika Burger and Uta

Ricken for technical assistance. We thank Sabine Wel-

ler and Hartmut Niemann for reading the manuscript.

We extend our thanks to three anonymous reviewers

Peroxisomal lipase Lpx1p S. Thoms et al.

512 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS

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who helped to improve the manuscript. This work

was supported by the Deutsche Forschungsgemeins-

chaft (Er178 ⁄ 2-4) and by the Fonds der Chemischen

Industrie.

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Seventh International Meeting on AAA Proteins 99

The AAA peroxins Pex1p and Pex6p function asdislocases for the ubiquitinated peroxisomalimport receptor Pex5pHarald W. Platta, Mykhaylo O. Debelyy, Fouzi El Magraoui and Ralf Erdmann1

Abteilung fur Systembiochemie, Medizinische Fakultat der Ruhr-Universitat Bochum, D-44780 Bochum, Germany

AbstractThe discovery of the peroxisomal ATPase Pex1p triggered the beginning of the research on AAA (ATPaseassociated with various cellular activities) proteins and the genetic dissection of peroxisome biogenesis.Peroxisomes are virtually ubiquitous organelles, which are connected to diverse cellular functions. Thehighly diverse and adaptive character of peroxisomes is accomplished by modulation of their enzymecontent, which is mediated by dynamically operating protein-import machineries. The import of matrixproteins into the peroxisomal lumen has been described as the ATP-consuming step, but the correspondingreaction, as well as the ATPase responsible, had been obscure for nearly 15 years. Recent work usingyeast and human fibroblast cells has identified the peroxisomal AAA proteins Pex1p and Pex6p asmechano-enzymes and core components of a complex which dislocates the cycling import receptorPex5p from the peroxisomal membrane back to the cytosol. This AAA-mediated process is regulated bythe ubiquitination status of the receptor. Pex4p [Ubc10p (ubiquitin-conjugating enzyme 10)]-catalysedmono-ubiquitination of Pex5p primes the receptor for recycling, thereby enabling further rounds of matrixprotein import, whereas Ubc4p-catalysed polyubiquitination targets Pex5p to proteasomal degradation.

IntroductionPex1p (formerly Pas1p), Sec18p [NSF (N-ethylmaleimide-sensitive factor)] and Cdc48p (cell division cycle 48 protein)[p97/VCP (valosin-containing protein)] represent the firstproteins that were recognized as belonging to a novel familyof ATPases [1], the AAA (ATPase associated with variouscellular activities) family [2], which later was extended tothe family of AAA+ proteins [3]. Belonging to the class ofP-loop (phosphate-binding) NTPases, the AAA+ proteinsare especially distinguished by the presence of at least oneevolutionarily conserved 200–250-amino-acid ATP-bindingdomain that contains Walker A and B motifs in additionto other structural features, such as the SRH (secondregion of homology), which distinguishes AAA proteinsfrom other AAA+ proteins [4]. Although the members ofthe AAA+ family display a high functional diversity, thecommon function of all seems to be the ability to catalysereactions that are associated with significant conformationalremodelling of substrate proteins or nucleic acids [5].

A lot of detailed information regarding the structure andmolecular mechanism of AAA proteins has been accumu-lated, but our understanding of the molecular function of

Key words: ATPase associated with various cellular activities (AAA), peroxin, PEX, protein

transport, ubiquitination.

Abbreviations used: AAA, ATPase associated with various cellular activities; Cdc48p, cell

division cycle 48 protein; NSF, N-ethylmaleimide-sensitive factor; NTD, N-terminal domain;

PTS, peroxisomal targeting signal; SRH, second region of homology; Ubc, ubiquitin-conjugating

enzyme; VCP, valosin-containing protein.1To whom correspondence should be addressed (email [email protected]).

Biochem. Soc. Trans. (2008) 36, 99–104; doi:10.1042/BST0360099

Pex1p and Pex6p, the second AAA peroxin in peroxisomalbiogenesis [6], remained incomplete for many years.

Peroxisomes are single-membrane-bound organelles ofvirtually all eukaryotic cells, which display a unique vari-ability in their enzyme content and metabolic functions thatare adjusted according to the cellular needs. Their matrixharbours at least 50 different enzymes that are linked todiverse biochemical pathways. The β-oxidation of fattyacids and the detoxification of hydrogen peroxide areregarded as the central function of peroxisomes. Theyare the source of signalling molecules such as jasmonatesin plants or lipid-derived ligands for PPARs (peroxisome-proliferator-activated receptors) in humans. Other functionsof peroxisomes include the final steps of penicillin biosyn-thesis in some filamentous fungi, the main reactions of photo-respiration in leaf peroxisomes and the synthesis of bile acidand ether lipids such as plasmalogens in mammals, whichcontribute more than 80 % of the phospholipid content ofthe white matter in the brain [7].

Because of the central role of peroxisomes in lipid meta-bolism, they are essential for normal human developmentand physiology. This is emphasized by a group of geneticdisorders collectively referred to as the peroxisome disorders,which, in most cases, lead to death in early infancy [8].Detailed analysis of the complementation groups finally re-vealed that the most common cause of peroxisomal biogenesisdisorders are mutations in Pex1p [9]. More than 80 % of allpatients with Zellweger syndrome, the most severe peroxi-some biogenesis disorder, carry mutations in Pex1p or Pex6p[10].

C©The Authors Journal compilation C©2008 Biochemical SocietyBio

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100 Biochemical Society Transactions (2008) Volume 36, part 1

Molecular architecture of the peroxisomalAAA complex

AAA proteins are characterized by a typical modulararchitecture as they contain an N-terminal non-ATPase do-main which is followed by at least one conserved AAAdomain. Each AAA cassette usually contains an ATP-bindingsite (Walker A) and an ATP-hydrolysis site (Walker B) alongwith other motifs, such as the SRH [4].

Pex1p and Pex6p are type II AAA proteins, which arecharacterized by two AAA domains (Figure 1). In both AAAperoxins, the second AAA domain is more conserved thanthe first one. Interaction and subsequent oligomerization ofPex1p and Pex6p is believed to be initiated in the cytosol andinvolves their first less conserved AAA domains (D1) [11,12].Although neither binding nor hydrolysis of ATP at D1 seemsto be essential for functionality in both yeast and humans,the interaction of human Pex1p and Pex6p is stimulated bybinding of ATP to D1 of human Pex1p and Pex6p [12,13].Furthermore, ATP binding, but not hydrolysis, at the secondAAA cassette (D2) of Pex1p is required for the Pex1p–Pex6pinteraction in both systems [11,12].

Pex1p and Pex6p are believed to form heterohexamericstructures in the cytosol and at the peroxisomal membrane[12,14–16]. However, it is not clear whether formation of aheteromeric assembly of the AAA peroxins is a prerequisitefor their function, as one population of Pex1p does notco-localize with Pex6p in mammalian cells [12,17]. Althoughthe formation of hexameric structures is common to AAAproteins, the formation of heterohexamers has been found infew other cases, such as the m-AAA (matrix AAA) complex,consisting of Yta10p and Yta12p, which is active at thematrix site of the inner mitochondrial membrane, [18] orthe six different Rpt ATPases from the 19S proteasome[19].

The recruitment of AAA complexes to peroxisomesis mediated by the tail-anchored peroxisomal membraneproteins Pex15p in Saccharomyces cerevisiae or its functionalorthologue Pex26p in human cells via binding of theN-terminal domain of Pex6p, stimulated by ATP bindingto the Walker A motif of Pex6p D1 [20,21]. In contrast,the Walker A and B motifs of Pex6p D2 are required foran efficient detachment from Pex15p/Pex26p [12,20,22].Although Pex15p and Pex26p have been described as adaptorproteins for the N-terminal part of Pex6p, no adaptor hasyet been identified for Pex1p.

The NTD (N-terminal domain) of murine Pex1p repre-sents the only available crystal structure of the AAA peroxins[23]. The NTD folds into two structurally independentglobular subdomains (N- and C-lobe), which comprisean N-terminal double-ψ fold and a C-terminal β-barrel,separated by a shallow groove. Similar grooves were found inthe adaptor-binding sites within the NTDs of VCP, NSF andVAT (VCP-like ATPase from Thermoplasma), suggestingfunctional similarity [23].

The Pex15p-anchored AAA complex itself is part of aneven larger protein complex at the peroxisomal membrane,

the peroxisomal matrix protein import machinery called theimportomer (Figure 1) [15].

To conclude, at least in S. cerevisiae, the Pex1p-boundnucleotides seem to influence the Pex1p–Pex6p interaction,while the different nucleotide states of Pex6p regulatethe dynamic Pex6p–Pex15p/Pex26p association. The non-conserved domains are responsible for oligomerization, whilethe conserved domains exhibit the main ATPase activity.

Pex1p and Pex6p: peroxins associatedwith diverse cellular activities?Besides their involvement in peroxisomal biogenesis, theAAA peroxins have been suggested to carry out other func-tions as well. Human Pex6p has been reported to interact spe-cifically with the nucleocytoplasmatic transcriptional regulat-ors Smad2, Smad3, Smad4 and Smad7 [24]. These proteins areinvolved in the signalling pathway of the plasma membranereceptor TGFβ (transforming growth factor β), which reg-ulates apoptosis. Furthermore, a suppressor screen for agingdefects in mitochondria revealed that Pex6p, but not Pex1p,complements an ATP2-caused import defect into mitochon-dria, indicating a novel, yet not understood, function of thisperoxin in mitochondrial inheritance and senescence [25].

In the context of peroxisomal biogenesis, the differentfunctions discussed are mostly linked to the modulationof membrane dynamics. On the basis of the finding thatPex1p and Pex6p can associate with membranous subcellularstructures distinct from mature peroxisomes in the yeastsPichia pastoris and Yarrowia lipolytica, these peroxins werethought to play a role in lipid or membrane transport [14,26].Utilizing in vitro fusion experiments, Pex1p and Pex6pwere shown to be required for the fusion of five differentpremature peroxisomal vesicle species in Y. lipolytica [26],a process which might play a role during the maturationof endoplasmic reticulum-derived peroxisomal structuresduring de novo synthesis of peroxisomes [27]. The stillputative functional relevance of the observed phospholipid-binding activity of the murine Pex1p NTD, which hasalso been described for VCP and NSF, might be linkedto this process [28]. Furthermore, the presence of Pex6pand Pex15p is required for peroxisomal localization of theGTPase Rho1p, which is thought to organize actin filamentson peroxisomes during proliferation [29].

The existence of a link between the AAA peroxins andmatrix protein import has been proposed previously [30],but has remained elusive for many years. Recently, theirfunctional role in peroxisomal protein import was discovered.The AAA peroxins are required for the dislocation of thecycling peroxisomal import receptors Pex5p and Pex20pfrom the peroxisomal membrane back to the cytosol in orderto complete their receptor cycle [31–34].

The AAA peroxins function as dislocases forthe ubiquitinated PTS1 (peroxisomaltargeting signal 1) receptor Pex5pImport of folded proteins into peroxisomes occurs in apost-translational manner and depends on ATP. The soluble

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Seventh International Meeting on AAA Proteins 101

Figure 1 Molecular organization of the AAA complex in S. cerevisiae

The AAA peroxins Pex1p and Pex6p are composed of an NTD, a non-conserved AAA domain (D1) and a conserved AAA

domain (D2). The AAA domains contain ATP-binding sites (A) and, with the exception of D1 of Pex6p, also ATP-hydrolysis

sites (B). Pex1p and Pex6p form a heteromeric complex, and oligomerization requires the presence of the D1 domains and

is stimulated by ATP binding to Pex1p D2. Recruitment of the AAA complex to peroxisomes occurs via binding of Pex6p NTD

to Pex15p and requires ATP binding at Pex6p D1, while detachment from Pex15p needs ATP binding and hydrolysis at Pex6p

D2. The peroxisomal AAA complex dynamically associates with the functional matrix protein-import machinery (importomer)

and Pex4p (Ubc10p) is supposed to be required for the disconnection of the AAA complex from the importomer.

PTS1 receptor Pex5p is the major signal-recognition factorof proteins destined for the peroxisomal matrix. The receptorcycle of Pex5p involves cargo recognition in the cytosol,docking of the receptor–cargo complex to the peroxisomalmembrane, translocation of the receptor–cargo complex tothe luminal side of the membrane, followed by release of thecargo into the matrix and retrotranslocation of the receptorback to the cytosol (Figure 2) [7].

Permeabilized cell systems of human fibroblasts providedthe first evidence that Pex5p accumulated reversibly at theperoxisomal membrane under ATP-modulated conditions[30]. Detailed in vitro studies revealed that the binding andtranslocation of Pex5p itself is ATP-independent while theexport of Pex5p back to the cytosol requires ATP [35]. Theidentity of the corresponding ATPase remained a matter ofdebate until in vitro systems in S. cerevisiae [34] and humanfibroblast cells [32] identified Pex1p and Pex6p as the motorproteins of Pex5p export. Their function in this processrequires the presence of their membrane-anchor proteins,Pex15p or Pex26p. The in vitro reconstitution of the completePex5p cycle revealed that ATP binding and hydrolysis at both

Pex1p D2 and Pex6p D2 is needed for receptor dislocation[34]. Interestingly, the Walker B motif of Pex1p D2 seemsto have no function in formation or targeting of the AAAcomplexes [11,12] and thus may be exclusively required forhandling of the substrate. The binding and consumption ofATP is believed to induce conformational changes withinthe AAA peroxins that generate the driving force topull the receptor out of the membrane by a mechanismpossibly similar to the one of Cdc48p (p97/VCP) in ERAD(endoplasmic reticulum-associated degradation) [36].

The mechanism of substrate recognition by the AAA per-oxins is not understood. Although Pex5p and the AAAproteins form a complex at the peroxisomal membrane[15,32,34], no direct interaction of the PTS1 receptor witheither Pex1p or Pex6p has been reported. This interactionseems to be regulated or mediated by a third factor, whichcould represent an unknown adaptor protein of the AAAperoxins or post-translational modification of the substrate.It is well known that both processes play a central role inthe function of Cdc48p (p97/VCP) [37,38], which is theclosest evolutionary relative of Pex1p and Pex6p [39,40].

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102 Biochemical Society Transactions (2008) Volume 36, part 1

Figure 2 Peroxisomal matrix protein import

The AAA peroxins export the ubiquitinated PTS1 receptor back to the cytosol. The PTS1-recognition factor Pex5p binds newly

synthesized PTS1-harbouring cargo proteins in the cytosol and ferries them to the docking complex (Pex13p, Pex14p, Pex17p)

at the peroxisomal membrane. The receptor–cargo complex reaches the luminal side of the membrane, where the cargo is

released in a process that possibly involves Pex8p. ATP-dependent retrotranslocation of the membrane-integrated Pex5p is

mediated by the Pex15p-anchored AAA complex consisting of Pex1p and Pex6p. Pex5p can either be mono-ubiquitinated

by the Pex22p-anchored Pex4p in order to be recycled (recycling pathway) or it can be polyubiquitinated by Ubc4p, Ubc5p

and Ubc1p, resulting in proteasomal degradation (proteolytic pathway). Both pathways rely on ATP-dependent activation of

ubiquitin by E1 and possibly require the RING (really interesting new gene) peroxins Pex2p, Pex10p and Pex12p as putative

E3 enzymes.

As a consequence, the question has to be addressed of howthe AAA peroxins can distinguish Pex5p forms destinedfor dislocation from cargo-loaded Pex5p species destined forcargo translocation. A possible solution may arise from thecrystal structure of Pex1p NTD, which displays similaritiesto the corresponding adaptor-binding domains of otherAAA proteins [23]. Data from p97 and Ufd1 have identifieda double-ψ β-barrel fold as a ubiquitin-binding domain withbinding sites for both mono- and poly-ubiquitin [41].

Most interestingly, the PTS receptors Pex5p, Pex18pand Pex20p have been demonstrated to be ubiquitinated[31,42–44]. The PTS1 receptor Pex5p of S. cerevisiae is mono-ubiquitinated in wild-type cells [45], whereas it has beenshown to be polyubiquitinated in mutants of the protea-some or cells affected in the AAA and Pex4p–Pex22pcomplexes of the peroxisomal protein-import machinery[42,43]. Polyubiquitination of Pex5p, requiring the ubiquitin-conjugating enzymes Ubc4p and the partly redun-

dant Ubc5p and Ubc1p, takes place exclusively at theperoxisomal membrane and marks the receptor forproteasomal degradation as part of a quality-control system[42,43,45]. Alternatively, Pex5p is the specific moleculartarget for mono-ubiquitination by Pex4p (Ubc10p) [33,46],which is essential for peroxisomal biogenesis [47] and isanchored via Pex22p to the peroxisomal membrane [48].

The functional role of ubiquitination in the dislocationprocess has been elucidated by in vitro export assays,revealing that mono-ubiquitination of Pex5p constitutesthe export signal under physiological conditions, whereaspolyubiquitination seems to provide an export signal for therelease of dysfunctional PTS1 receptors from the membraneand proteasomal degradation as part of the quality-controlpathway [33].

The direct mechanistic influence of this modification onthe export reaction remains to be investigated. The AAAperoxins may interact directly or indirectly via putative

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Seventh International Meeting on AAA Proteins 103

adaptors with the ubiquitin tag on Pex5p. Alternatively, theattachment of ubiquitin may induce local conformationalchanges within Pex5p to expose hidden binding sites. Thismode of interaction is also discussed for Cdc48p (p97/VCP),which binds ubiquitin via adaptor complexes such as Ufd1/Npl4 and via its N-terminal domain. This domain is capableof recognizing ubiquitin chains and also non-modifiedsegments of its substrates [49,50].

Notably, the AAA complex displays significantly in-creased association with the importomer in PEX4-deficientcells, indicating that the ATPase cycles of Pex1p and Pex6pare coupled to the mono-ubiquitination-dependent receptorcycle of Pex5p (Figure 1) [15].

ConclusionsPeroxisomes exhibit unique dynamics in their enzymecontent and metabolic functions. The accompanied changesare accomplished by elaborate protein-transport machineries.The energy requirement for peroxisomal protein import isdetermined by the ATP-dependent dislocation of the importreceptors, which probably represents the rate-limiting step.The energy is utilized by two enzyme activities: (i) mono-ubiquitination by Pex4p (recycling pathway) or polyubiquit-ination by Ubc4p (proteolytic pathway), as ubiquitin firsthas to be activated by E1; and (ii) ATP hydrolysis in theconserved AAA domains of Pex1p and Pex6p in order to pullthe primed PTS receptor out of the membrane.

These results bring together the previously disparate rolesof Pex4p and the AAA peroxins in one concerted reaction se-quence. For future research, it will be a challenge to elucidatehow AAA-mediated receptor dislocation is mechanisticallylinked to the peroxisomal import of folded proteins.

Note added in proof (received 13December 2007)After submission of the present paper, an article appearedconcerning the ubiquitination of mammalian Pex5p [51].This study demonstrates that this modification is requiredfor recycling and thus reveals that the mechanism of AAAperoxin function is highly censerved in evolution.

We apologize to those scientists whose work could not be cited due

to space limitations. We are grateful to Sigrid Wuthrich for technical

assistance and Wolfgang Girzalsky and Marion Witt-Reinhardt for

the reading of the manuscript. This work was supported by the

Deutsche Forschungsgemeinschaft (SFB642, Er178/2-4), the FP6

European Union Project ‘Peroxisome’ (LSHG-CT-2004-512018) and

by the Fonds der Chemischen Industrie.

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Received 28 August 2007doi:10.1042/BST0360099

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Ubp15p, an ubiquitin hydrolase associated with the peroxisomal export machinery

Mykhaylo O. Debelyy1, Harald W. Platta1,2, Delia Saffian1, Astrid Hensel1, Sven Thoms1,3, Helmut E. Meyer4, Bettina Warscheid4,5, Wolfgang Girzalsky1 and

Ralf Erdmann1§

1Abteilung für Systembiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany

2 Current address: Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

3Current address: Universitätsmedizin Göttingen, Abteilung für Pädiatrie und Neuropädiatrie, Georg-August-Universität, D-37099 Göttingen, Germany

4Medizinisches Proteom-Center, Ruhr-Universität Bochum, Universitätsstrasse 150, D-44780 Bochum, Germany

5Current address: Faculty of Biology and BIOSS Centre for Biological Signalling Systems, University of Freiburg, 79104 Freiburg, Germany

Running head: A role for Ubp15p in Peroxisome Biogenesis §Correspondence to: Dr. Ralf Erdmann, Institut für Physiologische Chemie, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Tel. 49-234-322-4943, Fax. 49-234-321-4266, email. [email protected] The peroxisomal matrix protein import is facilitated by cycling receptors shuttling between the cytosol and the peroxisomal membrane. One crucial step in this cycle is the ATP-dependent release of the receptors from the peroxisomal membrane. This step is facilitated by the peroxisomal AAA-proteins Pex1p and Pex6p with ubiquitination of the receptor being the main signal for its export. Here we report that the AAA-complex contains dislocase as well as deubiquitinating activity. Ubp15p, an ubiquitin hydrolase, was identified as novel constituent of the complex. Ubp15p partially localizes to peroxisomes and is capable to cleave off ubiquitin-moieties from the PTS1-receptor Pex5p. Furthermore, Ubp15p-deficient cells are characterized by a stress related PTS1-import defect. The results merge to a picture in which removal of ubiquitin of the PTS1-receptor Pex5p is a specific event and might represent a vital step in receptor recycling. Peroxisomes are organelles which carry out a wide variety of metabolic processes in eukaryotic organisms. As peroxisomes do not contain genetic material, their protein content is determined by the import of nuclear encoded proteins. Peroxisomes can multiply by division

(1) or de novo by budding from the ER (2,3). Without exception, peroxisomal matrix proteins are synthesized on free ribosomes and are subsequently imported in a post-translational manner (4,5). Like the sorting of proteins to other cellular compartments, protein targeting to peroxisomes depends on signal sequences. Peroxisomal matrix proteins contain a C-terminal type I peroxisomal targeting sequence (PTS1) or an N-terminal PTS2 (4). These PTSs are recognized by conserved receptors, Pex5p and Pex7p, respectively. Based on the concept of cycling receptors (6,7), the matrix protein import can be divided into four steps: 1) receptor-cargo recognition in the cytosol, 2) docking at the peroxisomal membrane, 3) cargo-translocation and release, and 4) receptor release from the membrane and recycling. With respect to the PTS1-receptor Pex5p, recent reports demonstrated that its dislocation from the peroxisomal membrane to the cytosol at the end of the receptor cycle is ATP-dependent and catalyzed by the AAA-peroxins Pex1p and Pex6p (8,9). The main signal for the export process is the attachment of a monoubiquitin moiety or, alternatively, the anchoring of a polyubiquitin chain (10,11). While receptor monoubiquitination occurs on a conserved cysteine, polyubiquitin chains are

http://www.jbc.org/cgi/doi/10.1074/jbc.M111.238600The latest version is at JBC Papers in Press. Published on June 10, 2011 as Manuscript M111.238600

Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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attached to two lysine residues (10,12). In general, conjugation of ubiquitin to a target protein or to itself is regulated by the sequential activity of ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligating (E3) enzymes, and it typically results in the addition of an ubiquitin moiety either to the ε-amino group of a Lys residue or to the extreme amino terminus of a polypeptide (13). In a very few cases, including Pex5p, also attachment to a Cys residue has been reported (12,14). Whereas the addition of a single ubiquitin to a target protein can alter protein activity and localization, the formation of a diverse array of ubiquitin chains is implicated as targeting to the 26S proteasome (15). In line with these findings, polyubiquitination of Pex5p makes the receptor available for proteasomal degradation as part of a quality control system for the disposal of dysfunctional Pex5p (16-18). Modification of Pex5p by a single ubiquitin on a conserved Cys residue provides the signal for the AAA-peroxin mediated release of the receptor from the peroxisomal membrane (10,11,19). This is of special importance as this ATP-dependent dislocation of the receptor is supposed to be responsible for the overall energy-requirement of the protein import cascade and thus might be mechanistically linked to the cargo translocation as proposed by the export-driven import model (20). The ubiquitination-cascade acting on Pex5p has been elucidated with the identification of Pex4p and the Ubc1p/Ubc4p/Ubc5p-family as responsible E2s (10,12,17,18,21). The peroxisomal RING-finger peroxins Pex2p, Pex10p and Pex12p have been identified as E3-enzymes responsible for the poly- and monoubiquitination of Pex5p (22,23). After export of the functional receptor to the cytosol, the ubiquitin-moiety has to be removed. This cleavage of ubiquitin from a substrate protein is generally carried out by ubiquitin hydrolases also known as deubiquitinating enzymes (DUBs) (24). S. cerevisiae contains genes coding for 18 DUBs (25,26). Recent in vitro data obtained from rat indicated that the monoUb moiety of Pex5p might be cleaved off in two different ways. A minor portion of the thioester-bound monoUb could be released in a non-enzymatic manner by a nucleophilic attack of glutathione while the major fraction of monoUb-Pex5p is deubiquitinylated enzymatically by a still to be identified ubiquitin hydrolase (27).

Here we report on the correlation of the ATP-dependent export of Pex5p and ubiquitin cleavage. The AAA-complex of the peroxisomal protein import machinery turned out to possess export as well as deubiquitinating activity. Ubp15p was identified as novel constituent of the complex which binds to the first AAA domain of Pex6p (D1 domain). Ubp15p exhibits ubiquitin hydrolase activity and is capable to cleave off ubiquitin-moieties from the PTS1-receptor Pex5p. The function of Ubp15p in peroxisome biogenesis is supported by a stress related PTS1-import defect of ubp15Δ cells. A scenario evolves in which receptor deubiquitination might be functionally linked to its AAA-peroxin mediated export and represents an important step in the receptor cycle which makes Pex5p available for a new round of matrix protein import.

EXPERIMENTAL PROCEDURES Yeast strains and culture conditions - The Saccharomyces cerevisiae strain UTL-7A (MATa, ura3-52, trp1, leu2-3/112) was used as wild-type strain for the generation of several isogenic deletion strains by the `short flanking homology` method as described previously (28). The resulting deletion strains were pex5Δ (29) ubp14Δ, ubp15Δ, ubp14Δ/ubp15Δ, doa4Δdoa4Δ/ubp15Δ (this study). cl3-ABYS-86 (30) served as wild-type strain for isolation of His6-Pex6p- and His6-GST-Ubp15p-complex. The yeast reporter strain L40 (MATa trp1 leu2 his3 LYS2::lexA-HIS3, URA3::lexA-lacZ) (31) was used for two-hybrid assays. Yeast media have been described previously (32). Inhibit of proteasomal degradation by the addition of MG132 to liquid cultures was performed according to (33) Plasmids and cloning strategies - Sequences of oligonucleotides available upon request. Two-hybrid plasmids expressing Gal4p-fusions with Pex1p, Pex6p or variants thereof were described previously (34). For expression of His6-Ubp15p in bakers yeast, two overlapping PCRs were performed using genomic S. cerevisiae DNA as template. PCRI (primers RE1813/ RE1749) amplified the 5`- half of UBP15 (NTP-UBP15) introducing an NcoI site to the 5`end. PCRII (primers RE1746/RE1730) amplified the 3`-half of UBP15 (CTP-UBP15) introducing an XhoI site to the 3`-end. Both PCR-products were subcloned into EcoRV

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digested vector pGEM®-T (Promega, Mannheim, Germany) resulting in vectors pGEM®-T-NTP-UBP15 and pGEM®-T-CTP-UBP15, respectively. Next, the introduced fragments were cut out of the pGEM® vectors (pGEM®-T-NTP-UBP15, NcoI/BamHI pGEM®-T-CTP-UBP15, BamHI/XhoI) and cloned together into NcoI/XhoI sites of pYES263 (35) leading to pYES263-UBP15. For the expression of GST-fusions of Ubp15p in E. coli, the vector pGEX-4T-2 (GE Healthcare, Freiburg, Germany) was digested with BamHI followed by a Klenow-refill reaction and the subsequent cleavage with XhoI. The Ubp15p coding region was obtained by cleaving pYES263-UBP15 with NcoI, Klenow-based refilling and subsequent XhoI treatment. The UBP15 fragment was then cloned into equally treated pGEX-4T-2 resulting in pGEX-4T-2-UBP15. In order to introduce a C214A amino-acid residue substitution into Ubp15p, the QuickChange mutagenesis kit (Agilent Technologies, Waldbronn, Germany) was used combined with pGEX-4T2-UBP15 as template and primers RE2274/RE2275 for the reaction. GFP-Ubp15p expression plasmid pUG36-UBP15 was constructed as follows. UBP15 was amplified by PCR using primers RE3196/RE3198 and plasmid pYES263-UBP15 as template. The SpeI/SalI digested PCR product was cloned into SpeI/SalI site of pUG36 (36). To obtain N-terminal His6-TEV-tagged Pex6p under the control of the GAL1-promotor for expression in yeast, the coding region for the N-terminal half of Pex6p was amplified by PCR using primers KU1549/KU1550 and plasmid pMB34 (37) as template. In a second step, PEX6 was amplified by PCR (primers KU1339/KU698, plasmid pMB34) and cloned into NcoI/SpeI site of pYES2.1V5-His-TOPO (Invitrogen, Darmstadt, Germany) leading to vector pYQ6/1. Finally, the first PCR (N-terminal Pex6p half) was digested with PvuII/SacI and the fragment was introduced into PvuII/SacI digested vector pYQ6/1, leading to pJK-5. Plasmids for expression of PTS2-dsRed or high expression of Pex15p were described elsewhere (38,39). Two-hybrid assay - The yeast reporter strain L40 was transformed with two-hybrid plasmids pPC86 and pPC97 (40) or derivates thereof and grown on synthetic medium lacking tryptophane and leucine for 3 days at

30 °C. Obtained double transformants were grown at 30 °C for 8 h in liquid synthetic medium. Lysates from these cells were prepared and subsequently subjected to ß-galactosidase assays as described by (41). Purification of Pex6p from S. cerevisiae cells - Recombinant His-tagged Pex6p or Ubp15p were expressed in S. cerevisiae strain cl3-ABYS-86 (30) transformed with pJK-5 or pYES263-UBP15, respectively. Galactose-grown cells were harvested, resuspended in lyses buffer (1.7 mM KH2PO4, 5.2 mM Na2HPO4, 300 mM NaCl, 1 mM DTT, 22.5 µg/ml DNase I) with protease inhibitors cocktail (8 µM antipain, 0.3 µM aprotinin, 1 µM bestatin, 10 µM chymostatin, 5 µM leupeptin, 1.5 µM pepstatin (Roche Diagnostics, Mannheim, Germany), 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 5 mM NaF). Cells were disrupted by glass bead lyses. Lysate was cleared by centrifugation and 0.22 µm filtration and loaded on Ni-Sepharose (GE Healthcare, Munich, Germany) columns equilibrated with washing buffer (1.7 mM KH2PO4, 5.2 mM Na2HPO4, 300 mM NaCl, 1 mM DTT, 40 mM imidazol).The column was washed until no more protein eluted. Pex6p was then eluted by a continuous imidazol gradient up to 500 mM imidazol in elution buffer (1.7 mM KH2PO4, 5.2 mM Na2HPO4, 300 mM NaCl, and 1 mM DTT, 500 mM imidazol). Fractions containing high protein concentration were combined and concentrated by VivaSpin concentrators (10,000 MWCO) (Sigma, Munich, Germany). Isolation of peroxisomes-Preparation of yeast spheroplasts, cell homogenization, preparation of post-nuclear supernatants and determination of the suborganellar localization of proteins were performed according to (42). Density gradient centrifugation was essentially performed as described (43), in particular, postnuclear supernatants (10 mg protein) were prepared and loaded onto preformed 2.25-24% (w/v) Optiprep (Iodixanol) gradients. Peroxisomes were separated from other organelles in a vertical rotor (Sorvall TV 860, 1.5 h at 48,000xg, 4°C). Fractions were collected from the bottom and subjected to enzyme and refractive index measurements as well as immunoblot analysis. Gel filtration of cell lysates and purified proteins - Analytical gel filtration was carried

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out on a SMART System (Amersham Pharmacia Biotech, Uppsala, Sweden) equipped with a Superose6 PC 3.2/30 column in running buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM β-mercaptoethanol, 2 mM ATP). Samples were cleared by centrifugation (15 min, 20,000g) and aliquots of 50 µl purified protein were separated at 40 µl/min. Fractions of 80 µl were collected form 0.8 to 1.6 ml after injection. The column was calibrated using ferritin (440 kDa), aldolase (158 kDa), and BSA (66 kDa) as markers. In vivo ubiquitination assays - Oleate-induced yeast cells were harvested, washed twice and resuspended in lysis-buffer (0.2 M HEPES, 1 M potassium acetate and 50 mM magnesium acetate, pH 7.5) and protease inhibitors cocktail (see above), 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 5 mM NaF). To accumulate monoubiquitinated Pex5p from wild-type cells, 20 mM NEM (Sigma, Munich, Germany) was added. Cells were disrupted by glass bead lysis and centrifuged at 1,500xg (Eppendorf rotor A-4-81) for 10 min. Supernatants were normalized for protein and volume, and membranes were sedimented by centrifugation at 100,000xg, (30 min, Sorvall AH650 rotor) followed by trichloroacetic acid precipitation and sample preparation (18). Deubiquitination assay - Deubiquitinating activity of Ubp15p was analyzed according to (44). In detail, 1 µg of Ubp15p/Ubp15pC214A and 250 ng of appropriate polyUb(K63)-chains (Biomol, Loerrach, Germany) were diluted in reaction buffer (50 mM Tris-HCl, pH 7.4, and 300 mM NaCl) to a total volume of 30 µl. Reactions were incubated for 2 h at 37 °C. Before and after reaction, 15 µl of each sample were charged with 3x SDS sample buffer and boiled for 5 min for further analysis. Five µl of each reaction were loaded onto a 15 % tris-glycin gel and subsequently subjected to immunoblot analysis. Protein Identification by Mass Spectrometry - Proteins in polyacrylamide gels were visualized by Coomassie staining according to (45). Destaining of proteins, in-gel tryptic digestion and subsequent peptide extraction was performed as described (46). Peptide samples were separated by online reversed-phase nano-HPLC using the Dionex LC

Packings HPLC systems (Dionex LC Packings, Idstein, Germany). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a Bruker Daltonics HCTplus ion trap instrument (Bremen, Germany) and subsequent protein identification by bioinformatics using the yeast NCBI database was performed as described (46). Miscellaneous - Immunopurification of ProtA-tagged Pex1p/Pex6p-complexes from yeast cells using IgG-Sepharose was described in (47). Immunoprecipitation of denatured proteins was carried out according to (22). Membranes containing monoubiquitinylated Pex5p were prepared according to (22) and incubated with purified yeast AAA-complex according to (8). Recombinant GST-fusion-proteins were expressed in E. coli BL21 (DE3) according to manufactures protocols (GE Healthcare, Freiburg, Germany). Immuno-reactive complexes were visualized using anti-rabbit or anti-mouse IgG-coupled horseradish peroxidase in combination with the ECL™ system from Amersham Pharmacia Biotech (Uppsala, Sweden). Alternatively, primary antibody was detected with a IRDye 800CW goat anti-rabbit IgG secondary antibody (LI-COR Bioscience, Bad Homburg, Germany) followed by a detection using the “Infrarot Imaging System“ (LI-COR Bioscience, Bad Homburg, Germany). Polyclonal rabbit antibodies were raised against Pex5p (48), Pex13p (29), and ubiquitin (Sigma, Munich, Germany). Monoclonal mouse antibodies were raised against GST (Sigma, Munich, Germany) and ubiquitin (clone FK2, Biomol, Hamburg, Germany). GFP- and dsRed-tagged proteins were monitored by life cell imaging with a Zeiss Axioplan 2 fluorescence microscope and AxioVision 4.8 software (Zeiss, Jena, Germany). Electron transmission microscopy, spheroplasting of yeast cells, homogenization and differential centrifugation at 25,000 x g of homogenates were performed as described previously (8,42,49).

RESULTS The peroxisomal AAA-complex exhibits deubiquitinating activity Dislocation of the PTS1 receptor Pex5p from the peroxisomal membrane to the cytosol depends on the peroxisomal AAA-proteins Pex1p and Pex6p (8,9) and ubiquitination of Pex5p is a prerequisite for this process (10).

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The main signal for the export process is the attachment of a monoubiquitin moiety (10,11). In order to gain more insight into the principal export mechanism of monoubiquitinated Pex5p, membranes were prepared from wild-type cells in the presence of NEM (N-ethyl-maleimid), which is a suitable inhibitor of de-ubiquitinating enzymes (DUBs) (50) and results in the accumulation of monoubiquitinated Pex5p at the peroxisomal membrane (Kragt et al., 2005; Platta et al., 2007). As NEM proved to be a competent inhibitor of the export machinery, membranes were washed extensively and NEM was avoided upon purification of the AAA-complexes. The membranes containing mono-ubiquitinated Pex5p were incubated with buffer alone or buffer containing purified cytosolic AAA-complex of S. cerevisiae in the presence of an ATP-regenerating system (ARS, (8)) for 30 min at 37 °C. Interestingly, the presence of the AAA-complex did result in the disappearance of the monoUb-Pex5p (Fig. 1, lanes 1 and 2) indicating that the peroxisomal AAA-complex does not only harbor the known dislocase activity of Pex1p/Pex6p but is also capable to facilitate receptor deubiquitination in addition. This assumption is supported by the result that incubation of the AAA-complex with DUB-inhibitors NEM (Fig. 1, lane 3) or ubiquitin-aldehyde (Fig. 1, lane 4) prior the assay blocks Pex5p deubiquitination. Ubp15p is associated with the AAA-complex The data described above indicate that the isolated AAA-complex contains export- and deubiquitinating activity also in the absence of the cytosol. Thus, the suspected additional factor is supposed to be part of the yeast AAA-complex. To identify the unknown factor, we isolated the cytosolic AAA-complex with Pex6p as overexpressed bait protein. For this purpose, a plasmid encoding N-terminal His6-tagged Pex6p under the inducible GAL1-promotor was transformed into the protease deficient yeast strain cl3-ABYS-86 (30). Transformants were precultured on glucose rich media and expression of the tagged Pex6p was induced by shifting to galactose media. His-Pex6p was isolated by affinity chromatography on NiNTA and analyzed by SDS-PAGE followed by silver stain. Two dominant protein bands were visible (Fig. 2A) which have been excised and analysed by mass spectrometry. The fast migrating protein was

identified as the bait protein Pex6p. The band with an approximate size of 140kDa consisted of three proteins, Clu1p, Ubp15p and Ecm21p. Clu1p is a subunit of translation initiation factor eIF3 that functions in AUG scanning in translation which is also required to maintain the morphology of mitochondria (51,52). Ubp15p is an ubiquitin-specific processing protease (53). Ecm21p is an arrestin-related protein which acts as an adaptor in ubiquitin ligation (54). As a second approach to identify AAA-peroxin associated proteins, we genomically tagged Pex1p with Protein A, isolated the complex as previously described (8), separated proteins of the isolated complex by SDS-PAGE and subjected selected protein bands to mass spectrometric analysis. The band marked in Fig. 2B contained Ubp15p, which indicated its association with the AAA-complex and moved the protein into the focus of our interest. To validate the Pex6p-Ubp15p-interaction, the complex isolation was performed vice versa using Ubp15p as bait. His6-GST-tagged Ubp15p was expressed in wild-type cl3-ABYS-86 strain, isolated by immuno-purification and the constituents of the complex were analyzed by immunoblotting. Pex6p was identified as a component of the Ubp15p-complex (Fig. 2C) and a minor portion of the PTS1-receptor Pex5p also co-eluted with the Ubp15p-complex. The soluble fructose 1,6-bisphosphatase (Fbp1p, (55)) was not retained by the chromatography, an indication for the specificity of the isolation procedure (Fig. 2C). A portion of the ubiquitin hydrolase Ubp15p localizes to peroxisomes Our results demonstrate that yeast Ubp15p possesses the ability to interact with Pex6p. As Pex6p is localized in the cytosol and at peroxisomes, the subcellular localization of Ubp15p was analyzed under peroxisome-inducing conditions. To this end, a cell free homogenate of oleic acid-induced wild-type cells expressing a genomically tagged UBP15 gene coding for a Ubp15p-protein A fusion protein (Ubp15p-TEV-ProtA) was prepared and organelles were separated by density gradient centrifugation (Fig. 3A). The presence of organelle marker proteins in fractions was assayed either by determination of enzyme activities or by immunoblotting. As indicated by the segregation behaviour of the peroxisomal membrane marker Pex13p and activity measurements of the peroxisomal

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catalase, peroxisomes migrated to the bottom fractions and showed a clear peak in fractions 3, clearly separated from the mitochondrial marker (porin). In line with the reported cytosolic localization (56), the majority of Ubp15p remained in the top gradient fractions. However, a significant portion of Ubp15p is detected at a higher density, co-localizing with peroxisomal marker proteins (Fig. 3A, lane 3). To support this finding, we monitored localization of GFP-tagged Ubp15p by fluorescence microscopy. GFP-Ubp15p was co-expressed with the synthetic peroxisomal marker protein PTS2-dsRed in wild-type cells. PTS2-dsRed exhibits punctate fluorescence pattern, typically for a peroxisomal localization (Fig. 3B, (38)). In line with our results obtained by cell fractionation (Fig. 3A), GFP-Ubp15p was predominantly localized to the cytosol which leads to overall cellular fluorescence (Fig. 3B). However, a portion of GFP-Ubp15p co-stained with PTS2-dsRed positive structures demonstrating its peroxisomal localization. Next we tried to increase the amount of peroxisomal Ubp15p by overexpression of Pex15p. The overexpression of this peroxisomal membrane protein leads to an increased recruitment of Pex6p to peroxisomes (37). We assumed that the consequence thereof should be an increased amount of Ubp15p bound to the peroxisomal membrane, as it is a binding partner of the Pex6p-complex. Indeed, upon Pex15p-overexpression only a small portion of GFP-Ubp15p was found cytosolic, whereas the major fraction was found co-localized with peroxisomal marker PTS2-dsRed (Fig. 3B). Taken together, the localization studies indicate that a portion of Ubp15p is associated with peroxisomes. Ubp15p interacts with the first AAA-domain of Pex6p In order to analyze the Ubp15p-interaction with the peroxisomal AAA-complex in more detail, we applied the yeast two-hybrid system. Plasmids expressing full-length Pex1p, Pex6p or ubiquitin fused to the Gal4p activation domain or the Gal4p-DNA-binding domain were transformed in the S. cerevisiae strain L40, and reporter gene expression was analyzed by assaying β-galactosidase activity. In line with previous findings (57), co-expression of Ubp15p with ubiquitin leads to significant reporter-gene activity as judged by determined ß-galactosidase activity, which

indicated the known Ubp15p-ubiquitin interaction (Fig. 4). The enzyme activities differed in dependence of whether Ubp15p was fused to the DNA-binding or trans-activation domain of Gal4p, however, in case of an interaction the enzyme activity was significantly higher than the controls of the empty vector versus bait plasmids. Comparison of the different assays revealed that Pex6p interacts with Ubp15p while the monitored β-galactosidase activity was only slightly above the control level when Pex1p was tested for interaction with Ubp15p. To determine the Pex6p-region that contributes to the Ubp15p interaction, we analyzed the interaction of Ubp15p with the N-terminal region (N, aa1–428), the first AAA-cassette (D1, aa421–716) and the second AAA-cassette (D2, aa704–1030) of Pex6p and combinations thereof. As shown in Fig. 3, neither the N-domain nor the second AAA-domain is capable to interact with Ubp15p. In contrast, the first AAA-domain of Pex6p alone or fused to the N-domain led to ß-galactosidase activity in the same range as observe with full-length Pex6p. Thus, the first AAA-domain of Pex6p is involved in the interaction with Pex1p (34,58) as well as with Ubp15p. Ubp15p facilitates deubiquitination of Pex5p in vitro UBPs represent a subclass of the deubiquitinating enzymes (DUB) comprising 18 putative members in S. cerevisiae, including Ubp15p (53). The UBP-family is highly divergent, but all members contain several short consensus sequences, the Cys- and the His- boxes that are likely to form a part of the active site (59). Within Ubp15p, the Cys-box covers the amino acids 206 to 223, whereas the His-box is localized between amino acids 449 and 533 (60). Sequence alignment of Ubp15p with other UBPs indicated that Cys214 of Ubp15p most likely represents an amino acid residue which is crucial for the deubiquitinating activity (60). Accordingly, a Cys214 to Ala substitution was introduced into the full-length protein by site directed mutagenesis and recombinant wild-type or mutant Ubp15p (Ubp15pC214A) fused to GST were expressed in E. coli and isolated by affinity chromatography. The tag was removed by thrombin cleavage. To demonstrate that recombinant Ubp15p exhibits deubiquitinating activity and is thus biologically active, in vitro ubiquitin-cleavage assays were performed. To

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this end, the isolated proteins were incubated with Ub-chains and the reaction was stopped after zero (control) or 120 min by adding SDS-sample buffer and subsequent boiling. Cleavage of the Ub-chain was monitored by immunoblot analysis with an antiserum against ubiquitin. Incubation of the Ub-chain with wild-type Ubp15p resulted in a decrease of higher molecular Ub-species and accumulation of monoUb as cleavage product (Fig. 5A, lane 2). When the assay was performed with mutated Ubp15p, no difference between the control sample and the sample incubated for 120 min (Fig. 5A, lanes 3 and 4) was observed. Thus, our data are clear in that Ubp15p acts as ubiquitin hydrolase on Ub-chains and that an enzyme harboring the Cys214Ala replacement is enzymatically inactive. This finding is not due to a dramatic influence of the mutation on the structure of the protein as both wild-type as well as mutated Ubp15p exhibit same behavior when analyzed by size exclusion chromatography (data not shown). Pex5p can be monoubiquitinated (21) or polyubiquitinated (17,18). In Fig. 1, we showed that the AAA-peroxin complex harbors deubiquitinating activity. Now, we addressed the question whether mono- or polyubiquitinated Pex5p can function as molecular target for deubiquitination by Ubp15p. To this end, we prepared membranes from wild-type cells in the presence of NEM which results in the accumulation of monoUb-Pex5p. These membranes were incubated with recombinant Ubp15p, followed by co-immuno-isolation of Pex5p. MonoUb-Pex5p was visible when the membranes were incubated with buffer alone but disappeared upon incubation with Ubp15p (Fig. 5B). Next, we assayed whether Ubp15p also acts on polyubiquitinated Pex5p. We isolated whole cell membranes from a pex1Δpex6Δ strain. These membranes show accumulation of polyUb-Pex5p species (17,18), Fig. 5C, lane 1). Incubation of these membranes with recombinant Ubp15p resulted in disappearance of modified Pex5p, indicating that Ubp15p can also cleave off ubiquitin from polyUb-Pex5p (Fig. 5C, lane 4). In line with this finding, no cleavage of Pex5p was observed when Ubp15p activity was blocked by NEM or Ub-aldehyde (Fig. 5C, lanes 2 and 3). Ub-aldehyde inhibits ubiquitin hydrolases by the formation of an extremely tight complex, in which the inhibitor is bound to the active site of DUBs (61). Taken

together, the data demonstrate that recombinant Ubp15p exhibits ubiquitin hydrolase activity and facilitates deubiquitination of mono- and poyUb-Pex5p. Clustered peroxisomes in ubp15∆ cells Ubp15p is a cytosolic protein, which is associated with the yeast AAA-complex. Pex1p and Pex6p are both required for peroxisomal matrix protein import, leading to the question whether also Ubp15p contributes to peroxisomal function in vivo. To address this question, growth test were performed on plates containing oleic acid as sole carbon source, which will support cell growth only if peroxisomal ß-oxidation is functional, which requires an intact organelle biogenesis. In contrast to wild-type, PEX5-deficient cells are unable to grow on this medium, which is in accordance with the literature (62) and typical for peroxisomal mutant strains of S. cerevisiae (42). Cells deficient in Ubp15p did not exhibit a growth defect on oleic acid medium (Fig. 6A). As a partial defect in peroxisome biogenesis does not inevitably lead to a complete destruction of peroxisome function, we analyzed the matrix protein import in wild-type and mutants in more detail. To this end, the subcellular localization of GFP fused to the peroxisomal targeting signal 1 (GFP-PTS1) as marker for the Pex5p dependent import, and PTS2-dsRed, an artificial substrate for the Pex7p dependent matrix protein import were monitored by live cell imaging. Fluorescence microscopy inspection of oleic acid-induced wild-type cells revealed a punctuate staining pattern for both marker proteins, typical for a peroxisomal labeling (Fig. 6B). Mutant pex5Δ cells that are affected in peroxisomal protein import of PTS1-proteins (62) exhibited a cytosolic fluorescence pattern for GFP-PTS1 as typical for these cells. In contrast, the PTS2-pathway is not affected in pex5Δ cells which results in a punctuate staining pattern for PTS2-dsRed. The fluorescence microscopy pattern observed for the ubp15∆ strain was similar to the one visible in the wild-type strain (Fig. 6B), suggesting that ubp15Δ cells are still able to import both, PTS1- and PTS2-containing peroxisomal matrix proteins. Interestingly, in contrast to wild-type peroxisomes, which are well separated, peroxisomes of ubp15Δ cells appeared to form clusters (Fig. 6B). This observation was corroborated by electron microscopic

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inspection of wild-type and mutant cells (Fig. 6C). ubp15Δ-cells exhibit lower steady state concentration of Pex5p but higher rate of ubiquitinated Pex5p. To investigate the consequence of a deletion of UBP15 on turnover of Pex5p, we estimated Pex5p steady state concentration in wild-type and ubp15Δ-cells. Whole cell lysates of oleic-acid induced wild-type and ubp15Δ-cells were subjected to immunoblot analysis with mitochondrial porin as loading control. Although porin concentration was same in both strains, Pex5p level differed (Figure 7A, left panel). For quantification of the observed difference, the signal intensity was analyzed by densitometry. It turned out that the Pex5p amount in ubp15Δ cells is reduced to approximately half of the wild-type level (Figure 7A, right panel). Next we analyzed whether the lower amount of Pex5p in ubp15Δ-cells is accompanied by a higher ubiquitination rate. To this end, we monitored receptor ubiquitination in cells treated with the MG132, which inhibits proteasomal protein degradation and leads to accumulation of ubiquitinated Pex5p (22). Accordingly, , ubiquitinated Pex5p was visible in both MG132-treated wild-type and ubp15Δ-cells (Figure 7B, left panel). However, while the level of unmodified Pex5p in ubp15Δ cells was half of wild-type level as described before (Figure 7B), ubp15Δ exhibits three times more polyubiquitinated Pex5p than the wild-type strain. Taken together, our results indicate a higher polyubiquitination rate of Pex5p in stains lacking Ubp15p which most likely causes a reduced steady state concentration of the PTS1-receptor. ubp15Δ-cells exhibit oxidative-stress related import deficiencies and growth on oleic acid None of the genes encoding ubiquitin hydrolases are essential for viability, suggesting that many of these enzymes have overlapping functions (53). As ubp15Δ exhibits normal growth and matrix protein import under oleic acid conditions, we suspected such a redundancy and tested double-deletion strains for growth on oleic acid medium and peroxisomal protein import. Cells lacking Doa4p are characterized by decreased free ubiquitin levels and these mutants display a strongly reduced turnover of several proteins that are targeted to degradation via ubiquitination. In line with this observation, Doa4p was shown the be involved in cleavage

of ubiquitin chains (59). Doa4p is required for turnover of the PTS2-co-receptor Pex18p, which also is ubiquitinated (63). Interestingly, Doa4p interacts with Ubp15p as well as Ubp14p (64,65). Thus, Ubp15p might be part of a ternary complex of ubiquitin hydrolases with overlapping functions. For this reason, we analyzed single mutants of UBP15, UBP14 and DOA4 as well as combination thereof for their capacity to import GFP-PTS1. As judged by fluorescence microscopy, neither the single- nor the double deletion strains exhibited an import deficiency for GFP-PTS1 (Fig. 8A, left panel). In all strains tested, the GFP-SKL exhibited a clear punctuate staining, demonstrating its localization in the peroxisomal matrix. It is well known that the function of redundant protein sometimes becomes essential when cells are under stress (66). To test for this possibility, we examined oleic acid induced wild-type and mutant cells for matrix protein import upon oxidative-stress conditions (0.2 mM H2O2 (67)). Under this condition, neither wild-type nor ubp14Δ cells showed an import defect for GFP-SKL as indicated by the clear punctuate staining with no background labeling (Fig. 8A, right panel). In contrast, doa4Δ and ubp15Δ cells showed a punctuate staining of peroxisomal matrix marker GFP-SKL but also a background labeling indicative for a partial mislocalization of the marker protein to the cytosol. This finding indicated that these mutants exhibit a partial peroxisomal protein import defect upon oxidative-stress. In this respect, it is worth to note that expression of Ubp15p and Dao4p but not of Ubp14p is induced by oleic acid (68). Moreover, induction of Doa4p and Ubp15p is also induced upon oxidative-stress by H2O2 (69). Our data indicate that deficiency in both, Ubp15p or Doa4p, affects proper peroxisomal protein import under oxidative-stress condition. To support this observation of an impaired peroxisomal function under oxidative-stress, we monitored the growth behaviour of UBP15-affected cells in comparison with wild-type and doa4Δ-cells. Cells were grown on either glucose or oleic acid as sole carbon source in the absence or presence of 0.2 mM H2O2. As judged by optical density measurements, wild-type as well as ubp15Δ cells exhibit similar growth rates when glucose served as energy source (Fig. 8B, lower panel). When H2O2 was added to the media, growth rates of these

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strains were nearly the same as without oxidative-stress. In contrast, doa4Δ cells showed the lowest growth rate of monitored strains already without H2O2, but growth was significantly delayed in the presence of H2O2.

When cells were cultured on oleic acid medium, growth of doa4Δ cells was drastically reduced (Fig. 8B, lower panel), which is in line with known pleiotropic effects of the deletion of this protein as Doa4p is involved in many Ub-dependent processes in the cell (70). The growth rate of wild-type and ubp15Δ cells was similar on glucose medium also under oxidative-stress conditions. Both strains also grew equally well on oleic acid medium without H2O2. However, oxidative-stress affected growth on oleic acid medium of both wild-type and ubp15Δ cells but while the wild-type still did grow reasonable well, growth of the ubp15Δ cells was severely affected (Fig. 8B, lower panel). Taken together, analysis of the mutant phenotypes disclosed a peroxisome- and stress-related defect of ubp15Δ cells.

DISCUSSION The AAA-complex of Pex1p and Pex6p is responsible for the release of the ubiquitinated PTS1-receptor Pex5p from the peroxisomal membrane, which has been regarded as the final step of the peroxisomal protein import cascade. In this work, we show that the AAA-complex is also responsible for receptor deubiquitination, which is supposed to be an important step in receptor recycling. We have identified the corresponding ubiquitin hydrolase Ubp15p as a novel factor that accompanies the AAA-complex in peroxisomal protein import. PolyUb-Pex5p (17,18) as well as monoUb-Pex5p (21,27) are solely found at the peroxisomal membrane fraction in wild-type yeast and rat liver cells, indicating that Pex5p ubiquitination exclusively takes place at the peroxisomal membrane. Interestingly, exported Pex5p appears to be unmodified, indicating that the Ub-moiety is removed during or directly after receptor export (10,11,21). However, published data on the deubiquitination of Pex5p so far have focused on in vitro assays with mammalian Pex5p. Soluble monoUb-Pex5p is formed when the in vitro export reaction is performed in presence of DUB inhibitors (11,27). Accordingly, it was concluded that deubiquitination of Pex5p occurs predominantly in the cytosol after

release from the membrane. It also was suggested that a small fraction of the dislocated Ub-Pex5p in vitro can already be deubiquitinated by reducing reagents like glutathione, while most of the Ub-Pex5p is deubiquitinated via an enzymatic pathway (27). However, cleavage of the Ub-moiety from mammalian Pex5p was thought to be catalyzed by an unspecific reaction that could be carried out by any DUB in the cytosol or may even function via a non-enzymatic reaction. Our data indicate that deubiquitination of yeast Pex5p represents a specific and important event for the optimal functionality of the export machinery. With Ubp15p, we have identified a deubiquitinating enzyme that is dedicated for this deubiquitination event in baker`s yeast. The deubiquitinating activity found to be associated with the endogenous AAA-complex was the first indication for the presence of such an enzyme. Mass spectrometry analysis of the AAA-complex derived from endogenous proteins as well as overexpressed Pex6p revealed a stable association of Ubp15p. The interaction with Pex6p was confirmed by yeast two-hybrid analysis and the interaction site could be mapped to the D1 domain of Pex6p. While the evolutionarily related AAA-protein Cdc48p(p97/VCP) utilizes several co-factors (71), Ubp15p is only the second known co-factor that accompanies the function of Pex6p, with its membrane-anchor Pex15p (Pex26p in mammals) being the first one (37). Pex6p acts in concert with Pex1p as dislocase complex for the ubiquitinated Pex5p in order to facilitate the export of the PTS1-receptor back to the cytosol (8,9). This leads to the intriguing question, how the activity of the deubiquitinating enzyme Ubp15p is coordinated with the Ub-dependent dislocation of Pex5p from the membrane and release into the cytosol. The finding that the deletion of UBP15 does not result in a complete peroxisomal biogenesis defect, can either be explained by the model, that deubiquitination has only modulating activity or it may indicate the existence of additional factors which may accompany the AAA-complex in its function. This situation could well be explained by redundant DUBs acting on Ub-Pex5p. Possible candidates are the known Ubp15p-binding partners Ubp14p and Doa4p (Ubp4p) (64,65). However, the characterization of the single deletion strains suggested that these two DUBs do not have a peroxisome-specific function

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similar to Ubp15p. The single deletion strain of Ubp14p had no significant effect on peroxisome morphology or cargo import, both under oleate as well as under H2O2 stress conditions. Previous studies have suggested a role for Ubp14p in the disassembly of unanchored polyubiquitin chains (64). The deletion of Doa4p had an effect on the efficiency of peroxisomal cargo import. However, it has to be taken into account that the deletion of Doa4p is known to result in pleiotropic effects on many Ub-dependent processes in the cell, as Doa4p influences the homeostasis of free ubiquitin (70). Possibly related to this function, DOA4 is a stress regulated gene, giving an alternative explanation for the oleate induction reported by (68). Thus, although we cannot fully exclude that Doa4p exhibits a peroxisome-related overlapping function with Ubp15p, the partial import defect observed for the doa4Δ strain might well be explained by the pleiotropic phenotype of this mutant. The observation that ubp15Δ cells contain more clustered peroxisomes than wild-type cells is puzzling. Earlier work correlated a reduced level of imported matrix proteins such as catalase and the occurrence of clustered peroxisomes (72). Slowing of Pex5p cycling is most certainly associated with reduced import rates. Interestingly, induction of oxidative stress by treating cells with hydrogen peroxide causes Pex5p to amass on the organelle membrane and significantly reduces PTS1 protein import (73-75). As our data are clear in that Ubp15p can deubiquitinate Pex5p and as the ubiquitination status of the PTS1-receptor directly influences its cycling (10,11), it is conceivable that the deletion of Ubp15p influences the import process of PTS1-proteins like catalase and thus possibly also morphology and clustering of peroxisomes. Although Ubp15p is not essential for peroxisomal biogenesis under normal conditions, its regulative function gains significantly more weight when the cells are stressed with H2O2 and require an efficient import of matrix proteins into peroxisomes. Thus, the findings that 1) Ubp15p is stably associated with the export machinery by interacting with Pex6p, 2) the fact that a small portion of the protein is associated with peroxisomes, and 3) the partial protein import defect for PTS1 proteins observed in ubp15Δ cells upon oxidative stress suggest that the

deubiquitination, at least in bakers yeast, is not an unspecific event that takes place at any location in the cytosol, as suggested by the mammalian study (27), but supports the notion that the detachment of the Ub-moiety is a regulated event. Ubiquitination of the receptor is a precondition for its export (10,11). In this respect, it is likely that the Pex1p/Pex6p-complex recognizes the Ub-moiety. This, however, still needs to be shown. The in vitro data demonstrate that the exported import receptor is deubiquitinated. This reflects the in vivo situation which is clear in that the cytosolic receptor is not ubiquitinated. Thus, the accumulating evidence indicates that the ubiquitin moiety is cleaved off from the import receptor during or shortly after export. There are several possible advantages to favor a peroxisome-associated deubiquitination of Ub-Pex5p. This could protect Pex5p from unspecific ubiquitination by detaching Ub-moieties from lysine residues or preventing the formation of a poly-ubiquitin chain at the crucial cysteine residue dedicated to mono-ubiquitination. This function would ensure an optimal protection and presentation of monoUb-Pex5p to the export machinery. Another possible explanation might be that the deubiquitination step may trigger the efficient release of Pex5p from the export-machinery by cleavage of the complex-bound Ub-moiety. Furthermore, this mechanism could prevent the monoUb-Pex5p to be recognized by the proteasome system to ensure an efficient recycling of the receptor for new matrix protein import cycles. The finding that both ubiquitinating and deubiquitinating activities are required for the transport of proteins from a membrane to the cytosol finds an examples in the ERAD pathway. The AAA-type ATPase p97(Cdc48/VCP) is evolutionary related to the peroxisomal AAA-proteins Pex1p and Pex6p (76). Most interestingly, among the growing number of known co-factors and adaptor proteins that p97 utilizes to carry out its different functions are also several deubiquitinating enzymes (71). The mammalian deubiquitinating enzymes YOD1 and Ataxin-3 are p97-associated proteins and function in the ERAD pathway (77-79). Most of the published literature defines both DUBs as a positive regulator of the p97-driven dislocation of the ERAD-substrates, most likely by editing the poly-Ub chains on the

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substrates themselves in order to ensure the best fit to downstream Ub-receptor proteins. Ubp15p acts in concert with the AAA-peroxins in the matrix protein import cycle of the PTS1-receptor. Pex5p deubiquitination occurs as a highly specific event in yeast and removal of ubiquitin of the PTS1-receptor Pex5p turns out to be a vital step in the receptor cycle in its own right. Thus, removal of the ubiquitin seems to complete the receptor cycle of Pex5p in order to make the receptor available for another round of matrix protein import.

ACKNOWLEDGMENTS We are grateful to Ulrike Freimann for technical assistance and to Wolfgang Schliebs for the reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB642). H.W.P was supported by an EMBO Long term Fellowship.

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FIGURE LEGENDS Figure 1. The yeast AAA-complex possesses ubiquitin hydrolase activity. MonoUb-Pex5p-containing wild-type membranes were incubated with the AAA-complex purified from a cytosolic fraction of pex5Δ cells using the TEV-ProtA tag. Where indicated, the AAA-complex was pre-incubated with NEM or with Ub-aldehyde to inhibit the observed ubiquitin hydrolase activity. Figure 2. Ubp15p forms a complex with the AAA-peroxins. Protein complexes were isolated by affinity chromatography from soluble fractions of (A) protease deficient yeast strain cl3-ABYS-86 with His6-Pex6p or (B) from UTL-7A strain with endogenously encoded Pex1p fused to TEV-ProtA tag. For the latter, the untransformed strain served as control for the specificity of the isolation. Isolated proteins were visualized by silver stain or colloidal Coomassie as indicated. (C) His6-GST-Ubp15p was isolated form soluble fraction of cl3-ABYS-86 strain and analyzed by immunoblotting. Equal volumes of load and the 100 x-concentrated eluate fractions were probed with antibodies raised against indicated proteins. The detection of cytosolic fructose1,6-bisphosphatase (Fbp1p) served as control for unspecific binding. Figure 3. Ubp15p is partially localized to peroxisomes. (A) A cell-free extract of oleate-induced wild-type cells expressing genomically tagged Ubp15p (Ubp15p-TEV-ProtA) was separated by density gradient centrifugation (2.25%-24% Optiprep, 18% sucrose). Fractions were subjected to measurements of the activity of catalase and cytochrom c oxidase as peroxisomal or mitochondrial marker, respectively (upper panel). Equal portions of fractions were probed by immunoblotting (lower panel) with antibodies against the protein A tag, Pex13p (peroxisomes); Porin (mitochondria) as well as Fbp1p (cytosol). (B) Wild-type cells expressing both the PTS2 marker protein PTS2-dsRed as well as GFP-Ubp15p, with and without overexpression of Pex15p, were grown on oleic acid plates for two days and examined by fluorescence microscopy. While only a small portion of GFP-Ubp15p is localized to peroxisomes in cells containing normal levels of Pex15p, a higher fraction of the fusion protein was recruited to peroxisomes upon overexpression of Pex15p, indicated by the co-localization of GFP-Ubp15p and the peroxisomal dsRed-marker. Figure 4. The first AAA-domain of Pex6p mediates the Ubp15p-interaction. The L40 reporter yeast cells were cotransformed with empty two-hybrid plasmids pPC86 and pPC97 (~) or plasmids expressing indicated proteins. Double-transformants were lysed and subjected to liquid ß-galactosidase assay. ß-galactosidase activities (expressed in arbitrary units) indicate binding and are represented as mean values of three independent experiments performed in duplicate. Error bars denote SEM (standard error of the mean). Abbreviations: Ub= ubiquitin; N= amino-terminal domain; D1= first AAA domain; D2= second AAA domain. Figure 5. Ubp15p is an ubiquitin hydrolase acting on poly- as well as monoubiquitinated Pex5p. (A) PolyUb-chains were incubated with recombinant wild-type Ubp15p or mutant Ubp15p(C214) harbouring a substitution of the supposedly active site cysteine. At indicated time-points reactions were stopped by adding SDS-sample buffer. Equal amounts of the samples were subjected to SDS-PAGE. The presence of the indicated proteins was monitored by immunoblotting with antibodies against ubiquitin or Ubp15p as indicated. Membranes isolated from (B) NEM-treated wild-type cells which harbour monoubiquitinated Pex5p were incubated with recombinant Ubp15p followed by Pex5p-immunoisolation or (C) pex1Δpex6Δ cells which contain poly-ubiquitinated Pex5p were incubated with recombinant Ubp15p without further purification steps. The presence of either NEM or

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Ub-aldehyde inhibits hydrolase activity of Ubp15p and serves as control. Samples were subjected to SDS-PAGE and immunoblot analysis and with antibodies against ubiquitin or Pex5p-specific antibodies as indicated to monitor the presence of ubiquitinated Pex5p. Figure 6. ubp15Δ-cells contain functional but clustered peroxisomes (A) Indicated strains were spotted as a series of 10-fold dilutions on media containing oleic acid as sole carbon source and incubated for 5 days at 30°C. In contrast to pex5Δ, ubp15Δ grew at wild-type rate, suggesting that the cells are not affected in peroxisome function. (B) The PTS1 marker protein GFP-SKL and the PTS2 marker protein PTS2-dsRed were co-transformed in wild-type, pex5∆ and ubp15∆-cells. The transformed strains were grown on oleic acid plates for two days and examined by fluorescence microscopy. Mutant pex5∆ cells are capable to import PTS2-proteins properly (indicated by the punctuate pattern) but are impaired in PTS1-dependent matrix protein import and accordingly mislocalize the marker protein to the cytosol. Both wild-type and ubp15∆ exhibit a punctuate congruent staining for both peroxisomal markers, indicative for normal peroxisomal protein import. The peroxisomes of ubp15∆ cells form clusters. (C) Ultrastructural appearance of clustered peroxisomes in ubp15Δ cells. Wild-type and ubp15∆-mutant cells were grown on oleic acid medium and analyzed by electron microscopy. In wild-type cells, peroxisomes are separated and distributed within the cell, whereas the ubp15Δ mutant cells are characterized by peroxisome clusters. Peroxisomes are marked with an asterisk. Size bar: 2.5 µm. Figure 7. ubp15Δ-cells exhibit a lower steady state concentration of Pex5p but higher rate of ubiquitinated Pex5p. (A) Whole cell lysates of oleic acid-induced wild-type as well as ubp15Δ cells were prepared and subjected to immunoblot analysis with antibodies specific for Pex5p and mitochondrial porin, which served as loading control (left). Signal intensity was estimated by densitometric analysis (right). (B) Indicated strains were grown for 10h under oleic-acid conditions and for additional 4 h under same conditions in the presence of MG132 to inhibit proteasomal degradation. Whole cell lysates were prepared and equal portion were subjected to immunoblot analyses with Pex5p antibodies (left). Signal intensity of modified Pex5p in ubp15pΔ cells and unmodified Pex5p in wild-type was quantified by densitometry (right). Figure 8. ubp15Δ-cells exhibit oxidative-stress related protein import deficiencies and defective growth on oleic acid (A) The PTS1 marker protein GFP-PTS1 was transformed into wild-type and indicated mutant strains. The transformed strains were grown in liquid oleic acid media in the absence or presence of 0.2 mM H2O2. All strains exhibit normal import of the marker protein GFP-PTS1 on oleic acid medium without oxidative stress. Upon supplementation of the oleic acid medium with H2O2, the marker protein was partially mislocalized to the cytosol in both doa4∆ and upb15Δ cells whereas wild-type and ubp14∆ remained unaffected. (B) Indicated strains were cultured in either oleic acid or glucose as sole carbon source in the absence (closed symbols) and presence (open symbols) of 0.2 mM H2O2. At different time points samples were taken and optical density was estimated at 600 nm.

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EUKARYOTIC CELL, June 2011, p. 770–775 Vol. 10, No. 61535-9778/11/$12.00 doi:10.1128/EC.05038-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

The Putative Saccharomyces cerevisiae Hydrolase Ldh1pIs Localized to Lipid Droplets�

Sven Thoms,1† Mykhaylo O. Debelyy,1 Melanie Connerth,2 Gunther Daum,2 and Ralf Erdmann1*Abteilung fur Systembiochemie, Institut fur Physiologische Chemie, Medizinische Fakultat der Ruhr-Universitat Bochum,

D-44780 Bochum, Germany,1 and Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria2

Received 18 March 2011/Accepted 30 March 2011

Here, we report the identification of a novel hydrolase in Saccharomyces cerevisiae. Ldh1p (systematic name,Ybr204cp) comprises the typical GXSXG-type lipase motif of members of the �/�-hydrolase family and sharessome features with the peroxisomal lipase Lpx1p. Both proteins carry a putative peroxisomal targeting signaltype1 (PTS1) and can be aligned with two regions of homology. While Lpx1p is known as a peroxisomal enzyme,subcellular localization studies revealed that Ldh1p is predominantly localized to lipid droplets, the storagecompartment of nonpolar lipids. Ldh1p is not required for the function and biogenesis of peroxisomes, andtargeting of Ldh1p to lipid droplets occurs independently of the PTS1 receptor Pex5p.

Peroxisomes and lipid droplets (LDs) are ubiquitous eukary-otic organelles involved in lipid metabolism. LDs appear asoleosomes in plants, as adiposomes in mammals, or as lipidparticles/bodies/droplets in yeasts and constitute a family ofmorphologically and biogenetically similar organelles (19).LDs are bound by a phospholipid monolayer and serve as themain storage sites for nonpolar lipids, mainly triacylglycerols(TAG) and cholesteryl ester (CE) (6, 7). LDs derive from theendoplasmic reticulum (ER), possibly by inclusion of nonpolarlipids between the two ER leaflets, eventually leading to thebudding of nascent LDs (1, 6, 24, 27, 36). A large number ofLD proteins have been identified by proteomic studies (12). Inrecent years, it has become evident that LDs, rather than beingsolely lipid storage sites, play a dynamic role in lipid biosyn-thesis, metabolism, degradation, and trafficking (6). Peroxi-somes are particularly engaged in the �-oxidation of long- andvery long-chain fatty acids (16). Notably, in yeast, peroxisomesare the only site of fatty acid �-oxidation (37). In mammals,peroxisomes are also involved in bile acid and plasmalogensynthesis, as well as amino acid metabolism (37, 38). Defectiveperoxisome biogenesis can lead to severe heritable diseases inhumans (32). Such biogenesis defects are caused by mutationsin PEX genes coding for proteins required for peroxisomebiogenesis, collectively called peroxins (25, 34). The majority ofperoxisomal matrix proteins are directed to peroxisomes by aperoxisomal targeting signal type1 (PTS1). The three aminoacids SKL (serine-lysine-leucine) at the very C terminus of aprotein represent the first PTS1 discovered. Generally, PTS1comprises tripeptides with the consensus sequence [SAC][KRH][LM]. The PTS1 is recognized in the cytosol by thecycling import receptor Pex5p (8). Masking of the PTS1 by theaddition of protein tags interrupts PTS1-Pex5p association and

prevents peroxisomal localization (40). A peroxisomal target-ing signal type 2 (PTS2) is located within the first 20 aminoacids of the N terminus of some peroxisomal proteins. Perox-isomal proteins with a PTS2 are recognized by the importreceptor Pex7p (20, 21, 42).

Here, we report the identification of a novel hydrolase in S.cerevisiae. The gene sequence of LDH1 predicts a GXSXG-type motif that is typical of �/�-hydrolases and/or lipases (31).Bioinformatics analysis suggests that LDH1 (YBR204C) en-codes a novel peroxisomal protein, due to its putative PTS1(17). In the present study, however, we show that Ldh1p is notrequired for the function and biogenesis of peroxisomes andthat Ldh1p primarily localizes to LDs, independently of theperoxisomal protein import machinery.

MATERIALS AND METHODS

Strains and plasmids. S. cerevisiae strains BY4742, BY4742 �yor084w,BY4742 �ybr204c, BY4742 �pex5, and BY4742 �pex1 were obtained from EU-ROSCARF (Frankfurt). BY4742 ERG6-RFP was obtained from W. K. Huh(San Francisco, CA). BY4742 ERG6-RFP �ybr204c was constructed by genereplacement using kanMX6 from pUG6 and primers 5�-CTAGAAGAGATTGTTCAAAATGCAGAAAATGCAGCTGATTTGGTCGTACGCTGCAGGTCGAC-3� and 5�-GCACGAAAATCTAGTTACGCAATGTGAAATCTAGAAAACCTTCTAATCGATGAATTCGAGCTCG-3�. BY4742 �pex5�ldh1 andBY4742 �pex1�ldh1 were constructed from BY4742 �pex5 and BY4742 �pex1 bygene replacement using a pUG6 vector and primers 5�-GCTAGAAGAGATTGTTCAAAATGCAGAAAATGCAGCTGATTTGGTCGTACGCTGCAGGTCGAC-3� and 5�-GCACGAAAATCTAGTTACGCAATGTGAAATCTAGAAAACCTTCTAATCGATGAATTCGAGCTCG-3� after removal of loxP-kanMX6-loxP marker cassettes (13, 14). The yeast media have been describedpreviously (9, 10). For construction of pUG35-LDH1 (Ldh1p-GFP), PCR-am-plified YBR204c (primers RE2444 [5�-GCGCGGATCCATGAATATGGCAGAACGTGCA-3�] and RE2445 [5�-GCGCAAGCTTCAATTTGGAATTATCAATCAC-3�]) was introduced into BamH I and HindIII sites of pUG35. Forconstruction of pUG36-LDH1 (GFP-Ldh1p), PCR-amplified YBR204C (prim-ers RE2444 [5�-GCGCGGATCCATGAATATGGCAGAACGTGCA-3�] andRE2446 [5�-GCGCAAGCTTCTACAATTTGGAATTATCAATCAC-3�]) wasintroduced into BamHI and HindIII sites of pUG36. All constructs were con-firmed by DNA sequencing. The GFP-SKL plasmid has been described previ-ously (29).

Nile Red and Oil Red O staining. For Nile Red staining (39), yeast cells instationary phase were washed and resuspended in phosphate-buffered saline(PBS) (150 mM NaCl, 1.7 mM KH2PO4, 5.2 mM Na2HPO4). The cells werestained with Nile Red solution (0.0005% in PBS, diluted from a 0.01% stocksolution in acetone) for 15 min at room temperature in the dark. The cells were

* Corresponding author. Mailing address: Institut fur PhysiologischeChemie, Ruhr-Universitat Bochum, Universitatsstr. 150, D-44780 Bo-chum, Germany. Phone: 49 234 322 4943. Fax: 49 234 321 4266. E-mail:[email protected].

† Present address: Universitatsmedizin Gottingen, Abteilung fur Pa-diatrie und padiatrische Neurologie, Georg-August-Universitat Got-tingen, D-37099 Gottingen, Germany.

� Published ahead of print on 8 April 2011.

770

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then washed six times with PBS to remove surplus dye. For Oil Red O staining(26, 39), yeast cells in stationary phase were washed twice, fixed by 4% formal-dehyde in PBS for 20 min, and washed twice again. The cells were then stainedwith Oil Red O (0.2% in a water-isopopanol [1:1] mixture) for 15 min at roomtemperature in the dark and washed six times before microscopic analysis.

Image acquisition. Samples were fixed with 0.5% (wt/vol) agarose on micro-scope slides. Fluorescence microscopic images were recorded on an AxioPlan 2microscope (Zeiss) equipped with a �Plan-FLUAR 100�/1.45 oil objective andan AxioCam MRm camera (Zeiss) at room temperature. If necessary, contrastwas linearly adjusted using the image acquisition software AxioVision 4.8(Zeiss).

Subcellular fractionation and organelle isolation. Subcellular fractionationand gradient centrifugation for the analysis of peroxisomes and mitochondria of�ldh1 were carried out as described previously (29, 33). Cell fractionation andLD isolation for the subcellular localization of Ldh1p have been describedpreviously (5, 11, 28).

RESULTS

Ldh1p and Lpx1p: two similar hydrolases. Ldh1p sharessome features with the peroxisomal lipase Lpx1p (33) (Fig. 1).Both proteins have almost the same predicted molecular mass,namely, 43 kDa for Ldh1p and 44 kDa for Lpx1p. Both pro-teins carry a putative PTS1, the prototypical SKL in Ldh1p,and glutamine-lysine-leucine (QKL) in Lpx1p (Fig. 1A). Fur-thermore, both proteins can be aligned with two regions ofhomology (Fig. 1A and B), with one in the central domain,

comprising the lipase motif GHSMG (4, 35), indicative ofmembers of the �/�-hydrolase family. In the case of Ldh1p, theamino acids adjacent to the active-site serine are identical inthe two proteins, namely, histidine (H) and methionine (M).Hydropathy plots indicated a pronounced hydrophobic regionin the centers of both proteins. Amino acids 130 to 154 ofLdh1p comprise a hydrophobic core region, 138VVELIFVLV146, and amino acids 154 to 177 of Lpx1p comprise the coreregion, 164LLILIEPVVI173 (Fig. 1C).

Absence of a synthetic phenotype of �ldh1 and �lpx1 inperoxisome biogenesis. Ldh1p carries the prototypical yet pu-tative PTS1 and has been speculated to be a peroxisomal ma-trix protein (17). Therefore, we first tested the effect of anLDH1 deletion on peroxisome biogenesis. Postnuclear super-natants (PNS) were prepared from wild-type and �ldh1 strainsand analyzed by density gradient centrifugation. The gradientfractions were assayed for peroxisomal catalase and mitochon-drial cytochrome c oxidase activity (Fig. 2A). The distributionof neither of these proteins indicated a significant change inthe abundance or density of peroxisomes or mitochondria,suggesting that peroxisomal and mitochondrial biogenesis re-main functional after deletion of LDH1. As a defect in perox-isome biogenesis would affect peroxisome presence or density,we conclude that Ldh1p is not a peroxin. Altogether, the avail-

FIG. 1. Ldh1p and Lpx1p from S. cerevisiae are similar proteins with a hydrolase/lipase motif. (A) Similarities between Lpx1p (predicted mass,43.7 kDa; 387 amino acids; theoretical pI, 8.16) and Ldh1p (predicted mass, 43.3 kDa; 375 amino acids; theoretical pI, 6.36) are indicated: tworegions of homology, the first of which contains the GHSMG hydrolase/lipase motif of the GXSXG consensus. Both proteins carry a (putative)PTS1, QKL, or SKL. (B) Alignment of the two regions of homology of Lpx1p and Ldh1p exhibiting 28% (region A) and 27% (region B) aminoacid identities. Asterisk, histidine of the probable catalytic triad; arrowhead, aspartate of the probable catalytic triad in Ldh1p. The GXSXGhydrolase/lipase motif is underlined; similar amino acids are indicated by a plus symbol. (C) Hydropathy plots of Ldh1p. The Kyte-Doolittle plotwas calculated with a window size of 11. Values greater than 1.8 indicate very hydrophobic regions. (D) C terminus of Ldh1p. The amino acidsin positions �2 and �5 are likely to interfere with peroxisomal targeting.

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able evidence suggested that Lpx1p and Ldh1p might be pro-teins exerting similar or redundant functions. Most mutantswhose peroxisome biogenesis or functions are affected arecharacterized by a growth defect on oleic acid (9). We there-fore tested the single and double knockouts of LPX1 andLDH1 for growth on oleate as the only carbon source (Fig. 2B).Neither of these knockouts had its growth on oleic acid af-fected, suggesting that Lpx1p and Ldh1p do not form a redun-dant pair in peroxisome function.

Ldh1p localizes to the lipid droplet membrane. Next, weinvestigated the subcellular distribution of Ldh1p. Ldh1p wasexpressed from a plasmid as N-terminally or C-terminallytagged green fluorescent protein (GFP) fusion proteins thatlocalized to a particular organelle about 1 to 2 �m in diameterwith several copies in a cell (Fig. 3A). Ldh1p specifically local-

ized to the surface membranes of these organelles. We rea-soned that the organelles were fragmented vacuoles, endo-somes, or LDs. Thus, we coexpressed marker proteins for theorganelles together with the Ldh1p fusion proteins and foundthat Ldh1p perfectly colocalized with Erg6p, the �(24)-sterolmethyl transferase (Fig. 3A, top), which is a major and promi-nent LD protein (18). Both proteins localize to the surface mem-brane of LDs. Ldh1p colocalized with Erg6p when GFP waslocalized at the N terminus or the C terminus of the protein (Fig.3A). Localization of Ldh1p in LDs was also confirmed by Oil RedO staining (Fig. 3B). Ldh1p contains a perfect consensus for aPTS1 at its extreme C terminus. The fact that some LD proteinscontain a C-terminal localization signal (22) and the possibility ofa common origin of peroxisomes and LD encouraged us to testwhether the PTS1 of Ldh1p is required for LD targeting. Wefound that neither masking of the SKL by expression of the GFPat the C terminus of Ldh1p nor deletion of the PTS1 receptorprotein Pex5p interfered with targeting of Ldh1p (Fig. 3C). Thus,the PTS1-like C terminus of Ldh1p does not function as a clas-sical peroxisomal targeting signal, nor does it interfere with tar-geting of the polypeptide to LD.

To verify the localization of Ldh1p, we performed cell frac-tionation analysis with a yeast strain that expressed plasmid-encoded Ldh1p-GFP. LDs were isolated by flotation on adensity gradient (5, 28). Subcellular fractions of the gradientwere analyzed by immunoblotting with polyclonal antibodiesagainst GFP and organelle-specific marker enzymes (Fig. 4).These data revealed that Ldh1p-GFP was highly enriched inLD, as represented by the LD marker proteins Erg1p(squalene epoxidase) and Erg6p, but Ldh1p-GFP also cofrac-tionated to some extent with the peroxisomal marker proteinFox1p (fatty-acyl coenzyme A oxidase) and the mitochondrialmarker protein Por1p (mitochondrial porin) (Fig. 4). It hasbeen shown that some LD proteins are not exclusively found inthis compartment but also localize to the ER; in contrast,Ldh1p appears to localize to LD and, possibly to a lesserextent, to mitochondria and peroxisomes.

The biogenesis of peroxisomes and lipid droplets does notrequire LDH1. To test whether deletion of LDH1 influencesthe intracellular distribution or morphology of peroxisomes,we analyzed wild-type and �ldh1 strains expressing the perox-isomal marker protein GFP-SKL by fluorescence microscopy.Microscopic inspection of the LD was performed by Oil Red Ostaining (Fig. 5). These results showed that the morphologicalappearance of peroxisomes, as well as the frequently observedproximity to LD, was not affected by deletion of LDH1. Havingshown that Ldh1p is targeted to LD independently of thesoluble PTS1 receptor, we investigated whether Ldh1p is re-quired for the biogenesis of LDs. After introducing a �ldh1knockout into the genomically tagged ERG6-red fluorescentprotein (RFP) marker strain for LD, we found that LD couldstill be formed in the absence of Ldh1p (Fig. 6A). We con-firmed these findings by LD staining with Nile Red (Fig. 6B)and Oil Red O (Fig. 6C). Taking these data together, it ap-pears that Ldh1p is not required for the formation of LD.

DISCUSSION

Ldh1p is a lipid droplet hydrolase with an SKL terminus.Ldh1p contains the consensus sequence for a classical peroxi-

FIG. 2. Ldh1p is dispensable for peroxisome biogenesis and func-tion. (A) Postnuclear supernatants prepared from oleate-induced wild-type and �ldh1 strains were fractionated by density gradient centrifu-gation, and each fraction was analyzed for catalase (peroxisome) andcytochrome c oxidase (mitochondria) activities. The absence of Ldh1phas no influence on the apparent densities of peroxisomes and mito-chondria. (B) Growth on oleate is not affected by deletion of the lipasegene LDH1 or LPX1 or both. Single or double deletions of LDH1 andLPX1 were spotted on oleate and ethanol plates with equal cell num-bers in a series of 10-fold dilutions and grown for 3 days at 30°C.

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somal targeting signal, but the protein is primarily targeted toLD and not to peroxisomes. Peroxisomal exclusion of Ldh1p islikely due to the upstream sequences with charged amino acidsin positions �2 and �5 (Fig. 1D). These positions are adverseto Pex5p binding and peroxisomal localization, for which polar/hydrophilic or positively charged amino acids in position �2

are preferred. In our case, the negatively charged amino acid isnot even counteracted by neighboring amino acids, giving thelikely explanation for dominating peroxisomal exclusion. Theclassical PTS1, SKL, is not completely sufficient to target pro-tein to peroxisomes if the upstream sequences are not support-ive. We show that the majority of Ldh1p is an LD protein thatis targeted independently of the PTS1-binding Pex5p. Thisview is confirmed by applying a PTS1 prediction algorithm

FIG. 3. Ldh1p primarily localizes to lipid droplets, and its localization is independent of the peroxisomal import receptor Pex5p. (A) Ldh1pcolocalizes with the LD marker protein Erg6p [�(24)-sterol methyl transferase]. GFP-Ldh1p and Ldh1p-GFP were coexpressed in a yeast strainwith genomically tagged Erg6p-RFP. Bar, 1 �m. (B) Ldh1p colocalizes with the LD marker dye Oil Red O. GFP-Ldh1p and Ldh1p-GFP werecoexpressed in a wild-type yeast strain. Bar, 1 �m. (C) Ldh1p localization is independent of the peroxisomal PTS1 pathway. GFP was fused to eitherthe C terminus (top images) or the N terminus (bottom images) of Ldh1p. Also, in a �pex5 deletion mutant, Ldh1p localization to LD was notcompromised (right). In both cases Ldh1p colocalizes with the LD marker dye Oil Red O.

FIG. 4. Subcellular localization of Ldh1p. (A) Organelles from thewild-type strain carrying Ldh1p-GFP were isolated from cells grown tostationary phase in oleic acid-containing medium. Proteins from thesubcellular fractions were precipitated, and the same amounts wereseparated by SDS-PAGE and analyzed by Western blotting using pri-mary antibodies against marker enzymes, as indicated. The sameamounts of proteins were loaded; therefore, the intensity of the GFPband does not represent the relative distribution of Ldh1p betweenLDs, mitochondria, and peroxisomes. The presence of organelles wasdetected with primary antibody against marker enzymes, as indicated.Erg1p, squalene epoxidase; Erg6p, �(24)-sterol methyl transferase(lipid droplets); Fox1p, fatty-acyl coenzyme A oxidase (peroxisomes);Por1p, porin (mitochondria); Wbp1p (endoplasmic reticulum); H, ho-mogenate; C, cytosol; 40g, 40,000 � g microsomes (endoplasmic retic-ulum); 100g, 100,000 � g microsomes (endoplasmic reticulum); Mt,mitochondria; Px, peroxisomes.

FIG. 5. The association of lipid droplets and peroxisomes is notaffected by deletion of LDH1. Shown is fluorescence microscopy ofwild-type yeast and the �ldh1 strain transformed with pGFP-SKL. LDswere stained with Oil Red O (ORO). BF, bright field. Bar, 1 �m.

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(http://mendel.imp.ac.at/pts1/) (23) that does not predict per-oxisomal localization for Ldh1p.

LD localization signals are only poorly characterized. It hasbeen suggested that LD localization signals are constituted ofhydrophobic residues at the C terminus of a protein (22, 41). AKyte-Doolittle plot of Ldh1p indicated a region with particu-larly high hydrophobicity from amino acids 130 to 154 (Fig.1C). This stretch might be required to target and/or to attachLdh1p to LDs. Indeed, our data show that LD targeting is notabrogated when GFP is added to the C terminus or the Nterminus of Ldh1p. Thus, targeting information within centralparts of Ldh1p, rather than at its termini, is sufficient for theLD localization. Interestingly, the Lpx1p stretch of high hydro-phobicity is in a similar location in the primary sequence,namely, at amino acids 154 to 177. The hydrophobic stretchesin Ldh1p are likely not classical transmembrane domains(TMD), because LDs are bound by a single monolayer mem-brane of phospholipids.

Extended localization studies of Ldh1p-GFP showed that atleast a portion of the polypeptide is targeted to peroxisomesand mitochondria. While this triple localization may reflect thetrue cellular scenario, we also have to take into account thatpartial targeting of Ldh1p to peroxisomes and mitochondriamay be due to the overexpression of Ldh1p-GFP.

We were able to show that Ldh1p and the lipase Lpx1p arenot redundant, provided that other enzymes, probably withsomewhat lower homology, cannot compensate for a defect inthe two enzymes. Both peroxisomes and LD function in con-cert in lipid metabolism. LDs require the action of triacylglyc-erol lipases to metabolize nonpolar lipids, while peroxisomesrepresent the sole cellular site for fatty acid oxidation. It is thuspossible that the peroxisomal Lpx1p and the LD Ldh1p play aphysiological role in lipid metabolism by mobilizing fatty acidsand channeling them to their site of degradation. LDs, as fattyacid depot organelles, can be the storage sites for nonpolarlipids that are further metabolized in peroxisomes. For thisreason, and not surprisingly, LDs have been found in proximityto peroxisomes in different organisms (2, 15, 30). It was alsoshown that S. cerevisiae peroxisomes attach to LDs or evenproject into LDs, which was interpreted as an intimate inter-action between the two compartments (3). Our work on Ldh1pand Lpx1p shows that, beyond a metabolic collaboration, per-oxisomes and LDs may be equipped with similar hydrolases.

ACKNOWLEDGMENTS

We thank Elisabeth Becker, Monika Burger, and Uta Ricken fortechnical assistance; Robert Rucktaschel for scientific input; and Wolf-gang Girzalsky for reading the manuscript.

This work was supported by the Deutsche Forschungsgemeinschaft(SFB642, ER178/4-1).

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EUKARYOTIC CELL, June 2011, p. 776–781 Vol. 10, No. 61535-9778/11/$12.00 doi:10.1128/EC.05040-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Involvement of the Saccharomyces cerevisiae Hydrolase Ldh1pin Lipid Homeostasis�

Mykhaylo O. Debelyy,1 Sven Thoms,1† Melanie Connerth,2 Gunther Daum,2 and Ralf Erdmann1*Abteilung fur Systembiochemie, Institut fur Physiologische Chemie, Medizinische Fakultat der Ruhr-Universitat Bochum,

D-44780 Bochum, Germany,1 and Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria2

Received 18 March 2011/Accepted 30 March 2011

Here, we report the functional characterization of the newly identified lipid droplet hydrolase Ldh1p.Recombinant Ldh1p exhibits esterase and triacylglycerol lipase activities. Mutation of the serine in thehydrolase/lipase motif GXSXG completely abolished esterase activity. Ldh1p is required for the maintenanceof a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of theSaccharomyces cerevisiae �ldh1 strain is the appearance of giant lipid droplets and an excessive accumulationof nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source.Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipidand nonpolar lipid levels.

Lipid droplets (LDs) are remarkable dynamic subcellularorganelles of globular shape with a size range from 20 to 100�m, depending on the cell type (9, 12, 15, 31). LDs are depotsof neutral lipids with a complex biology that exist in virtuallyany kind of cell, ranging from bacteria to yeasts, plants, andhigher mammals (3, 13, 15). In many cells, LDs occupy aconsiderable portion of the cell volume and weight (35). As themajor intracellular storage organelles, LDs were first describedin the works of R. Altmann and E. B. Wilson in the 19thcentury (1, 37). In contrast to the vesicular organelles, whichhave the aqueous content enclosed by a phospholipid bilayermembrane (12, 13), mature LDs have a unique physical struc-ture: they have a neutral lipid core consisting of triacylglycerols(TG) and sterol esters (SE) surrounded by a phospholipidmonolayer (3, 24, 38) that contains numerous peripheral orembedded proteins (26, 33). TG as well as SE play crucial rolesfor the cell: TG is the main energy store, and both TG and SEare depots of membrane lipid components (35). LDs cantightly regulate the level of intracellular free cholesterol byhydrolyzing sterol ester (26). The LD core also contains otherendogenous neutral lipids, like monoacylglycerol, diacylglyc-erol, free cholesterol, and retinol ester, and xenobiotic hydro-phobic compounds, such as polycyclic aromatic hydrocarbons(15, 17, 29, 32, 33). A number of proteins are specificallytargeted to the LD surface (18), where they can regulate LDdynamics and the turnover of stored lipids (24). Lipid-metab-olizing enzymes, including hydrolases and lipases, are the ma-jor class of LD enzymes (9). LDs play crucial roles in cellularenergy homeostasis and lipid metabolism (35). LDs can pro-vide a rapidly mobilized lipid source for many important bio-logical processes. Neutral lipids may be mobilized for the gen-

eration of energy by �-oxidation or for the synthesis ofmembrane lipids and signaling molecules (9). It has beenshown that nearly all cell types have the ability to generate LDsin response to elevated fatty acid levels and to subsequentlymetabolize and disperse these LDs when conditions are re-versed (26), thereby providing an emergency energy pool forcell survival (3). Due to their unique architecture, LDs canprotect cells from the effects of potentially toxic lipid species,such as unesterified lipids (23, 24) or toxic free fatty acids (3),by depositing them inside the LD’s core. In addition to thislipid-scavenging function, LDs can transiently store certainproteins, which may be released or degraded at later timepoints (9, 13, 14, 36).

Here, we report the functional characterization of the newlyidentified LD hydrolase Ldh1p (34a). We demonstrate thatrecombinant Ldh1p exerts esterase and triacylglycerol lipaseactivities. The enzyme activity was abolished upon mutation ofthe conserved GXSXG-type lipase motif of the protein. TheSaccharomyces cerevisiae �ldh1 strain is characterized by theappearance of giant LDs and the accumulation of nonpolarlipids and phospholipids in LDs, indicative of a role of Ldh1pin maintaining lipid homeostasis.

MATERIALS AND METHODS

Strains and plasmids. S. cerevisiae strains BY4742, BY4742 �ybr204c, BY4742�yor084w, BY4742 �ybr204c �yor084w, BY4742 ERG6-RFP, and BY4742ERG6-RFP �ybr204c are described in reference 34a. DNA plasmids pUG35-LDH1 (Ldh1p-GFP) and pUG36-LDH1 (GFP-Ldh1p) are described in reference34a. Yeast media have been described previously (10, 11). pUG35-LDH1-M1[Ldh1p-(S177A)-GFP] and pUG36-LDH1-M1 [GFP-Ldh1p-(S177A)] were clonedfrom pUG35-LDH1 and pUG36-LDH1 using a QuikChange Site-Directed Mu-tagenesis Kit (Agilent Technologies) (primers RE2400 [5�-ATAGTGCTTGTAGGGCATGCTATGGGTTGTTTTCTGGCA-3�] and RE2401 [5�-TGCCAGAAAACAACCCATAGCATGCCCTACAAGCACTAT-3�]). pET21d-LDH1 wasconstructed by introducing PCR-amplified YBR204c (primers OST248 [5�-GCGAATTCCATATGAATATGGCAGAACGTGCAG-3�] and OST217 [5�-GCTGCGGCCGCCAATTTGGAATTATCAATCACC-3�]) into NdeI and NotIsites of pET21b (EMD Chemicals). pET21d-LDH1-M1 [Ldh1p-(S177A)-His6]was cloned from pET21d-LDH1 using the QuikChange Site-Directed Mutagen-esis Kit (Agilent Technologies) (primers RE2400 [5�-ATAGTGCTTGTAGGGCATGCTATGGGTTGTTTTCTGGCA-3�] and RE2401 [5�-TGCCAGAAAA

* Corresponding author. Mailing address: Institut fur Physiolo-gische Chemie, Ruhr-Universitat Bochum, Universitatsstraße 150,D-44780 Bochum, Germany. Phone: 49 234 322 4943. Fax: 49 234321 4266. E-mail: [email protected].

† Present address: Universitatsmedizin Gottingen, Abteilung fur Pa-diatrie und padiatrische Neurologie, Georg-August-Universitat Got-tingen, D-37099 Gottingen, Germany.

� Published ahead of print on 8 April 2011.

776

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CAACCCATAGCATGCCCTACAAGCACTAT-3�]). All constructs wereconfirmed by DNA sequencing.

Protein expression. Ldh1p was expressed from plasmid pET21b-LDH1 inEscherichia coli BL21(DE3). Cells were harvested by centrifugation and dilutedin buffer A (1� phosphate-buffered saline [PBS], 300 mM sodium chloride, 1mM dithiothreitol, 40 mM imidazole) containing a protease inhibitor mixture (8�M antipain-dihydrochloride, 0.3 �M aprotinin, 1 �M bestatin, 10 �M chymo-statin, 5 �M leupeptin, 1.5 �M pepstatin), together with 50 �g/ml lysozyme, 22.5�g/ml DNase I, and 40 mM imidazole. The cells were sonicated using a 250DBranson (Danbury, CT) Digital Sonifier. After removal of cell debris by centrif-ugation, the supernatant was clarified by 0.22-�m filtration and loaded on His-Trap columns (GE Healthcare Life Sciences) equilibrated with buffer A. Thecolumn was washed in buffer A, and recombinant Ldh1p was eluted by acontinuous 40 to 500 mM imidazole gradient. Peak fractions were identifiedby SDS-PAGE and pooled, and the isolated protein was concentrated withVivaSpin concentrators (30-kDa cutoff; Sartorius). The concentrated Ldh1p wassubjected to size exclusion chromatography on an AKTA Purifier FPLC System withSuperdex 200 (GE Healthcare Life Sciences). Peak fractions of Ldh1p were iden-tified by SDS-PAGE and pooled, and the isolated protein was concentrated withVivaSpin (30-kDa cutoff; Sartorius).

Enzyme assays. Esterase activity was determined with p-nitrophenyl butyrate(PNB) (Sigma) in PBS (pH 7.4) in a total volume of 200 �l at 37°C. Freep-nitrophenol was determined at 410 nm in 96-well plates. Michaelis-Mentenkinetics was analyzed using GraphPad Prism 5 (GraphPad Software). Triacyl-glycerol lipase (TGL) activity was determined using 1,2-dioleoyl-3-pyrenede-canol-rac-glycerol (DPG) (Marker Gene) in 0.1 M glycine, 19 mM sodiumdeoxycholate, pH 9.5, in a total volume of 200 �l at 37°C. Hydrolysis of DPG wasfollowed in 96-well plates at 460 nm with 360-nm excitation in a Sirius HTfluorescence plate reader (MWG Biotech). The TGL activity of Ldh1p towardDPG was compared with the TGL activity of Candida rugosa triacylglycerollipase (Lipase AT30 Amano; 1,440 units/mg; Sigma) as a control. We alsoadapted a specific and sensitive TGL assay originally developed for the mea-surement of bacterial TGLs (22). The TGL activity of Ldh1p on rhodamine Bagar plates was determined by using agar plates containing trioleoylglycerol andrhodamine B. The agar (1% [wt/vol]) was dissolved in PBS, adjusted to pH 7.4,autoclaved, and cooled to 60°C. Then, trioleoylglycerol (2.5% [wt/vol]) andrhodamine B (0.001% [wt/vol]) were added to the agar medium with vigorousstirring for 1 min. The medium was kept for 10 min at 60°C to reduce foaming,and 20 ml of medium was poured into plastic petri dishes. To detect triacylglyc-erol lipase activity, holes with a diameter of 6 mm were punched into the agarand filled with 200 �l protein solution. Ldh1p and C. rugosa lipase (CRL) werediluted in PBS (pH 7.4). The plates were incubated for 48 h at 30°C. After 48 h,the plates began to show an orange fluorescence visible under UV light (350 nm).

Lipid extraction and TLC. The lipids were extracted by the method of Blighand Dyer (4). The organic layer was washed three times with 1 M KCl, and thesolvent was removed by evaporation in a vacuum. The lipids were dissolved in asmall volume of chloroform and separated on thin-layer chromatography (TLC)plates (TLC Silica gel 60 F254; 20 by 20 cm; Merck) using chloroform-methanol-water (65:25:4 [vol/vol/vol]) as the developing solvent. Lipid classeswere visualized with iodine vapor and identified according to TLC standard18-5A (Nu-Chek Prep, Elysian, MN).

Electron microscopy. The ultrastructure of yeast cells was studied with oleate-induced cells that had been fixed with 1.5% KMnO4 and processed as describedpreviously (10).

Miscellaneous. Oil Red O staining, image acquisition, and the isolation of LDsare described in reference 34a. LD purification for lipid extraction was per-formed as described previously (8, 27). The weight of LDs was estimated gravi-metrically in 1.5-ml reaction tubes (Eppendorf).

RESULTS

Enzymatic activity of Ldh1p. Characteristic GXSXG motifsand similarities to �/�-hydrolases in the predicted protein se-quences of Ldh1p suggest that the protein is an esterase orlipase (5, 28, 34). Indeed, Ldh1p was identified as a serinehydrolase by computational and chemical proteomics methods(2). We expressed Ldh1p as hexahistidine-tagged fusions inE. coli (Fig. 1A) and tested the isolated protein for esteraseactivity using PNB as a substrate. We found Ldh1p to be an

active esterase hydrolyzing the model substrate PNB with a Km

of 0.77 mM and a Vmax of 0.041 �mol/min/mg (Fig. 1B).Phospholipase A, C, and D activities were not detected (data

not shown). For the analysis of phospholipase A activity, weused the fluorogenic phospholipase A substrate bis-BODIPYFL C11-PC (B7701; Invitrogen) [1,2-bis-(4,4-difluoro-5,7-di-methyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine]. For analysis of phospholipase C

FIG. 1. Protein expression, purification, and enzymatic activity ofLdh1p. (A) Ldh1p was expressed as a fusion protein with a hexahisti-dine tag and purified by affinity chromatography. (B) Esterase activityof Ldh1p toward PNB. Km and Vmax values were calculated usingMichaelis-Menten approximations. (C) TGL activity of Ldh1p towardDPG. (D) Purified Ldh1p and CRL were incubated on plates contain-ing 2.5% trioleoylglycerol and 0.001% rhodamine B and, after 48 h,imaged at 350 nm. The numbers indicate the concentrations in mg/ml.In total, 200 �l was loaded per agar slot. Hydrolysis of trioleoylglycerolwas identified by fluorescent halos.

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activity, we used the Amplex Red Phosphatidylcholine-SpecificPhospholipase C Assay Kit (A12218; Invitrogen). Phospho-lipase D activity was assayed with the Amplex Red Phospho-lipase D Assay Kit (A12219; Invitrogen).

Next, we assayed TGL activity using DPG as a substrate(Fig. 1C). One of the acyl residues of DPG contains the exi-mer-forming pyrene decanoic acid. Upon hydrolytic cleavage,the released pyrene decanoic acid leads to a decrease in eximerfluorescence. We found Ldh1p to be an active triacylglycerollipase hydrolyzing the model substrate DPG with a Km of 3.3mM and a Vmax of 1 �mol/min/mg (Fig. 1C). TGL activity wasalso confirmed by an assay with fluorescein dilaurate as thesubstrate (not shown). We also adapted a specific and sensitiveTGL assay originally developed for the measurement of bac-terial TGLs (22). This assay is based on the hydrolysis oftrioleoylglycerol and the formation of orange fluorescent rho-damine B halos. The results shown in Fig. 1D revealed thatLdh1p exerts a weak TGL activity. In summary, the purifiedLdh1p exerts esterase and TG lipase activities.

Mutational analysis of the GXSXG-type lipase motif. Thecharacteristic GXSXG motif of �/�-hydrolases is present inLdh1p and is thought to contribute to the active site of theenzyme (Fig. 2A). To test this experimentally, we introduced apoint mutation into the putative active site of Ldh1p (S177A)and analyzed the mutated protein for esterase activity. Re-placement of serine with alanine in the hydrolase/lipase motifof Ldh1p completely abolished hydrolase activity. The mutatedprotein (Ldh1m1p) still localized to LDs, suggesting that thecatalytic activity is not required for its topogenesis (Fig. 2B).

The �ldh1 mutant is characterized by the accumulation oflipids. Ldh1p has been shown to be predominantly localized toLDs (34a). To characterize the function of Ldh1p in moredetail, we investigated whether the enzyme is involved in thebiogenesis of LDs. To this end, LDs were isolated from oleicacid-induced wild-type and �ldh1 mutant cells and appeared as

a thick layer on top of a gradient of the mutant (Fig. 3A). Thetotal weight of LDs was drastically increased in the �ldh1 yeaststrain in comparison to the wild type (Fig. 3B). These datawere corroborated by TLC separation of extracted lipids from

FIG. 2. Hydrolase activity is not required for Ldh1p targeting to LDs. (A) Ldh1p is a hydrolytically active serine hydrolase with a classicalcatalytic triad containing a conserved serine (GXSXG motif), histidine, and aspartate (grey shading). (B) GFP-Ldh1m1p and Ldh1m1p-GFP werecoexpressed in a yeast strain with genomically tagged Erg6p-RFP. Ldh1m1p colocalizes with the LD marker protein Erg6p [�(24)-sterol methyltransferase]. Ldh1p containing a mutation of the active site (S177A) still localizes to LDs, indicating that the lipid targeting is independent of itscatalytic activity. Bar, 1 �m. BF, bright field.

FIG. 3. The �ldh1 yeast strain exhibits excessive accumulation ofnonpolar and polar lipids in LDs during growth on medium containingoleic acid as a sole carbon source. (A) LDs were isolated from wild-type and �ldh1 mutant cells and appeared as a thick layer on top of agradient of the mutant. (B) The total weight of LDs was stronglyincreased in the �ldh1 yeast strain in comparison to the wild type. Theerror bars indicate standard deviations. (C) TLC separation of ex-tracted nonpolar and polar lipids from purified LDs, which showed theincrease in nonpolar lipids and phospholipids in the �ldh1 yeast strain.PC, phosphatidylcholine; PE, phosphatidylethanolamine; NPL, non-polar lipids.

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purified LDs, which showed the increase in nonpolar lipids andphospholipids in the �ldh1 yeast strain (Fig. 3C).

Giant lipid droplets in �ldh1 mutant cells. To analyzewhether the accumulation of lipids in mutant cells lacking theLD protein Ldh1p is accompanied by changes in LD morphol-ogy, the LDs of oleic acid-induced wild-type and �ldh1 knock-out cells expressing genomically encoded Erg6p-red fluores-cent protein (RFP) were visualized by fluorescence microscopy(Fig. 4A), and the LDs of oleic acid-induced wild-type and�ldh1 knockout cells were stained with Oil Red O and in-spected by fluorescence microscopy (Fig. 4B). The data dem-onstrate that LDs can still be formed in the absence of Ldh1p,indicating that Ldh1p per se is not required for the formationof LDs. However, the morphological appearance of LDs in�ldh1 mutant cells differed significantly from that in wild-typecells. The LDs of the mutant exhibited brighter fluorescence,indicating the existence of bigger LDs. These data were cor-roborated by electron microscopic inspection of wild-type andmutant cells, which revealed the presence of giant LDs in the�ldh1 mutant (Fig. 4C).

Esterase activity of Ldh1p is required for lipid homeostasis.The �ldh1 yeast strain exhibits an excessive accumulation oflipids in LDs during growth on medium containing oleic acid asa sole carbon source. To test whether the loss of hydrolaseactivity of Ldh1p is responsible for the observed phenotype, wetested complementation of the mutant with functional andcatalytic dead Ldh1p harboring a substitution of the active-siteserine (Ldh1m1p). LDs were isolated from oleic acid-induced

wild-type cells, �ldh1 mutant cells, and mutant cells expressingplasmids encoding either wild-type Ldh1p or the mutantLdh1m1p. LDs appeared as a thick layer on top of the gradi-ent, and comparison of the gradients revealed a thin lipid layeron top of the gradient for the wild type and the �ldh1 mutantcomplemented with wild-type Ldh1p. A thicker layer, which istypical of the �ldh1 mutant, was monitored for mutant cellsthat contained the catalytic dead Ldh1p (Fig. 5A). These datawere corroborated by determination of the total weight of LDs,which was increased in the �ldh1 strain and remained increasedupon expression of the mutant protein (not shown). Accordingly,staining with Oil Red O and inspection of the cells by fluores-cence microscopy (Fig. 5B), as well as by electron microscopy(Fig. 5C), revealed that the giant-LD phenotype of the �ldh1strain could be complemented with wild-type Ldh1p, but not withthe catalytic dead mutant Ldh1p. These data demonstrate thatfunctional complementation of the �ldh1 mutant phenotype re-quires expression of enzymatically active Ldh1p, indicating thatthe hydrolase activity of the enzyme is required for its function inlipid homeostasis.

DISCUSSION

Ldh1p is a hydrolytically active serine hydrolase with a clas-sical catalytic triad containing a serine (GXSXG motif). Aconserved histidine was revealed by profile hidden Markovmodels (9a), and the aspartate of the probable triad was de-rived from an alignment with canine gastric triacylglycerollipase (Fig. 2A). The putative active-site serine of Ldh1p islocated next to the regions of highest hydrophobicity, suggest-ing that Ldh1p is a membrane-active hydrolase. We demon-strated that the hydrolase activity of Ldh1p could be com-pletely abolished by the replacement of the active-site serine byalanine. Fluorescence microscopy analysis indicated thatLdh1p targets to the boundary of the LD monolayer mem-brane, supporting the idea that Ldh1p is involved in metabolicprocesses. Taken together, these features characterize Ldh1pas an active LD hydrolase. Mutation of the active site of Ldh1pdoes not lead to protein mislocalization, indicating that thelipase active site of Ldh1p is not involved in LD targeting.

Cells deficient in Ldh1p are characterized by giant LDsaccompanied by the accumulation of nonpolar lipids and phos-pholipids. Thus, Ldh1p seems to be required for the mobiliza-tion of LD-stored lipids, which would also explain the depen-dency of the Ldh1p function on its hydrolase activity. Wespeculate that Ldh1p plays a role in maintaining lipid homeo-stasis by regulating both phospholipid and nonpolar lipid lev-els. Interestingly, the �ldh1 (�ybr204c) strain has been re-ported to exhibit resistance to the lipophilic drug camptothecin(16, 19, 20). Camptothecin is a cytotoxic quinoline alkaloid thatinhibits the DNA enzyme topoisomerase I. The resistance tocamptothecin might be explained by increased detoxificationproperties of LDs with an excessive amount of nonpolar lipids,which may serve as a reservoir for hydrophobic toxic molecules(3, 7, 21, 35). Global genomic screening research recently dis-closed the transient induction of LDH1 by growth on oleatemedium (30). It was shown that the level of Ldh1p increasedwithin the first 3 h of induction, followed by a decrease withinthe subsequent 6 h and complete reduction to basal levelswithin the next 17 h. Such an expression profile might hint at a

FIG. 4. Giant LDs in the �ldh1 mutant. (A) Comparison of LDmorphologies of the wild type (BY4742 Erg6p-RFP) and a deletionstrain (BY4742 �ldh1 Erg6p-RFP) by fluorescence microscopy. Bar, 1�m. (B) Localizations and morphologies of Oil Red O-stained wild-type (BY4742) and deletion strain (BY4742 �ldh1) LDs. Bar, 1 �m.(C) Absence of LDH1 leads to the formation of giant LDs, as well tothe reduction of the total LD number in a cell. Shown are electronmicroscopic images of cells: the wild type (BY4742) and a deletionstrain (BY4742 �ldh1). Bars, 1 �m.

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regulatory or signaling function instead of direct involvementof the enzyme in lipid metabolism. Interestingly, LDH1 expres-sion is also induced upon sporulation (6), which is mildly af-fected in cells deficient in Ldh1p (25). Our data clearly showthat Ldh1p per se is not required for the biogenesis of LDs, butthe severe accumulation of lipids and the corresponding ap-pearance of the giant LDs in �ldh1 mutant cells strongly sug-gest a role for the enzyme in LD lipid homeostasis.

ACKNOWLEDGMENTS

We thank Elisabeth Becker, Monika Burger, and Uta Ricken fortechnical assistance; Robert Rucktaschel for scientific input; and Wolf-gang Girzalsky for reading the manuscript.

This work was supported by the Deutsche Forschungsgemeinschaft(SFB642 and ER178/4-1).

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FIG. 5. The esterase activity of Ldh1p is required for lipid homeostasis. The �ldh1 yeast strain exhibits excessive accumulation of lipids in LDsduring growth on medium containing oleic acid as a sole carbon source. LDs were isolated from oleic acid-induced wild-type cells and �ldh1 mutantcells expressing plasmids encoding either wild-type Ldh1p or the mutant Ldh1m1p. (A) LDs appeared as a thick layer on top of the gradient, andcomparison of the gradients revealed a thin lipid layer on top of the gradient for the wild type (WT) and the �ldh1 mutant complemented withwild-type Ldh1p. A thicker layer, typical of the �ldh1 mutant, was monitored for mutant cells that contained the catalytic dead Ldh1p. (B) Stainingwith Oil Red O and inspection by fluorescence microscopy revealed that the giant-LD phenotype of the �ldh1 strain could be complemented withwild-type Ldh1p, but not with the catalytic dead mutant Ldh1p. Bar, 1 �m. (C) Electron microscopy revealed that the giant-LD phenotype of the�ldh1 strain could be complemented with wild-type Ldh1p, but not with the catalytic dead mutant Ldh1p. Bar, 1 �m.

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CHAPTER 3. DISCUSSION_________________________________________________

77

CHAPTER 3. DISCUSSION

The novel hydrolases and peroxisome related ubiquitin-specific protease of yeast

S. cerevisiae are characterized in this chapter. Lpx1p and Ldh1p are hydrolases of

peroxisome and lipid droplets, respectively. Ubp15p is a peroxisome related

deubiquitinating enzyme. Triacylglycerol lipase and hydrolase activities were shown for

both recombinant proteins Lpx1p and Ldh1p as well as oligoubiqutin-hydrolase and

Ub-Pex5p deubiquitinating activities were shown for recombinant Ubp15p in vitro. It is

demonstrated that the Lpx1p protein is not required for wild-type-like steady-state

function of peroxisomes and that Δlpx1 mutants have an aberrant morphology

characterized by intraperoxisomal vesicles or invaginations. Morover, Ldh1p is not

required for the function and biogenesis of peroxisomes, but is essential for the

maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. In

line with this finding, the Δldh1 strain is characterized by appearance of giant lipid

droplets and an excessive accumulation of nonpolar lipids and phospholipids upon

growth on medium containing oleic acid as a sole carbon source.

It is demonstrated that the peroxisomal AAA-complex contains Pex5p dislocase

and Ub-Pex5p deubiquitinating activites and that Ubp15p is a novel constituent of this

complex. Δubp15 mutant is characterized as a strain which has a stress related PTS1-

import defect.

3.1 Novel hydrolases of yeast S. cerevisiae

Lpx1p as well as Ldh1p, a novel hydrolase of S. cerevisiae (35, 209), comprises the typical

GXSXG-type lipase motif of members of the α/β-hydrolase family (189). LPX1 is one of the

most strongly induced genes following a shift from glucose to oleate, as determined by serial

analysis of gene expression (SAGE) experiments (103). The oleate-induced increase in

mRNA abundance is abolished in the Δpip2 Δoaf1 double deletion strain, indicating that its

induction is dependent on the transcription factor pair Pip2p and Oaf1p (103). It was shown

by use of an antibody raised against Lpx1p that this protein itself is induced by oleic acid

(210). Besides, it was determined, by using a Protein A tag, that Lpx1p protein is strongly

induced by oleic acid (196). Moderate induction by oleic acid was also demonstrated for

Ldh1p protein (196).

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CHAPTER 3. DISCUSSION_________________________________________________

78

Both proteins Lpx1p as well as Ldh1p carry a putative peroxisomal targeting signal

type-1 (PTS1) (126) and can be aligned with two regions of homology by WUBLAST-2

search (134) (Fig. 3.1.1).

Fig. 3.1.1 Ldh1p and Lpx1p from S. cerevisiae are similar proteins with a α/β-hydrolase/lipase motif. Alignment of the two regions of homology of Lpx1p and Ldh1p exhibiting 28% (region A) and 27% (region B) amino acid identities. The GXSXG hydrolase/lipase motif is underlined; similar amino acids are indicated by a plus symbol. Taken with modifications from (209).

Lpx1p does not conform with its QKL motife to the general PTS1 consensus. Three

other proteins with an QKL on their extreme C-terminus are known in S. cerevisiae, which

are probably not peroxisomal: Efb1p (systematic name: Yal003wp) is the elongation factor

EF-1b (82), Rpt4p (Yor259cp) is a mostly nuclear 19S proteasome cap AAA protein (149),

and Tea1p (Yor337wp) is a nuclear Ty1 enhancer activator (70). However, QKL is sufficient

to sponsor Pex5p binding (124). Why are these QKL proteins not imported into peroxisomes?

This is probably due to the upstream sequences. Lpx1p has a lysine at position -1 (relative to

the PTS1 tripeptide) and a hydrophobic amino acid at position -5. These features promote

Pex5p binding and are not found in the other three QKL proteins (124).

Ldh1p contains the consensus sequence for a classical peroxisomal targeting signal

type-1 (PTS1), but the protein is primarily targeted to lipid droplets and not to peroxisomes.

Peroxisomal exclusion of Ldh1p is likely due to the upstream sequences with charged amino

acids in positions - 2 and - 5. These positions are adverse to Pex5p binding and peroxisomal

localization, for which polar/hydrophilic or positively charged amino acids in position - 2 are

preferred. The negatively charged amino acid is not even counteracted by neighbouring amino

acids, giving the likely explanation for dominating peroxisomal exclusion. The classical

PTS1, SKL, is not completely sufficient to target protein to peroxisomes if the upstream

sequences are not supportive. It was shown that the majority of Ldh1p is a lipid droplets

protein that is targeted independently of the PTS1-binding Pex5p.

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Moreover, it was shown by applying a PTS1 prediction algorithm

(http://mendel.imp.ac.at/pts1/) (156, 157), which predicted peroxisomal localization, that only

Lpx1p but not Ldh1p as well as Efb1p, Rpt4p, and Tea1p, is localized in peroxisome.

It was shown dimerization of Lpx1p in the context of piggyback protein import into

peroxisomes (210). Self-interaction (dimerization) is frequently found in regulation of the

enzymatic activity of other lipases such as Candida rugosa lipase or human lipoprotein lipase

(63, 164).

Lipid droplets localization signals are only poorly characterized. It has been suggested

that lipid droplets localization signals are constituted of hydrophobic residues at the C-

terminus of a protein (154, 237). A Kyte-Doolittle plot of Ldh1p indicated a region with

particularly high hydrophobicity from amino acids 130 to 154. This stretch might be required

to target and/or to attach Ldh1p to lipid droplets. Indeed, it was shown that lipid droplets

targeting are not abrogated when GFP is added to the C-terminus or the N-terminus of Ldh1p.

Thus, targeting information within central parts of Ldh1p, rather than at its termini, is

sufficient for the lipid droplets localization. Interestingly, the Lpx1p stretch of high

hydrophobicity is in a similar location in the primary sequence, namely, at amino acids 154 to

177 (210). The hydrophobic stretches in Ldh1p are likely not classical transmembrane

domains, because lipid droplets are bound by a single monolayer membrane of phospholipids.

Extended localization studies of Ldh1p-GFP showed that at least a portion of the polypeptide

is targeted to peroxisomes and mitochondria. While this triple localization may reflect the true

cellular scenario, it has to be taken into account that partial targeting of Ldh1p to peroxisomes

and mitochondria may be due to the overexpression of Ldh1p-GFP.

The putative active-site serine of Lpx1p is located next to the region of highest

hydropathy, suggesting that Lpx1p is a membrane-active lipase that contributes to metabolism

or the membrane shaping of peroxisomes. Peroxisomes are sites of lipid metabolism (223). It

is thus not surprising to find a lipase associated with peroxisomes. It was demonstrated that

Lpx1p has triacylglycerol lipase activity; however, activities towards the artificial test

substrates DPG (1,2-dioleoyl-3-(pyren-1-yl) decanoyl-rac-glycerol) and DGR (1,2-O-dilauryl-

rac-glycero-3-glutaric acid (6-methyl resorufin) ester) were low (210). The evidence for

phospholipase A activity of the enzyme (substrate: 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-

3a,4a-diaza-sindacene-3-undecanoyl)-sn-glycero-3-phosphocholine), together with the

electron microscopy phenotype, suggest that Lpx1p has a more specialized role in modifying

membrane phospholipids (210). A mammalian group VIB calcium-independent

phospholipase A2 (iPLA2c) was identified that possesses a PTS1 SKL and a mitochondrial

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targeting signal (140, 234). The enzyme is localized in peroxisomes and mitochondria, and is

involved, among others, in arachidonic acid and cardiolipin metabolism (139, 155). Knockout

mice of iPLA2c show mitochondrial ⁄ cardiac phenotypes (141). It will be exciting to

determine whether human iPLA2c and yeast Lpx1p are functionally related. It was shown that

peroxisomes are still functional in the absence of LPX1. This suggests a non-essential

metabolic role for Lpx1p in peroxisome function (210). The morphological defect found in

electron microscopic images of a deletion of Lpx1p (peroxisomes containing inclusions or

invaginations) is symptomatic of a yeast peroxisomal mutant, and is reminiscent of the

phenotypes found in human peroxisomal disorders (56, 151). All these data suggest that

Lpx1p is required to determine the shape of peroxisomes (210).

Lipase activity and cellular function of Lpx1p could be involved in various processes:

(a) detoxification and stress response, (b) lipid mobilization, or (c) peroxisome biogenesis. As

Lpx1p expression may be regulated by Yrm1p and Yrr1p (135), a transcription factor pair that

mediates pleiotropic drug resistance effects, it was speculated that Lpx1p is required for a

multidrug resistance response (210). The epoxide hydrolase activity for Lpx1p was, however,

excluded because hydrolysis of the epoxide hydrolase test substrate was not affected by a

specific epoxide hydrolase inhibitor (210).

It was demonstrated that recombinant Ldh1p exerts an esterase and triacylglycerol

lipase activities. The enzyme activity was abolished upon mutation of the conserved GXSXG-

type lipase motif of the protein. The S. cerevisiae Δldh1 strain is characterized by the

appearance of giant lipid droplets and the accumulation of nonpolar lipids and phospholipids

in lipid droplets, indicative of a role of Ldh1p in maintaining lipid homeostasis (35). Ldh1p is

a hydrolytically active serine hydrolase with a classical catalytic triad containing a serine

(GXSXG motif). A conserved histidine was revealed by profile hidden Markov models (40),

and the aspartate of the probable triad was derived from an alignment with canine gastric

triacylglycerol lipase (209). The putative active-site serine of Ldh1p is located next to the

regions of highest hydrophobicity, suggesting that Ldh1p is a membrane-active hydrolase. It

was demonstrated that the hydrolase activity of Ldh1p could be completely abolished by the

replacement of the active-site serine by alanine. Fluorescence microscopy analysis indicated

that Ldh1p targets to the boundary of the lipid droplets monolayer membrane, supporting the

idea that Ldh1p is involved in metabolic processes.

Taken together, these features characterize Ldh1p as an active lipid droplets hydrolase.

Mutation of the active site of Ldh1p does not lead to protein mislocalization, indicating that

the lipase active site of Ldh1p is not involved in lipid droplets targeting.

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Cells deficient in Ldh1p are characterized by giant lipid droplets accompanied by the

accumulation of nonpolar lipids and phospholipids. Thus, Ldh1p seems to be required for the

mobilization of lipid droplets-stored lipids, which would also explain the dependency of the

Ldh1p function on its hydrolase activity. It was speculated that Ldh1p plays a role in

maintaining lipid homeostasis by regulating both phospholipid and nonpolar lipid levels (35).

Interestingly, the Δldh1 (Δybr204c) strain has been reported to exhibit resistance to the

lipophilic drug camptothecin (89, 110, 111). Camptothecin is a cytotoxic quinoline alkaloid

that inhibits the DNA enzyme topoisomerase I. The resistance to camptothecin might be

explained by increased detoxification properties of lipid droplets with an excessive amount of

nonpolar lipids, which may serve as a reservoir for hydrophobic toxic molecules (15, 32, 112,

221).

Global genomic screening research recently disclosed the transient induction of LDH1

by growth on oleate medium (196). It was shown that the level of Ldh1p increased within the

first 3 h of induction, followed by a decrease within the subsequent 6 h and complete

reduction to basal levels within the next 17 h. Such an expression profile might hint at a

regulatory or signalling function instead of direct involvement of the enzyme in lipid

metabolism.

Interestingly, LDH1 expression is also induced upon sporulation (29), which is mildly

affected in cells deficient in Ldh1p (142). The data clearly show that Ldh1p per se is not

required for the biogenesis of lipid droplets, but the severe accumulation of lipids and the

corresponding appearance of the giant LDs in Δldh1 mutant cells strongly suggest a role for

the enzyme in lipid droplets lipid homeostasis (35).

While Lpx1p was shown to be a peroxisomal enzyme, subcellular localization studies

revealed that Ldh1p is predominantly localized to lipid droplets. It was shown that Lpx1p

import is dependent on the PTS1 receptor Pex5p. Moreover, it was shown that Lpx1p is

piggyback-transported into peroxisomes. But it was demonstrated that targeting of Ldh1p to

lipid droplets occurs independently of the PTS1 receptor Pex5p. Triacylglycerol lipase as well

as hydrolase activities were shown for both recombinant proteins Lpx1p and Ldh1p in vitro. It

was shown that the Lpx1p protein is not required for wild-type-like steadystate function of

peroxisomes, which might be indicative of a metabolic rather than a biogenetic role. It was

clearly shown that peroxisomes in Δlpx1 mutants have an aberrant morphology characterized

by intraperoxisomal vesicles or invaginations.

It was demonstrated that Ldh1p is not required for the function and biogenesis of

peroxisomes. Ldh1p is required for the maintenance of a steady-state level of the nonpolar

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82

and polar lipids of lipid droplets. A characteristic feature of the Δldh1 strain is the appearance

of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids

upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to

play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and

nonpolar lipid levels.

Ldh1p hydrolase and the Lpx1p lipase are not redundant proteins; other enzymes,

probably with somewhat lower homology, cannot compensate for a defect in the two

enzymes. Both peroxisomes and lipid droplets function in concert in lipid metabolism. Lipid

droplets require the action of triacylglycerol lipases to metabolize nonpolar lipids, while

peroxisomes represent the sole cellular site for fatty acid oxidation. It is thus possible that the

peroxisomal Lpx1p and the lipid droplets Ldh1p play a physiological role in lipid metabolism

by mobilizing fatty acids and channeling them to their site of degradation. Lipid droplets, as

fatty acid depot organelles, can be the storage sites for nonpolar lipids that are further

metabolized in peroxisomes. For this reason, and not surprisingly, LDs have been found in

proximity to peroxisomes in different organisms (14, 78, 187). It was also shown that S.

cerevisiae peroxisomes attach to lipid droplets or even project into lipid droplets, which was

interpreted as an intimate interaction between the two compartments (18). Ldh1p and Lpx1p

shows that beyond a metabolic collaboration, peroxisomes and lipid droplets may be equipped

with similar hydrolases (209).

3.2 Ubp15p, a novel compound of AAA-complex

It was proposed, at least for Pex1p, that it can fulfil its function by unfoldase activity,

using its N-terminal putative adaptor-binding domain (193).

So far, it is possible only speculate, that both AAA peroxins, Pex1p and Pex6p are

highly substrate specific unfoldases/foldase (chaperons), enzymes that unfold/fold protein

substrate in ATP-hydrolysis dependent manner. In this case, ubiquitinated Pex5p could be a

substrate for such activity. On the first step of the Ub-Pex5p release from the peroxisome it

could be unfolded by one of AAA peroxin, probably by Pex6p. On the next step, Pex5p could

be folded by second AAA peroxin Pex1p, with the following release of Pex5p to the cytosol.

Such hypothesis could explain a requirement of two AAA ATPases, instead of just one.

It was recently shown that the AAA-complex is responsible for receptor

deubiquitination, which is supposed to be an important step in receptor recycling (34).

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It was shown that yeast S. cerevisiae has 19 deubiquitinating enzymes (DUB) (Table

3.2.1). All of them have catalytic regions with three evolutionary conserved amino acids:

cysteine, hystedine and tryptophan (Fig 3.2.1).

Fig. 3.2.1 Highest homology region of S. cerevisiae deubiquitinating enzymes. Moderately conserved residues are shaded in grey whereas the conserved histidine (H) and tryptophan (D), two aminoacids of the catalytic triad CHD, as well as highly conserved tyrosine (Y) are highlighted in black. ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) algorithm was used for yeast S. cerevisiae DUBs alignment. The corresponding deubiquitinating enzymes (DUB) Ubp15p was identified as a novel factor

that accompanies the AAA-complex in peroxisomal protein import (34). It was demonstrated

that Ubp15p share common features conserved among other UBP members (90) (Fig 3.2.2).

All members of the ubiquitin-specific processing protease family (UBP) of deubiquitinating

enzymes (DUB) share strong homology in the Cys and His Boxes. The Cys Box contains the

catalytic cysteine residue, which is thought to undergo deprotonation and to unleash a

nucleophilic attack on the carbonyl carbon atom of the ubiquitin Gly76 at the scissile peptide

bond. In analogy with other cysteine proteases, the deprotonation of this cysteine residue most

likely is assisted by an adjacent His residue, which, in turn, is stabilized by a nearby side

chain from an Asn or Asp residue. Together, these three residues constitute the so-called

catalytic triad (90). Previous mutagenesis studies on several UBPs have provided evidence

that these residues have critical roles in catalysis (9, 61, 90, 91).

PolyUb-Pex5p (106, 171) as well as monoUb-Pex5p (71, 116) are solely found at the

peroxisomal membrane fraction in wild-type yeast and rat liver cells, indicating that Pex5p

ubiquitination exclusively takes place at the peroxisomal membrane.

Interestingly, exported Pex5p appears to be unmodified, indicating that the Ub-moiety

is removed during or directly after receptor export (27, 116, 169). However, published data on

the deubiquitination of Pex5p so far have focused on in vitro assays with mammalian Pex5p.

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Table 3.2.1 Deubiquitinating enzymes of yeast Saccharomyces cerevisiae

DUB

Description

Cellular component

References

1

Ubp1p

Ubiquitin-specific protease that removes ubiquitin from ubiquitinated proteins; cleaves at the C terminus of ubiquitin fusions irrespective of their size; capable of cleaving polyubiquitin chains.

cytoplasm, endoplasmic reticulum

(1, 4, 12, 23, 34, 77, 109, 115, 125, 130, 133, 214)

2

Ubp2p

Ubiquitin-specific protease that removes ubiquitin from ubiquitinated proteins; interacts with Rsp5p and is required for MVB sorting of membrane proteins; can cleave polyubiquitin and has isopeptidase activity.

cytoplasm (12, 23, 122, 125)

3

Ubp3p

Ubiquitin-specific protease that interacts with Bre5p to co-regulate anterograde and retrograde transport between the ER and Golgi; inhibitor of gene silencing; cleaves ubiquitin fusions but not polyubiquitin; also has mRNA binding activity.

cytoplasm (12, 30, 31, 148)

4

Doa4p

Ubiquitin isopeptidase, required for recycling ubiquitin from proteasome-bound ubiquitinated intermediates, acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.

endosome, membrane fraction, proteasome complex, mitochondrion

(1, 3, 5, 6, 109, 161, 162, 174, 202)

5

Ubp5p

Putative ubiquitin-specific protease, closest paralog of Doa4p but has no functional overlap; concentrates at the bud neck.

cellular bud neck, incipient cellular bud site

(3, 5, 115, 161)

6

Ubp6p

Ubiquitin-specific protease situated in the base subcomplex of the 26S proteasome, releases free ubiquitin from branched polyubiquitin chains; works in opposition to Hul5p polyubiquitin elongation activity; mutant has aneuploidy tolerance.

proteasome complex, proteasome regulatory particle

(1, 23, 74)

7

Ubp7p

Ubiquitin-specific protease that cleaves ubiquitin-protein fusions. cytoplasm (5, 83)

8

Ubp8p

Ubiquitin-specific protease that is a component of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) acetylation complex; required for SAGA-mediated deubiquitination of histone H2B.

DUBm complex, SAGA complex, SLIK (SAGA-like) complex

(5, 79, 83)

9

Ubp9p

Ubiquitin carboxyl-terminal hydrolase, ubiquitin-specific protease that cleaves ubiquitin-protein fusions.

cytoplasm (77, 115)

10

Ubp10p

Ubiquitin-specific protease that deubiquitinates ubiquitin-protein moieties; may regulate silencing by acting on Sir4p; involved in posttranscriptionally regulating Gap1p and possibly other transporters; primarily located in the nucleus.

nucleus (5, 101, 102, 194)

11

Ubp11p

Ubiquitin-specific protease that cleaves ubiquitin from ubiquitinated proteins.

UNKNOWN (5, 125)

12

Ubp12p

Ubiquitin carboxyl-terminal hydrolase, ubiquitin-specific protease present in the nucleus and cytoplasm that cleaves ubiquitin from ubiquitinated proteins.

cytoplasm, nucleus (5, 23, 92)

13

Ubp13p

Putative ubiquitin carboxyl-terminal hydrolase, ubiquitin-specific protease that cleaves ubiquitin-protein fusions.

UNKNOWN (5, 77, 83)

14

Ubp14p

Ubiquitin-specific protease that specifically disassembles unanchored ubiquitin chains; involved in fructose-1,6-bisphosphatase (Fbp1p) degradation; similar to human isopeptidase T.

cytoplasm (1, 4, 130)

15

Ubp15p

Ubiquitin-specific protease that may play a role in ubiquitin precursor processing.

cytoplasm, peroxisome (1, 23, 34, 115, 133)

16

Ubp16p

Deubiquitinating enzyme anchored to the outer mitochondrial membrane, probably not important for general mitochondrial functioning, but may perform a more specialized function at mitochondria.

cytoplasm, mitochondrial outer membrane

(109, 133)

17

Yuh1p

Ubiquitin C-terminal hydrolase that cleaves ubiquitin-protein fusions to generate monomeric ubiquitin; hydrolyzes the peptide bond at the C-terminus of ubiquitin; also the major processing enzyme for the ubiquitin-like protein Rub1p.

cytoplasm (12, 23, 125, 131, 182, 214)

18

Otu1p

Deubiquitylation enzyme that binds to the chaperone-ATPase Cdc48p; may contribute to regulation of protein degradation by deubiquitylating substrates that have been ubiquitylated by Ufd2p; member of the Ovarian Tumor (OTU) family.

cytoplasm, nucleus (22, 59, 98, 137, 181)

19

Rpn11p

Metalloprotease subunit of the 19S regulatory particle of the 26S proteasome lid; couples the deubiquitination and degradation of proteasome substrates; involved, independent of catalytic activity, in fission of mitochondria and peroxisomes.

cytosol, mitochondrion, nucleus, proteasome regulatory particle, lid subcomplex, proteasome storage granule

(16, 74, 85, 220, 235)

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Fig 3.2.2 Sequence alignment of Ubp15p with six representative UBP family proteins Conserved residues are shaded in yellow whereas the catalytic triad is highlighted in red. Residues that are involved in direct inter-molecular hydrogen bond interactions using their side chains and main chains are marked with purple and green arrows, respectively. Residues that are involved in van der Waals contact with ubiquitin aldehyde (Ubal) are labelled with blue squares. Residues that coordinate the oxyanion through hydrogen bonds are identified with blue triangles above the alignment. The secondary structural elements above the sequences are indicated for the free HAUSP (lower) and the ubiquitin-bound HAUSP (upper), respectively. Taken with modifications from (90).

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CHAPTER 3. DISCUSSION_________________________________________________

86

Soluble monoUb-Pex5p is formed when the in vitro export reaction is performed in

presence of DUB inhibitors (27, 71).

Accordingly, it was concluded that deubiquitination of Pex5p occurs predominantly in

the cytosol after release from the membrane. It also was suggested that a small fraction of the

dislocated Ub-Pex5p in vitro can already be deubiquitinated by reducing reagents like

glutathione, while most of the Ub-Pex5p is deubiquitinated via an enzymatic pathway (71).

Cleavage of the Ub-moiety from mammalian Pex5p was originally thought to be

catalyzed by an unspecific reaction that could be carried out by any DUB in the cytosol or

may even function via a non-enzymatic reaction (71). Later it was shown that

deubiquitination of yeast Pex5p represents a specific and important event for the optimal

functionality of the export machinery (34). Ubp15p has been identified as deubiquitinating

enzyme that is dedicated for this deubiquitination event in baker’s yeast. The deubiquitinating

activity found to be associated with the endogenous AAA-complex was the first indication for

the presence of such an enzyme. Mass spectrometry analysis of the AAA-complex derived

from endogenous proteins as well as overexpressed Pex6p revealed a stable association of

Ubp15p. The interaction with Pex6p was confirmed by yeast two-hybrid analysis and the

interaction site could be mapped to the D1 domain of Pex6p. While the evolutionarily related

AAA-protein Cdc48p (p97/VCP) utilizes several co-factors (98), Ubp15p is only the second

known co-factor that accompanies the function of Pex6p, with its membrane-anchor Pex15p

(Pex26p in mammals) being the first one (20).

Pex6p acts in concert with Pex1p as dislocase complex for the ubiquitinated Pex5p in

order to facilitate the export of the PTS1-receptor back to the cytosol (150, 172). This leads to

the intriguing question, how the activity of the deubiquitinating enzyme Ubp15p is

coordinated with the Ub-dependent dislocation of Pex5p from the membrane and release into

the cytosol. The finding that the deletion of UBP15 does not result in a complete peroxisomal

biogenesis defect, can either be explained by the model that deubiquitination has only

modulating activity or it may indicate the existence of additional factors which may

accompany the AAA-complex in its function. This situation could well be explained by

redundant DUBs acting on Ub-Pex5p.

Possible candidates are the known Ubp15p-binding partners Ubp14p and Doa4p

(Ubp4p) (4, 120). However, the characterization of the single deletion strains suggested that

these two DUBs do not have a peroxisome-specific function similar to Ubp15p. The single

deletion strain of Ubp14p had no significant effect on peroxisome morphology or cargo

import, both under oleate as well as under H2O2 stress conditions. Previous studies have

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CHAPTER 3. DISCUSSION_________________________________________________

87

suggested a role for Ubp14p in the disassembly of unanchored polyubiquitin chains (4). The

deletion of Doa4p had an effect on the efficiency of peroxisomal cargo import. However, it

has to be taken into account that the deletion of Doa4p is known to result in pleiotropic effects

on many Ub-dependent processes in the cell, as Doa4p influences the homeostasis of free

ubiquitin (202). Possibly related to this function, DOA4 is a stress regulated gene, giving an

alternative explanation for the oleate induction reported by (197). Thus, although it is not

possible to fully exclude that Doa4p exhibits a peroxisome-related overlapping function with

Ubp15p, the partial import defect observed for the Δdoa4 strain might well be explained by

the pleiotropic phenotype of this mutant.

The observation that Δubp15 cells contain more clustered peroxisomes than wild-type

cells is puzzling. Earlier work correlated a reduced level of imported matrix proteins such as

catalase and the occurrence of clustered peroxisomes (238). Slowing of Pex5p cycling is most

likely associated with reduced import rates. Interestingly, induction of oxidative stress by

treating cells with hydrogen peroxide causes Pex5p to amass on the organelle membrane and

significantly reduces PTS1 protein import (127, 165, 206). As data are clear in that Ubp15p

can deubiquitinate Pex5p and as the ubiquitination status of the PTS1-receptor directly

influences its cycling (27, 169), it is conceivable that the deletion of Ubp15p influences the

import process of PTS1-proteins like catalase and thus possibly also morphology and

clustering of peroxisomes. Although Ubp15p is not essential for peroxisomal biogenesis

under normal conditions, its regulative function gains significantly more weight when the

cells are stressed with H2O2 and require an efficient import of matrix proteins into

peroxisomes. Thus, the findings that 1) Ubp15p is stably associated with the export

machinery by interacting with Pex6p, 2) the fact that a small portion of the protein is

associated with peroxisomes, and 3) the partial protein import defect for PTS1 proteins

observed in Δubp15 cells upon oxidative stress suggest that the deubiquitination, at least in

baker’s yeast, is not an unspecific event that takes place at any location in the cytosol, as

suggested by the mammalian study (71), but supports the notion that the detachment of the

Ub-moiety is a regulated event.

Ubiquitination of the receptor is a precondition for its export (27, 169). In this respect,

it is likely that the Pex1p/Pex6p-complex recognizes the Ub-moiety. This, however, still

needs to be shown. The in vitro data demonstrate that the exported import receptor is

deubiquitinated. This reflects the in vivo situation which is clear in that the cytosolic receptor

is not ubiquitinated. Thus, the accumulating evidence indicates that the ubiquitin moiety is

cleaved off from the import receptor during or shortly after export.

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88

There are several possible advantages to favour a peroxisome-associated

deubiquitination of Ub-Pex5p. This could protect Pex5p from unspecific ubiquitination by

detaching Ub-moieties from lysine residues or preventing the formation of a poly-ubiquitin

chain at the crucial cysteine residue dedicated to mono-ubiquitination (Fig 3.2.3). This

function would ensure an optimal protection and presentation of monoUb-Pex5p to the export

machinery

Fig 3.2.3 The PTS1-receptor cycle. Hypothetical model describes a possible function of Ubp15p in Pex5p cycle. Ubp15p can protect Pex5p from unspecific polyubiquitination by ubiquitin-conjugating enzyme (E2) Ubc4p. Such activity of Ubp15p can prevent Pex5p proteasomal degradation and save it for the next round of recetor cycle. Red colored arrows show direction of the Pex5p cycle with participation of Ubp15p; blue colored arrow show direction of the Pex5p cycle without participation of Ubp15p. DUB, unknown deubiquitinating enzyme; P, proteasome; Ub, ubiquitin.

Another possible explanation might be that the deubiquitination step may trigger the

efficient release of Pex5p from the export-machinery by cleavage of the complex-bound Ub-

moiety. Furthermore, this mechanism could prevent the monoUb-Pex5p to be recognized by

the proteasome system to ensure an efficient recycling of the receptor for new matrix protein

import cycles.

The finding that both ubiquitinating and deubiquitinating activities are required for the

transport of proteins from a membrane to the cytosol finds an examples in the ERAD

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89

pathway. The AAA-type ATPase p97 (Cdc48/VCP) is evolutionary related to the peroxisomal

AAA-proteins Pex1p and Pex6p (58). Most interestingly, among the growing number of

known co-factors and adaptor proteins that p97 utilizes to carry out its different functions are

also several deubiquitinating enzymes (98). The mammalian deubiquitinating enzymes YOD1

and Ataxin-3 are p97-associated proteins and function in the ERAD pathway (39, 45, 224).

Most of the published literature defines both DUBs as a positive regulator of the p97-

driven dislocation of the ERAD-substrates, most likely by editing the poly-Ub chains on the

substrates themselves in order to ensure the best fit to downstream Ub-receptor proteins.

Ubp15p acts in concert with the AAA-peroxins in the matrix protein import cycle of

the PTS1-receptor. Pex5p deubiquitination occurs as a highly specific event in yeast and

removal of ubiquitin of the PTS1-receptor Pex5p turns out to be a vital step in the receptor

cycle in its own right. Thus, removal of the ubiquitin seems to complete the receptor cycle of

Pex5p in order to make the receptor available for another round of matrix protein import.

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232. Williams, C., M. van den Berg, R. R. Sprenger, and B. Distel. 2007. A conserved cysteine is essential for Pex4p-dependent ubiquitination of the peroxisomal import receptor Pex5p. J Biol Chem 282:22534-22543.

233. Wilson, E. 1896. The Cell in Development and Inheritance. New York: Macmillan. 234. Yang, J., X. Han, and R. W. Gross. 2003. Identification of hepatic peroxisomal

phospholipase A(2) and characterization of arachidonic acid-containing choline glycerophospholipids in hepatic peroxisomes. FEBS Lett 546:247-250.

235. Yao, T., and R. E. Cohen. 2002. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419:403-407.

236. Ye, Y., H. H. Meyer, and T. A. Rapoport. 2003. Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains. J Cell Biol 162:71-84.

237. Zehmer, J. K., R. Bartz, P. Liu, and R. G. Anderson. 2008. Identification of a novel N-terminal hydrophobic sequence that targets proteins to lipid droplets. J Cell Sci 121:1852-1860.

238. Zhang, J. W., Y. Han, and P. B. Lazarow. 1993. Novel peroxisome clustering mutants and peroxisome biogenesis mutants of Saccharomyces cerevisiae. J Cell Biol 123:1133-1147.

239. Zweytick, D., K. Athenstaedt, and G. Daum. 2000. Intracellular lipid particles of eukaryotic cells. Biochim Biophys Acta 1469:101-120.

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CHAPTER 5. MISCELLANEOUS

5.1 Publications

1. Debelyy, M. O., H. W. Platta, D. Saffian, A. Hensel, S. Thoms, H. E. Meyer, B.

Warscheid, W. Girzalsky, and R. Erdmann. 2011. Ubp15p, an ubiquitin hydrolase

associated with the peroxisomal export machinery. Journal of Biological Chemistry. In

press

2. Debelyy, M. O., S. Thoms, M. Connerth, G. Daum, and R. Erdmann. 2011.

Involvement of the Saccharomyces cerevisiae Hydrolase Ldh1p in Lipid Homeostasis.

Eukaryot Cell 10:776-781.

3. Platta, H. W., M. O. Debelyy, F. El Magraoui, and R. Erdmann. 2008. The AAA

peroxins Pex1p and Pex6p function as dislocases for the ubiquitinated peroxisomal

import receptor Pex5p. Biochem Soc Trans 36:99-104.

4. Thoms, S., M. O. Debelyy, M. Connerth, G. Daum, and R. Erdmann. 2011. The

Putative Saccharomyces cerevisiae Hydrolase Ldh1p Is Localized to Lipid Droplets.

Eukaryot Cell 10:770-775.

5. Thoms, S., M. O. Debelyy, K. Nau, H. E. Meyer, and R. Erdmann. 2008. Lpx1p is

a peroxisomal lipase required for normal peroxisome morphology. Febs J 275:504-

514.

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5.2 Personal contribution to the papers 1. Debelyy, M. O., H. W. Platta, D. Saffian, A. Hensel, S. Thoms, H. E. Meyer, B.

Warscheid, W. Girzalsky, and R. Erdmann. 2011. Ubp15p, an ubiquitin hydrolase associated with the peroxisomal export machinery. Journal of Biological Chemistry. In press.

Planning: 70 % Experiments: 70 % Manuscript writing: 30 %

2. Debelyy, M. O., S. Thoms, M. Connerth, G. Daum, and R. Erdmann. 2011.

Involvement of the Saccharomyces cerevisiae Hydrolase Ldh1p in Lipid Homeostasis. Eukaryot Cell 10:776-781.

Planning: 80 % Experiments: 80 % Manuscript writing: 80 %

3. Platta, H. W., M. O. Debelyy, F. El Magraoui, and R. Erdmann. 2008. The AAA

peroxins Pex1p and Pex6p function as dislocases for the ubiquitinated peroxisomal import receptor Pex5p. Biochem Soc Trans 36:99-104.

Manuscript writing: 30 %

4. Thoms, S., M. O. Debelyy, M. Connerth, G. Daum, and R. Erdmann. 2011. The

Putative Saccharomyces cerevisiae Hydrolase Ldh1p Is Localized to Lipid Droplets. Eukaryot Cell 10:770-775.

Planning: 50 % Experiments: 50 % Manuscript writing: 20 %

5. Thoms, S., M. O. Debelyy, K. Nau, H. E. Meyer, and R. Erdmann. 2008. Lpx1p is

a peroxisomal lipase required for normal peroxisome morphology. Febs J 275:504-514.

Planning: 50 % Experiments: 30 % Manuscript writing: 20 %

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5.3 Conferences

1. Open European Peroxisome Meeting 2006. Leuven, Belgium, 18-19 September

2006. (Poster)

2. VAAM-Symposium: Biology of Yeast and Filamentous Fungi 2006. Bochum,

Germany, 12 October 2006. (Poster)

3. Seventh International Meeting on AAA Proteins 2007. Royal Agricultural College,

Cirencester, United Kingdom, 9—13 September 2007. (Poster)

4. The EMBO Meeting – Advancing the Life Sciences 2009. Amsterdam, Netherlands,

29 August – 1 September 2009. (Poster)

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5.4 Curriculum Vitae

PERSONAL DATA

Name Mykhaylo O. Debelyy

Date of Birth 11 March 1978

Place of Birth Dnepropetrovsk, Ukraine

Citizenship Ukrainian

Marital Status Married, one child

EDUCATION

July 2006 – July 2011 Ruhr-University Bochum

Institute of Physiological Chemistry

Department of System Biochemistry

Ph.D. student

Guidance by:

Prof. Dr. Ralf Erdmann

Dr. Wolfgang Girzalsky

September 1996 – August 2001 Dnepropetrovsk National University

Department of Biochemistry & Biophysics

Dipl.-Biol. &. Biochem.

Guidance by:

Prof. Dr. Natalia I. Shtemenko

Prof. Dr. Galina A. Ushakova

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CHAPTER 5. MISCELLANEOUS____________________________________________ 108

5.5 Acknowledgement

Prof. Dr. Ralf Erdmann

Prof. Dr. Wolf-H. Kunau

Prof. Dr. Günter Daum

PD Dr. Mathias Lübben

PD Dr. Wolfgang Schliebs

Dr. Wolfgang Girzalsky

Dr. Robert Rucktäschel

Dr. Harald W. Platta

Dr. Shirisha Nagotu

Dr. Christian Cizmowski

Dr. Pratima Bharti

Dr. David Managadze

Vishal Kalel

Alexander Neuhaus

Delia Saffian

Immanuel Grimm

Fouzi El Magraoui

Sohel Hasan

Sabrina Mindthoff

Sabrina Beck

Rezeda Mirgalieva

Imtiaz Ali

Christiane Sprenger

Sigrid Wuethrich

Monika Bürger

Ülrike Freimann

Elisabeth Becker

Frauke Albustin

Meike Möller

Britta Stickel

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5.6 Global scientific outlook for human race

Sociology:

1. Is it possible to create a classless society of equal possibilities for each human being,

where all people have an access to high quality food, safe environment,

accommodation, medical services, elementary and higher education and have the

possibility for individual development?

Biology:

1. What are life and death?

2. What is brain and what is the nature of consciousness?

3. Is it possible to improve the limited nature of human beings?

Physics:

1. What is the time?

1. What is the nature of gravity and momentum?

2. What is the nature of electric and magnetic fields?

3. What is space and what it is filled with?

Philosophy:

1. Is there a limit to the scientific exploration of the universe? If yes, then would it make

sense to improve the limited nature of human beings by the physical influence?