Recruitment of AP-1 Clathrin Adaptors to Liposomal Recruitment of AP-1 Clathrin Adaptors to...

Click here to load reader

  • date post

    24-Jun-2020
  • Category

    Documents

  • view

    0
  • download

    0

Embed Size (px)

Transcript of Recruitment of AP-1 Clathrin Adaptors to Liposomal Recruitment of AP-1 Clathrin Adaptors to...

  • Recruitment of AP-1 Clathrin Adaptors to

    Liposomal Membranes

    Inauguraldissertation

    Zur

    Erlangung der Würde eines Doktors der Philosophie

    vorgelegt der

    Philosophisch-Naturwissenschaftlichen Fakultät

    der Universität Basel

    von

    Daniel Markus Meyer

    aus Gebenstorf AG

    Basel, 2004

  • Genehmigt von der Philosophisch-

    Naturwissenschaftlichen Fakultät auf

    Antrag von

    Prof. Martin Spiess

    Prof. Jean Pieters

    Basel, den 10. Februar 2004

    Prof. Marcel Tanner

    Dekan

  • ACKNOWLEDGEMENTS Many people supported me with my research at the Biozentrum. I would

    especially like to thank the following:

    Martin Spiess, for his enormous scientific and personal support, guiding me

    through the recent years

    Pascal “Medline” Crottet, for the scientific advice and all the rest

    Jean Pieters and Hans-Peter Hauri, for participating on my thesis committee

    All the members of the Spiess-lab for the support and fun: Cristina Baschong,

    Nicole Beuret, Eric Dumermuth, Mike Friberg, Stefan Gander, Veit Goder,

    Marie Higy, Michael Jeske, Tina June, Szymon Kobialka, Vivienne Laird,

    Adriana Pagano, Anja Renold, Jonas Rutishauser, Michael Schneider, Hans

    Stettler, Gregor Suri

    Dan Cassel, for scientific advice

    Vreni Widmer, for helping with the Sf9 cells

    My parents, for their unconditional support

    My grandparents, for their help and encouragement

    My wife Sarah, for the invaluable comments she made on the style and

    language of this thesis, and for everything else

  • SUMMARY Protein and membrane traffic between organelles within the endocytic

    and exocytic pathway is mediated most prominently by coated vesicles.

    These vesicles are formed by the assembly of cytosolic coat proteins

    onto the donor membrane, which deform it into a bud so that vesicles

    can pinch off. Clathrin with its associated adaptors, COPI and COPII are

    the three major coats. Various in vitro studies allowed insight into the

    mechanism of coat formation. COPI and COPII vesicle budding from

    chemically defined liposomes has been reconstituted in vitro, using

    pure coat compounds. Further, it has been demonstrated that cargo is

    sorted into these vesicles. The mechanism of clathrin-coated vesicle

    formation appears to be more complicated.

    The AP-1 clathrin adaptor is involved in vesicle formation at the trans-

    Golgi network and endosomes. This work presents an in vitro assay

    where AP-1 is recruited to peptidoliposomes, presenting covalently

    linked peptides corresponding to sorting signals. In this system, AP-1

    recruitment depends on myristyolated ADP-ribosylation factor 1 (ARF1),

    GTP or GMP-PNP, tyrosine signals and a small amount of

    phosphoinositides, most prominently phosphatidyl inositol 4,5-

    bisphosphate. In such a minimal system AP-1 is recruited as a high-

    molecular weight complex indicating the formation of a precoat in the

    absence of clathrin. GTP hydrolysis, induced by ARF GTPase-activating

    protein 1 (ARFGAP1), disassembled this complex. Further, AP-1 is able

    to enhance the GAP activity of ARFGAP1 on myristoylated ARF1,

    suggesting a regulatory function of GTP hydrolysis in early steps of coat

    recruitment.

    This work provides insights into the mechanism of AP-1 clathrin coat

    formation which might also be used to investigate the recruitment of

    other coats.

  • Table of Contents

    ACKNOWLEDGEMENTS............................................................................... 3

    SUMMARY...................................................................................................... 4

    1 INTRODUCTION...................................................................................... 7

    1.1 Intracellular membrane transport ..................................................................................7

    1.1.1 The exocytic / biosynthetic pathway .........................................................................7

    1.1.2 The endocytic pathway............................................................................................10

    1.2 Coated vesicles...............................................................................................................11

    1.2.1 The clathrin coat ......................................................................................................12

    1.2.2 The COPI coat .........................................................................................................29

    1.2.3 The COPII coat ........................................................................................................30

    1.2.4 ARF GTPases in protein trafficking ........................................................................31

    1.2.5 Vesicle targeting ......................................................................................................34

    1.3 The role of inositol lipids as regulators of membrane traffic ..................................35

    1.4 Coat recruitment and vesicle formation in chemically defined systems...............37

    1.5 Aim of this thesis............................................................................................................39

    2 RESULTS .............................................................................................. 41

    2.1 ARF1·GTP, Tyrosine-based Signals, and Phosphatidylinositol 4,5-Bisphosphate Constitute a Minimal Machinery to Recruit the AP-1 Clathrin Adaptor to Membranes .....41

    2.1.1 Introduction ..............................................................................................................43

    2.1.2 Materials and methods............................................................................................46

    2.1.3 Results .....................................................................................................................50

    2.1.4 Discussion................................................................................................................64

    2.2 The AP-1 clathrin adaptor forms a high-molecular weight precoat which can be disassembled by ARFGAP1 .......................................................................................................70

    2.2.1 Introduction ..............................................................................................................72

    2.2.2 Materials and Methods............................................................................................75

    2.2.3 Results .....................................................................................................................78

    2.2.4 Discussion................................................................................................................81

    3 DISCUSSION......................................................................................... 89

    3.1 Coat formation – similar mechanisms, different players? .......................................89

    3.2 Outlook.............................................................................................................................92

    REFERENCES.............................................................................................. 93

  • Abbreviations AAK1 adaptor-associated kinase 1 AC-LL acidic cluster dileucine AP-1, -2, -3, -4 adaptor protein 1, 2, 3, 4 ARF ADP-ribosylation factor ARFGAP1 ADP-ribosylation factor GTPase-activating protein 1 BFA Brefeldin A CALM clathrin assembly lymphoid myeloid leukaemia protein CD cation-dependent CHC clathrin heavy chain CI cation-independent CK2 casein kinase II COPI / II coat protein I / II CSV constitutive secretory vesicle EEA1 early endosome antigen 1 EH Eps15 homology EM electron microscopy ENTH epsin N-terminal homology EPS15 Epidermal growth factor protein substrate 15 epsin1 EPS15 interacting protein ER endoplasmatic reticulum ERGIC ER-Golgi intermediate compartment EST expressed-sequence tags GAK cycling G-associated kinase GAP GTPase-activating protein GAT GGA and Tom GDI GDP dissociation inhibitor GEF guanine nucleotide exchange factor GGA golgi-localized, γ ear-containing, ADP ribosylation factor binding protein GMP-PNP guanylyl imidodiphosphate GTPγS guanosine 5'-O-(3-thiotriphosphate) ISG immature secretory granule Lamp1 lysosome-associated membrane protein-1 LAP like AP-180 MPR mannose 6-phosphate receptor MSG mature secretory granule NSF N-ethylmaleimide-sensitive-factor PA phosphatidic acid PC phosphatidylcholine PE phosphatidylethanolamine PI phosphatidylinositol PIP phosphoinositide PM plasma membrane PP2A protein phosphatase 2A PS phosphatidylserine PTEN phosphatase and tensin homology RER rough ER SER smooth ER SNAP soluble NSF attachment protein SNARE Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor TfR transferrin receptor TGN trans-Golgi network VHS Vps, Hrs, and STAM VSV-G vesicular stomatitis virus G protein

  • Introduction 7

    1 INTRODUCTION

    1.1 Intracellular membrane transport

    A cell consists of different organelles which have to fulfill various functions.

    They are delimited by membranes and contain specific proteins. All proteins

    are synthesized in the cytosol. Except for mitochondrias and cloroplasts, all

    organelles receive their prote