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1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu * , Stephen M.T. Hoke * , Julie Genereaux * , Carol Hannam * 1 , Katherine MacKenzie * , Olivier Jobin-Robitaille + , Julie Guzzo § , Jacques Côté + , Brenda Andrews § , David B. Haniford * and Christopher J. Brandl* * Department of Biochemistry Schulich School of Medicine and Dentistry, University of Western Ontario, London, Canada N6A5C1, + Laval University Cancer Research Center Hotel-Dieu de Quebec (CHUQ), Quebec City, Canada G1R-2J6 and § Department of Medical Genetics and Microbiology and the Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular & Biomolecular Research, University of Toronto, Toronto, Canada M5S 3E1 The first two authors contributed equally to this work. 1 Present address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 Microarray data from this article has been submitted to the GEO database at NCBI under accession number GSE6847 Genetics: Published Articles Ahead of Print, published on July 29, 2007 as 10.1534/genetics.107.074476

Transcript of Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function...

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Structure Function Analysis of the Phosphatidylinositol-3-kinase

Domain of Yeast Tra1

A. Irina Mutiu*, Stephen M.T. Hoke*, Julie Genereaux*, Carol Hannam*1,

Katherine MacKenzie*, Olivier Jobin-Robitaille+, Julie Guzzo§, Jacques Côté+,

Brenda Andrews§, David B. Haniford* and Christopher J. Brandl*

* Department of Biochemistry

Schulich School of Medicine and Dentistry,

University of Western Ontario, London, Canada N6A5C1,

+Laval University Cancer Research Center

Hotel-Dieu de Quebec (CHUQ), Quebec City, Canada G1R-2J6

and

§Department of Medical Genetics and Microbiology and the Banting and Best Department of

Medical Research, Terrence Donnelly Centre for Cellular & Biomolecular Research,

University of Toronto, Toronto, Canada M5S 3E1

The first two authors contributed equally to this work.

1 Present address: Department of Molecular and Cellular Biology, University of Guelph, Guelph,

Ontario N1G 2W1

Microarray data from this article has been submitted to the GEO database at NCBI under accession

number GSE6847

Genetics: Published Articles Ahead of Print, published on July 29, 2007 as 10.1534/genetics.107.074476

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Running title: Structure/function of S. cerevisiae Tra1

Keywords: Tra1, gene expression, PI3-kinase domain, SAGA complex, NuA4 complex

Running title: Structure/function of Tra1

Corresponding author: Dr. Christopher J. Brandl

Department of Biochemistry,

University of Western Ontario,

London, CANADA N6A5C1

(519) 661-2111 ext 86857

Fax: (519) 661-3175

[email protected]

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Tra1 is an essential component of the Saccharomyces cerevisiae SAGA and NuA4 complexes. Using

targeted mutagenesis, we identified residues within its C-terminal phosphatidylinositol-3-kinase

(PI3K) domain that are required for function. The phenotypes of tra1-P3408A, S3463A and SRR3413-

3415AAA included temperature sensitivity and reduced growth in media containing 6% ethanol,

Calcofluor white or depleted of phosphate. These alleles resulted in ≥2-fold change in expression of

~7% of yeast genes in rich media and reduced activation of PHO5 and ADH2 promoters. Tra1-SRR3413

associated with components of both the NuA4 and SAGA complexes and with the Gal4 transcriptional

activation domain similar to wild-type protein. Tra1-SRR3413 was recruited to the PHO5 promoter in

vivo but gave rise to decreased relative amounts of acetylated histone H3 and histone H4 at SAGA and

NuA4 regulated promoters. Distinct from other components of these complexes, tra1-SRR3413 resulted

in generation dependent telomere shortening and synthetic slow growth in combination with deletions

of a number of genes with roles in membrane related processes. While the tra1 alleles have some

phenotypic similarities with deletions of SAGA and NuA4 components, their distinct nature may arise

from the simultaneous alteration of SAGA and NuA4 functions.

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INTRODUCTION

In eukaryotic cells the post-translational modification of nucleosomes by multisubunit complexes is a

key aspect of transcriptional regulation (reviewed in Berger, 2002). Histone modifications including

acetylation, methylation, ubiquitylation and phosphorylation can directly alter chromatin structure or

act as a recruitment signal for additional factors (Strahl and Allis, 2000). As well as regulating

transcriptional initiation, nucleosome modifications affect transcriptional elongation and other nuclear

processes such as DNA replication, DNA repair and RNA export (Iizuka and Smith, 2003).

The Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex modifies chromatin

and provides an interface between DNA binding transcriptional regulators and the basal transcriptional

machinery (reviewed in Green, 2005). The structural core of SAGA is composed of a subset of the

TBP-associated factors (TAFs) (Grant et al., 1998a; Wu et al., 2004), with Spt7, Ada1 and Spt20 also

being required for the integrity of the complex (Horiuchi et al., 1997; Roberts and Winston, 1997;

Sterner et al., 1999). The histone acetyltransferase Gcn5/Ada4 activates and represses transcription by

modifying histones H3 and H2B (Brownell et al., 1996; Grant et al., 1997; Kuo et al., 1998; Wang et

al., 1998, Ricci et al., 2002). In turn, the Ada proteins, Ada2 and Ngg1/Ada3 regulate the activity and

substrate preference of Gcn5 (Balasubramanian et al., 2001). Further regulation is provided by the

interaction of Spt3 and Spt8 with the TATA-binding protein (Eisenmann et al., 1992, 1994; Dudley et

al., 1999). Recruitment of SAGA to promoters is mediated by Tra1, an essential 437 kDa protein

(Saleh et al., 1998; Grant et al., 1998b) that interacts directly with transcriptional activators (Brown et

al., 2001; Bhaumik et al., 2004; Fishburn et al., 2005; Reeves and Hahn, 2005). The mammalian

ortholog of Tra1, TRRAP is required for transcriptional regulation by myc, p53, E2F and E1A (Ard et

al., 2002; Bouchard et al., 2001, Deleu et al., 2001; Kulesza et al., 2002; McMahon et al., 1998). Its

deletion results in defects in cell cycle progression and early embryonic lethality (Herceg et al., 2001).

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Tra1 is also a component of the multisubunit NuA4 complex (Allard et al., 1999) that preferentially

acetylates histones H4 and H2A, the catalytic subunit being the essential protein Esa1 (Clarke et al.,

1999; Smith et al., 1998). NuA4 associates with acidic activation domains, probably through Tra1

mediated interactions, and activates transcription in an acetylation-dependent manner (Vignali et al.,

2000; Nourani et al., 2004). Acetylation by NuA4 is also critical for nonhomologous end-joining of

DNA double stand breaks and for replication coupled repair (Bird et al., 2002; Choy and Kron, 2002;

Downs et al., 2004). The role of NuA4 in repair was highlighted by the finding that it acetylates the

histone variant Htz1 which is intimately involved with these processes (Keogh et al., 2006).

Aside from its length, a distinguishing feature of Tra1 is its C-terminal domain of approximately 300

amino acids that is related to the phosphatidylinositol-3-kinase (PI3K) domain found in several key

cellular regulators including ATM, DNA-PK, and FRAP (Keith and Schreiber, 1999). The group also

shares less well-defined sequences flanking the PI3K domain called the FAT (FRAP-ATM-TRRAP)

and C-FAT domains (Bosotti et al., 2000). Unlike other members of the family, Tra1 and TRRAP lack

the signature motifs of kinases and kinase activity (Bosotti et al., 2000). The exact role of the PI3K

domain is thus unclear; though in human cells, the PI3K domain of TRRAP is required for cellular

transformation by myc and E1A (Park et al., 2001).

We have used a mutagenesis approach to determine the structure/function relationships of Tra1,

focusing in particular on the PI3K domain. We have identified eight mutations in the PI3K domain that

result in cellular inviability and three that result in temperature sensitive growth and reduced growth on

media containing 6% ethanol. Characterization of these temperature sensitive alleles at the permissive

temperature confirms a role for the PI3K domain in transcriptional regulation. These mutations confer

altered expression of ~7% of the yeast genome and were distinct from those affecting individual

components of either SAGA or NuA4 complexes.

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MATERIALS AND METHODS

Yeast strains and growth. Yeast strains are listed in Table 1. TRA1 alleles contained on TRP1

centromeric plasmids were transformed into CY1021 and the wild-type copy displaced by plasmid

shuffling (Sikorski and Boeke, 1991). CY1896 is a derivative of Y5565 that has been gene replaced

with tra1-SRR3413 and selected for through placement of Tn10LUK at the downstream BstB1 site.

CY2437 and CY2438 are derivatives of KY320 and similarly contain integrations of wild-type TRA1

and tra1-SRR3413, respectively, in a background lacking BamHI and SalI restriction sites within TRA1.

Growth comparisons were performed on selective plates after three to five days at 30o unless stated

otherwise. Assays were performed in duplicate on independently constructed strains. Scoring for PI3K

domain mutations was done in relation to CY1524, which contains TRA1SB, the background allele used

to construct mutations within the PI3K domain. TRA1SB is Flag-tagged and contains a BamHI site that

converts N3580A and a SalI site of nucleotides A9714G and T9717G. Scoring for the viability of non-PI3K

domain mutations was relative to CY1020 (Saleh et al., 1998).

Construction of DNA molecules. lacZ reporter constructs were cloned as his3-lacZ fusions into the

LEU2 centromeric plasmid YCp87 (Brandl et al., 1993). ADH2-lacZ contains promoter sequences (-

440 to -150; Saleh et al., 1998). PHO5 (-452 to +47), INO1 (-517 to +40) and HIS4 (-545 to + 29),

were engineered by PCR as BamHI-HindIII fragments and cloned into YCp87 (see supplemental Table

1 for a listing of oligonucleotides).

Construction of TRA1 alleles: Mutations of DSP171AAA, EFS184AAA, KEL345AAA, VLL837AAA,

VRL867AAA, KKK1686AAA, GLW2677AAA, GYH2939AAA, TLP2972AAA, HFQ3168AAA,

SRR3413AAA, and PFR3621AAA were engineered into myc-TRA1-Ycplac111 (Saleh et al., 1998). Each

allele was synthesized in two PCR reactions. Reactions contained a listed oligonucleotide and the

appropriate outside flanking primer with a unique cloning site (oligonucleotides 842, 2542-3, 2337-1,

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2323-2, 2323-1, 2542-4, 2337-2 and 2346). Mutagenized codons were replaced with a NotI site such

that 5’ and 3’ halves could be ligated sequentially into pUC vectors. Fragments were moved into myc-

TRA1-Ycplac111 using the outside restriction sites. Mutations within the coding region of the PI3K

domain (including additional alleles of EER3416AAA, and PFR3621AAA) were constructed in TRA1SB.

Flag epitopes were introduced at an N-terminal NotI site. Mutagenized alleles were constructed by

PCR and ligated as SalI-BamHI (for alterations N-terminal to N3580) or BamHI-SacI (for alterations C-

terminal to N3580). Flanking primers used in the PCR reactions were 4293-1 and 4225-3 or 4249-1 and

2346/4479-1 depending on the placement relative to N3580. YNG2 and GCN5 were similarly cloned into

this molecule.

TAP-tagged molecules were cloned into YCpDed-TAP, a derivative of the URA3-containing

centromeric plasmid YCplac33 (Gietz and Sugino, 1988) that contains a DED1 promoter driving

expression of a TAP epitope. The DED1 promoter was synthesized by PCR using oligonucleotides

4149-1 and 4149-2 and cloned as a PstI-BamHI fragment into YCplac33. The TAP epitope was

subsequently cloned as a BamHI to SacI fragment into this molecule after PCR using oligonucleotides

4149-4 and 4168-1 and pFA6a as the template (kindly provided by Kathy Gould). The coding

sequence for the Flag-epitope was cloned into this molecule at the engineered NotI site to give

YCpDed-TAP-Flag. TRA1 was introduced into this vector as a NotI-Sac1 fragment.

TAP purification. Tandem affinity purification was carried out as described by Rigaut et al. (1999).

One liter of cells grown in YPD to an A600 ~ 1.5 were ground in liquid nitrogen (Saleh et al., 1997).

Further steps were at 4°. Cell extract suspended in 5 ml of IPP150 with protease inhibitors (1 mM

phenylmethylsulfonylflouride, 0.1 mM benzamidine hydrochloride, 2 μg/ml pepstatin A, 2 μg/ml

leupeptin and 0.1 mg/ml trypsin inhibitor) was cleared by centrifugation at 147,000 g and incubated

with 600 μl of IgG-Agarose (Sigma-Aldrich Canada Ltd) for 2 h. After washing with 30 ml of IPP150

and 10 ml of TEV cleavage buffer, beads were suspended in 1 ml of cleavage buffer and incubated

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with 100 U TEV protease for 2 h. 3 μl of 1 M CaCl2 was added to the eluent, then diluted with 3 ml of

calmodulin binding buffer. The protein was incubated with 600 μl of Calmodulin-Sepharose

(Stratagene Inc) for 1 h, washed with 30 ml of calmodulin binding buffer and protein eluted.

β-galactosidase assays. For HIS4-lacZ cells grown in minimal media to an A600 of ~1.5 were pelleted,

washed in LacZ buffer and concentrated ~5-fold. β-galactosidase was determined using ο-nitrophenol-

β-D-galactosidase as substrate, standardizing to cell density (Ausubel et al., 1998). Analysis of PHO5-

lacZ, INO1-lacZ and ADH2-lacZ were performed similarly except that overnight cultures were washed

3-times in water then grown to an A600 ~1.5 in YPD depleted of phosphate (Han et al., 1988), depleted

of inositol or minimal media containing 3% ethanol (v/v) and 0.1% glucose.

Gal4 affinity chromatography. Amino acid residues 764 to 882 of Gal4 were expressed as a GST

fusion in the vector pGEX1. One liter of E. coli cells was induced with 0.6 mM isopropyl β-D-

thiogalactopyranoside for 6 hours at 37o. Harvested cells were resuspended in lysis buffer (40 mM

TRIS [pH 8.5], 20 mM NaCl, 10% glycerol, 0.08% NP-40, 0.2 mM EDTA [pH 8.0], 1 mM DTT) with

protease inhibitors, treated with 1 mg/ml lysozyme on ice for 1 h and broken by sonication. Cleared

extract was incubated on ice with one ml Glutathione Sepharose 4B (GE Healthcare), washed three

times with 10 ml of 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and once with 10

ml of lysis buffer. TAP-Tra1 purified on IgG-Agarose from 1.0 liter cultures of CY1962 and CY1963

containing HA-tagged Ada2 (Saleh et al., 1997) was applied to Gal4-containing Glutathione Sepharose

in 3 ml of lysis buffer and rotated at 4o for 60 minutes, washed with lysis buffer and bound protein

eluted with 10 mM reduced glutathione. 30 μl of the eluent was separated on a 10% SDS-PAGE gel,

transferred to PVDF membrane and blotted with anti-HA antibody.

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Genetic analyses. Identification of an intragenic suppressor of tra1-SRR3413. Approximately 2x106

cells of yeast strain CY1531 (tra1-SRR3413) were plated onto YPD containing 6% ethanol. Two

colonies were isolated after 5 days of growth and Tra1 expressing plasmids recovered (Hoffman and

Winston, 1987). The SalI-SacI fragment was re-cloned into TRA1SB and found sufficient for

suppression. Systematic genetic array analysis on yeast strain CY1896 (tra1-SRR3413) was performed

as described by Tong and Boone (2006).

RNA purification and microarray analysis. Yeast cells were grown in YP media containing 2%

glucose to an A600nm=2.0 at 30o. RNA was purified from 108 cells after glass bead disruption as

described previously (Mutiu and Brandl, 2005; Mutiu et al., 2007). Microarray analyses were

performed using the Agilent Yeast Oligo Array Kit at MOgene LC (St. Louis, MO). Comparisons were

made with wild-type strain CY1524 and performed in duplicate with dye reversal. Array data for the

confirmed set of protein coding genes was log transformed and averages obtained. Genes displaying

an expression change greater than 1.5-fold, where the direction of the response differed between

replicates were discarded. Profiles were normalized by subtracting the mean response of all genes

from the response of each gene, then dividing by the standard deviation of all responses within a

profile. An initial cluster analysis using a subset of the compendium data (Hughes et al., 2000) that

included deletion strains with gene ontology terms indicating gene expression, identified microarray

profiles of strains with deletions of cka2, ckb2, gcn4, hat2, isw1, mbp1, rpd3, rtg1, sap30, sin3, ste12

and yap3 as being most similar to tra1-SRR3413. Agglomerative hierarchical clustering based on the

average linkage of uncentered correlations was performed on the profiles from these strains and strains

with deletions of SAGA (Ingvarsdottir et al, 2005) and NuA4 (Krogan et al., 2004) components using

CLUSTER 3.0 software (Eisen et al., 1998). Genes not appearing in at least two of the microarray

profiles were excluded. The data was visualized using MAPLETREE

(http://rana.lbl.gov/EisenSoftware.htm). Gene ontology terms associated with gene clusters were

determined using FunSpec (Robinson et al., 2002).

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Chromatin Immunoprecipitation Assays. Assays were performed essentially as described by Kuo

and Allis, (1999). For assays comparing acetylated histone H3, 50 ml of culture was grown to an

A600nm ~ 2.0. To equalize amounts of input DNA, cell extract was reverse cross-linked and PCR

reactions performed on serial dilutions. For immunoprecipitations, DNA equal to that contained in 50

μg (protein) of cell extract from the wild-type sample, was incubated with Protein A Sepharose 4 Fast

Flow (Pharmacia Inc), transferred to clean tubes, and rotated overnight with one μl of antibody (anti-

H3: Abcam Inc., ab1791; anti-AcH3, Lys9: Upstate Biotechnology, 07-352). Precipitate was removed

by centrifugation and the supernatant incubated with Protein A beads. After washing and reversal of

crosslinks products were analyzed by PCR (Ricci et al., 2002) using primers for the PHO5 (-550 to -

300), ADE1 (-505 to 51), PHO84 (-199 to 74), PGK1 (-249 to 28) promoters or PHO5 gene (1035 to

1322). PCR conditions were 5 minutes at 95o, followed by 25 cycles of 30 seconds at 95o, 52o, and 72o,

then 5 minutes at 72o. Assays of acetylated histone H4 were performed with 250 ml cultures grown to

A600nm ~ 2.0 in YPD, 0.5 mg of chromatin (in a total volume 0.5 ml), and Protein A/G plus 2 μl of anti-

AcH4, Lys8 (Abcam Inc, ab1760). For immunoprecipitations using TAP-Flag-Tra1, chromatin was

prepared in RIPA buffer with washing and elution as described by Alekseyenko et al. (2006).

Western blotting. Yeast extract prepared by glass bead disruption (Brandl et al., 1993) or grinding in

liquid nitrogen (Saleh et al., 1997) was separated by SDS-PAGE, transferred to PVDF membranes

(Roche Applied Science) using a wet transfer system in 48mM Tris, 40mM glycine, 0.0375% SDS and

20% methanol for 1 h at 100V. Anti-Flag antibody (M2, Sigma-Aldrich Canada Ltd) and Anti-HA

antibody (ascites fluid from the 12CA5 cell line) were used at a ratio of 1:4000. Secondary antibody

(Anti-Mouse IgG HRP, Promega) used at a ratio of 1:10000 was detected using a 20% solution of

Immobilon Western (Millipore Corp). Densitometric scanning of films was performed using

AlphaImager 3400 software (Alpha Innotech, Inc).

Telomere length assays - Plasmid linearization assay. Strains were transformed with pVL106

(Lundblad and Szostak, 1989). Leu+ Ura+ colonies were grown to saturation in media lacking uracil

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then YPD. 10 μl was spotted onto leucine-depleted plates containing 5-FOA. Values reported are the

percent of 5-FOA resistant, Leu+ colonies in the mutant strain as compared to wild-type, normalized to

the number of cells plated. Terminal restriction fragment determination. TRA1 alleles were

transformed into CY1021 and plasmid shuffling performed (generation zero). 10 ml saturated cultures

were grown from these colonies at 30˚ or 34˚ and DNA prepared (Ausubel et al., 1998). Each culture

was also diluted 1:1000 into YPD and grown again to saturation, then repeated. Genomic DNA was

digested with XhoI and Southern blotting performed (Teng and Zakian, 1999).

Fractionation of Tra1 containing complexes on HiTrap Q Sepharose Fast Flow. 18 mg of whole

cell extract containing HA-Ada2p and Tra1SB or Tra1-SRR3413 was prepared in 40 mM Tris-HCl (pH

7.7) buffer containing 20 mM NaCl, 10% glycerol, 0.08% Nonidet P-40, 0.2 mM EDTA, and 0.1 mM

dithiothreitol. Extracts were treated with protamine sulphate (0.2%), rotated at 4o for 30 minutes,

cleared by centrifugation at 147,000 g and applied to a HiTrap Q Sepharose Fast Flow column (1.0 ml;

flow rate of 1.0 ml/min ; Amersham Pharmacia Biotech). After washing with 4 ml of extraction buffer,

protein was eluted with a 16-ml gradient of 0-1.0 M NaCl. 20-µl aliquots of 200-µl fractions was

separated by SDS-PAGE and analyzed by Western blotting with anti-HA antibody. 25 mg of whole

cell extract containing TAP-Flag-Yng2 and Tra1SB or Tra1-SRR3413 was blotted using anti-Flag

antibody.

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RESULTS

The size of Tra1 presents a significant challenge for analyses of structure and function using molecular

genetic approaches. Because of the difficulty in identifying and characterizing random mutations, we

undertook a targeted mutagenesis approach, initially constructing 12 triple alanine scanning mutations

scattered in regions that might be relevant for function (Table 2). These alleles were expressed on a

TRP1-containing centromeric plasmid and transformed into yeast strain CY1021 that contains a

disruption of the genomic copy of TRA1 complemented by wild-type TRA1 expressed on a URA3-

containing centromeric plasmid (Saleh et al., 1998). Each allele was analyzed for growth properties

after shuffling out wild-type TRA1 on media containing 5-fluoroorotic acid (5-FOA). Phenotypes

examined included: mating efficiency, temperature sensitivity (37o), growth on media depleted for

inositol, amino acids and phosphate, fermentation of galactose and raffinose, sensitivity to

hydroxyurea, methylmethanesulfonate (MMS), ultraviolet irradiation, γ-irradiation, high osmolarity,

cycloheximide and tunicamycin. Two of the alleles tested, DSP171AAA and EFS184AAA altered

potential phosphorylation sites. (The number indicates the first residue of the three. We will not

include the AAA for each allele.) VLL837 altered a putative LXXLL motif. Many of the other altered

residues are conserved in yeast and/or human Tra1 homologs. Only two of the 12 alleles, both with

mutations within the PI3K domain, had discernible phenotypes. tra1-PFR3621 would not support

growth on media containing 5-FOA and therefore is a nonfunctional allele. tra1-EER3416 showed

reduced growth on all media tested, as well as slight temperature sensitivity.

Targeted mutation of the PI3K-domain. Based on the above results, we directed additional

mutations to the PI3K domain. As we were concerned that mutations might simply alter the fold of the

PI3K domain, we focused on mutagenizing codons for amino acid residues conserved in the

Tra1/TRRAP family of molecules but not in the broader PI3K domain family. In the absence of a

structure for the Tra1 PI3K domain, the structure of PI3K-γ (Walker et al., 1999; see supplemental

Figure 1) allows an initial prediction of the positions of these residues. The PI3K domain consists of

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N-terminal and C-terminal lobes connected by a loop between β7 and β8. For PI3K-γ, ATP

interactions are found with the β3-β4 loop (P-loop), the end of β5 and the β7-β8 loop. The region

between α6 and β9 corresponds to the catalytic loop. The C-terminal lobe also contains an activation

loop that is required for substrate binding of the PI3K molecules (Bondeva et al., 1998) and likely

binds phosphatidylinositols in PI3K-γ.

The phenotypes of strains containing the mutagenized tra1 alleles are shown in Table 3. Of the

nineteen alleles with targeted mutations in the PI3K domain, eight did not support viability. Two of the

eight, R3456A and R3567A, were single amino acid substitutions; the others, WRR3317, DIE3351, RFL3374,

FRK3544, PFR3621 and RDE3668 were triple alanine scanning mutations. Five of the eight mutations were

within predicted loop regions (WRR3317, DIE3351, RFL3374, R3567 and PFR3621). F3443, which aligns with

the α3-β6 loop can be considered in this group since strains containing tra1- F3443A were marginally

viable in rich media but inviable under conditions of stress. If their positioning within loops is correct,

it is not likely that these residues are important for structure but instead may be involved in protein-

protein interactions or have regulatory roles. In agreement with this, R3567 of Tra1 aligns with R947, an

essential residue within the catalytic loop of PI3K-γ (Dhand et al., 1994; Stack and Emr, 1994).

Likewise it is required for the activity of Tra1, though for Tra1/TRRAP the size of the residue may be

the critical factor since human TRRAP contains a leucine at this position. Similarly, PFR3621 aligns

with the activation loop and despite the lack of kinase activity, these residues are required for the

activity of Tra1. (We note that the phenotype of PFR3621 may in part be due to reduced expression

levels, not shown). Three mutations giving rise to inviability were not in predicted loops. R3456 is in a

comparable position to G877 of PI3K-γ, which is found within β7 and conserved in many of the lipid

kinases (Walker et al., 1999). FRK3544 and RDE3668 align with α6 and α9, respectively, and are

uniquely conserved in the Tra1/TRRAP molecules.

Eleven alleles supported viability in rich media. Their growth characteristics under a variety of

conditions that reflect ada, spt or NuA4 phenotypes are shown in Table 3. Other conditions and assays

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tested but without substantial effects were growth on MMS (also see below), tert-butylhydroperoxide,

galactose, raffinose, 6-azauracil, 1.0 M NaCl, and tunicamycin. In addition, none of these alleles

significantly changed the rate of loss of a URA3-containing centromeric plasmid, after growth on

nonselective conditions or resulted in a significant defect in mating (not shown). K3571A and N3580A

had little effect on growth under any of the conditions tested. Four of the alleles, P3446A, DKL3491,

P3662A and N3617A, showed a very weak classic ada phenotype (Berger et al., 1992), being partially

resistant to overexpression of VP16. (Note that a deletion of gcn5 would score as ++++ on this scale.)

Some of these alleles were also slightly sensitive to 6% ethanol, characteristic of an ada2 disruption

(Takahashi et al., 2001).

Three alleles, tra1-SRR3413, tra1-P3408A and tra1-S3463A, resulted in a common and distinct phenotype.

(EER3416 had a similar phenotype but to a lesser extent.) Strains containing these alleles were

temperature sensitive for growth at 37o, had dramatically reduced growth in media containing elevated

concentrations of ethanol and grew poorly in media lacking phosphate. Plates showing the growth of

strains containing these alleles under some of the conditions are shown in Figure 1a. The two alleles

with the greatest temperature sensitivity (tra1-SRR3413 and tra1-P3408A) resulted in somewhat reduced

growth on media depleted of inositol or containing benomyl, a microtubule-destabilizing agent. These

latter two phenotypes are shared with deletions of some Spt components of SAGA (Roberts and

Winston, 1997) and for components of the NuA4 complex (LeMasson et al., 2003; Bittner et al., 2004;

Krogan et al., 2004), respectively. The tra1 alleles displayed similar sensitivity to ethanol as deletion

of ada2 (not shown) and like ada2 had reduced growth on Calcofluor white (Figure 1a) suggesting that

the effect results from changes in cell wall properties or the ability of the strains to respond to changes

in cell wall integrity (Takahashi et al., 2001). We examined the growth properties of strains containing

these alleles in more detail (Figure 1b). At 34o, the tra1-SRR3413, tra1-P3408A and tra1-S3463A alleles

resulted in a reduced growth rate in rich media relative to the TRA1SB allele. Upon heat shock the

mutant strains showed an immediate growth arrest but did recover; however, four hours after the shift

to 37o growth stopped. To ensure that the effects of these mutations was not due to expression on a

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plasmid or the background N3580A mutation from the BamHI site within TRA1SB, tra1-SRR3413 lacking

the N3580A mutation was integrated into the genome. As shown in Figure 1c, the integrated allele

showed similar slow growth at elevated temperature and in the presence of Calcofluor white as did the

plasmid version.

Since the NuA4 complex has a role in DNA double strand break repair (Bird et al., 2002), we

examined strains containing Tra1-SRR3413, P3408A and S3463A more closely for sensitivity to MMS

comparing to a deletion of the NuA4 component Vid21/Eaf1. tra1-SRR3413 showed a slight sensitivity

to MMS but dramatically less so than deletion of vid21/eaf1 (not shown). In support of only minor

changes in double strand break repair, none of the three alleles resulted in enhanced sensitivity to γ-

irradiation at 30o (not shown).

To evaluate whether the growth phenotypes might result from reduced expression of the altered forms

of Tra1, the levels of Flag3x-tagged Tra1-P3408A, Tra1-S3463A and Tra1-SRR3413 were examined by

Western blotting of crude yeast extracts (Figure 2a). Each of these forms was expressed at a level

approximately equivalent to Tra1SB, though we observed slightly elevated levels of Tra1-S3463A. We

focused additional studies on tra1-SRR3413 since it displayed the most dramatic phenotypes. To

determine if Tra1-SRR3413 associated with components of NuA4 and SAGA complexes, a TAP-tagged

version of the molecule was purified by tandem affinity purification and the presence of Spt7 and Flag-

tagged Yng2 determined by Western blotting (Figure 2b). SRR3413 interacted with Flag-Yng2 and with

two forms of Spt7 suggesting its presence in NuA4, SAGA and SAGA-like (SLIK) complexes. To

further examine if Tra1-SRR3413 is found within SAGA and NuA4 complexes, crude extracts

containing Tra1-SRR3413 were fractionated on a HiTrap Q Sepharose column and the elution profile of

HA-Ada2 and Flag-Yng2 compared to that found in a strain containing Tra1SB (Figure 2c and 2d). The

elution profiles for Tra1-SRR3413 varied little with Tra1SB suggesting that it associates in the

appropriate high molecular mass complexes.

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Gene expression in the conditional tra1 strains. To determine if the tra1-SRR3413, tra1-P3408A and

tra1-S3463A alleles resulted in changes in transcriptional regulation, we examined expression of PHO5,

HIS4, INO1 and ADH2 using lacZ-reporter fusions (Figure 3a). Assays were performed at 30o under

conditions that would induce each specific promoter. Strains deleted for gcn5, spt7 and yng2 were

examined for comparison. The tra1 mutations resulted in expression of PHO5 at a level from 3-17%

that seen in the wild-type (TRA1SB), the extent paralleling the severity of the growth defects.

Expression of HIS4 and INO1 were relatively unchanged in the tra1 backgrounds. ADH2 was

decreased on average to 60% wild-type levels for the three tra1 mutants with the extent again

paralleling the growth defect. The pattern of expression seen in the tra1 strains most closely resembled

that seen upon disruption of gcn5; expression of HIS4 is reduced upon disruption of spt7 and yng2,

unlike the tra1 strains and INO1 is reduced upon disruption of spt7. Elevated temperature did result in

enhanced effects of tra1-SRR3413 as expression of INO1-lacZ was reduced to ~ 50% that seen in

TRA1SB when cells were grown at 35o (Figure 3b).

A more detailed analysis of gene expression in the tra1-SRR3413, tra1-P3408A and tra1-S3463A strains

was obtained by performing microarray analysis. The Agilent Yeast Oligo array representing all

independent yeast ORFs was probed with cRNA produced from cells grown logarithmically at 30o in

YP media containing 2% glucose (note that this differs from the data shown in Figure 4 which were

performed in inducing conditions). Two-dye experiments were performed comparing expression in the

mutant strains to that found in a strain containing TRA1SB. For each mutant, the experiment was

performed in duplicate with dye-reversal. The tra1-SRR3413 allele resulted in a change in expression of

two-fold or greater of ~7% of the protein encoding genes with ~70% having decreased expression. The

25 genes showing the greatest increase and decrease in expression for each mutant is shown in Table 4

(the full data set has been submitted to the GEO database at NCBI under accession number GSE6847).

The genes affected in the three strains were highly similar, consistent with the mutant alleles having a

common defect. Gene ontology of those most affected indicate a group of upregulated genes are

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involved with ion transport and cell cycle regulation, while a number of the down-regulated genes have

roles in nucleotide and amino acid metabolism.

Hierarchical cluster analysis was performed with the array data for the tra1-SRR3413 strain, strains with

deletions of SAGA and NuA4 components and a subset of the compendium data set (Hughes et al.,

2000) including those genes linked to transcription. tra1-SRR3413 clustered in a leaf with a deletion of

ada2, though the depth of the branch is indicative of only weak similarity (Figure 4). Profiles of strains

with deletions of the NuA4 components (eaf5, yaf9, eaf1/vid21 and yng2) showed less similarity with

tra1-SRR3413, than ada2, spt3 or spt8. Overall, the relatively low level of similarity indicates that the

transcription pattern of tra1-SRR3413 results from the cumulative effects of this allele on both SAGA

and NuA4 complexes, and/or there being one or more independent roles for Tra1.

Brown et al. (2000) previously identified a tra1 allele that resulted in decreased binding to the Gal4

transcriptional activation domain. To address whether the transcriptional changes observed for tra1-

SRR3413 were due to a defect in its interaction with transcriptional activation domains, we compared the

ability of Tra1-SRR3413 and Tra1SB to associate with the Gal4 activation domain (Gal4AD). Tandem

affinity purified Tra1SB and Tra1-SRR3413, prepared from strains containing HA-tagged Ada2 were

chromatographed on GST-Gal4AD columns. Association of SAGA with Gal4 was detected by Western

blotting for HA-Ada2 (Figure 5a). SAGA containing Tra1-SRR3413 bound Gal4AD to the same extent as

SAGA-containing Tra1SB (compare lanes 4 and 6), suggesting that the mutation does not decrease

association with transcriptional activators. This is consistent with the region required for activator

interaction being contained within amino acid residues 2233 to 2836 (Brown et al., 2000). Recruitment

of Tra1-SRR3413 to the PHO5 promoter in vivo was examined by chromatin immunoprecipitation using

the TAP-tagged derivatives of Tra1. As shown in Figure 5b, Tra1-SRR3413 was recruited as efficiently

to the PHO5 promoter as Tra1SB (compare lanes 2 and 3 of the top panel; input DNAs are shown in the

lower panel).

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To determine if Tra1-SRR3413 alters acetylation of either histone H3 or histone H4, chromatin

immunoprecipitations were performed. We first compared the ratio of acetylated histone H4 (at lysine

8) to total histone H3 at the ADE1 and PHO5 promoters for the strain containing tra1-SRR3413 with

strains containing deletions of the NuA4 components Vid21/Eaf1 and Yng2. Figure 6a shows the

relative level of acetylated histone H4 as a percentage of that found in the wild-type strain. For both

ADE1 and PHO5 promoters, tra1-SRR3413 resulted in a decrease in relative acetylation to

approximately 60% of that found in the TRA1SB strain. This decrease was comparable to that found

upon deletion of yng2 and somewhat less than deletion of vid21/eaf1. For each mutant strain, a smaller

effect was observed for sequences at the 3’ end of the PHO5 gene. Acetylation of histone H3 (at

lysine 9) was then analyzed at PHO5, PHO84 and PGK1 promoters (Figure 6b); the latter being

SAGA-independent. tra1-SRR3413 resulted in a decrease in the ratio of acetylated histone H3 to total

histone H3 at PHO5 and PHO84 promoters that was approximately 35% of that found in the wild-type

tra1SB strain, approaching that seen in strains deleted for spt7 and ada2. At the PGK1 promoter, the

decrease in acetylation of histone H3 was less apparent (~70% of wild-type for tra1-SRR3413).

Although we can not exclude an indirect role of Tra1 on acetylation, the simplest interpretation of

these results is that the PI3K domain of Tra1 is required for optimal histone acetylation by both the

NuA4 and SAGA complexes.

Tra1-SRR3413 results in shortened telomere length. The similarity of Tra1 to many of the PI3K-

domain containing proteins involved in telomere biology (for example, Tel1 and Mec1) led us to test if

the tra1-SRR3413, tra1-P3408A and tra1-S3463A alleles affected telomere length. As an initial test we

assayed the effects of these alleles on the ability of strains to generate and stably maintain linearized

plasmids after transformation with circular plasmids containing inverted repeats of telomeric

sequences (Lundblad and Szostak, 1989). Strains containing tra1-SRR3413, tra1-P3408A and tra1-S3463A

were transformed with the LEU2 containing centromeric plasmid pVL106 (kindly supplied by Dr. V.

Lundblad), which contains URA3 flanked by inverted telomeric sequences. Cells were grown in

nonselective media and plated onto media containing 5-FOA and lacking leucine. 5-FOA

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resistant/Leu+ colonies were counted and normalized to the number of total cells plated. As shown in

Figure 7a, tra1-SRR3413 resulted in a reduced ability to linearize and maintain pVL106. To directly

examine telomere length, Southern blotting was used to compare the terminal restriction fragments

(TRFs) of chromosomes in the tra1-SRR3413 and TRA1SB strains. CY1021 containing wild-type TRA1

on a URA3-containing plasmid was transformed with TRA1SB or tra1-SRR3413 on TRP1-centromeric

plasmids. The wild-type TRA1 allele was lost after selection on 5-FOA, establishing generation zero.

Strains were grown through multiple generations, genomic DNA isolated and TRF length determined

after digestion of the genomic DNA with XhoI. Cells were grown at both 30o and 34o. The latter

significantly reduces growth rate and from the linearization assay appeared to suppress the effects of

SRR3413 on telomere shortening (not shown). As shown in Figure 7b, at 10 generations of growth, the

TRFs were of approximately equal lengths in TRA1SB and tra1-SRR3413 strains. By 50 generations,

TRFs were appreciably shorter in the tra1-SRR3413 strain, with a further shortening apparent at 100

generations when cells were grown at 30o. The median difference was approximately 120 and 140 base

pairs at 50 and 100 generations, respectively. The effect of SRR3413 was not apparent when cells were

grown at 34o, perhaps the result of increasing the period available for telomere extension.

Genetic analysis. To examine the molecular basis for the tra1-SRR3413 phenotype, we initiated a

suppressor analysis. tra1-SRR3413 was introduced into yeast strain CY1021 by plasmid shuffling.

Approximately 2 x 106 cells were plated onto YPD containing 6% ethanol. Growth of two colonies

was observed after 5 days. To determine if these were intragenic, the tra1 expressing plasmid was

isolated from both strains and the phenotype re-examined after plasmid shuffling in yeast strain

CY1021. In both cases the plasmid enabled growth in YPD containing 6% ethanol. The 3’ SalI-SacI

fragment of each allele was sequenced and found to contain the SRR3413 mutation and a common

mutation converting H3530Y. This mutation was shown to be sufficient for suppression by recloning the

SalI-SacI fragment into TRA1SB and plasmid shuffling. H3530Y suppressed the growth defects

associated with SRR3413 on both 6% ethanol containing plates (Figure 8a) and 37o (not shown). Allele

specificity was examined by introducing H3530Y into wild-type and tra1-P3408A containing

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backgrounds. H3530Y on its own had no effect on growth but did suppress the ethanol sensitivity due to

P3408A and therefore is a general suppressor of the ethanol-sensitive class of tra1 alleles. As shown in

Figure 8b, the effects of H3530Y are most likely due to suppression of transcriptional defects since this

mutation suppressed the slow induction of PHO5 seen in a tra1-SRR3413 strain.

To detect genes whose disruption in parallel with tra1-SRR3413 generates a synthetic slow growth

phenotype, we performed a systematic genetic array (SGA) analysis in which a strain containing tra1-

SRR3413 was crossed into the array representing the collection of ~4700 viable haploid deletion strains.

Candidate genetic interactions, scored as slow growing colonies through two independent screenings,

were examined individually after generation of double mutant strains by tetrad dissection. Growth for

these strains was examined on synthetic complete media at 34o. The twenty-three genes identified and

their functions are listed in supplemental Table 2. Consistent with the finding that tra1-SRR3413 resulted

in sensitivity to ethanol and Calcofluor white, ten of these appear involved in cell membrane and cell

wall processes. This number is greater if indirect roles, for example a contribution to

phosphatidylserine synthesis by SER1 and SER2, are considered. The other prevalent groups

represented were genes involved with transport and mitochondria function. A hierarchal cluster

analysis was performed with the tra1-SRR3413 interacting genes and the datasets of Tong et al. (2004)

and Pan et al. (2006). tra1-SRR3413 did not cluster closely with any of the strains used in these analyses.

In fact, no common synthetic growth defects were found amongst those listed in the Saccharomyces

Genome Database for NuA4 and SAGA components and those for tra1-SRR3413.

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DISCUSSION

Tra1 is one of the largest yeast proteins and is essential for viability. As a component of both NuA4

and SAGA complexes, Tra1 interacts directly with DNA-binding transcriptional regulators recruiting

these HAT complexes to promoters. Based upon the similarity of this domain with that found in other

cellular regulators it is likely that the PI3K domain of the molecule has a significant function.

Indicative of a key role for the PI3K domain, eight targeted mutations within this region resulted in

loss of viability. Although we lack detailed structural information for the Tra1/TRRAP subfamily, the

alignment of these residues in comparison to the structure of porcine PI3K-γ is interesting. Residues

R3567 and PFR3621 are not unique to Tra1/TRRAP and are found in the catalytic-loop and activation-

loop, respectively. As is the case for the kinase members of the PI3K family (Dhand et al., 1994; Stack

and Emr, 1994), R3567 is required for function of Tra1. Similarly, PFR3621 is generally conserved within

the activation loop of the PI3K family. The activation-loop plays a critical role in substrate binding for

these molecules (Bondeva et al., 1998), suggesting a similar essential role for this region in Tra1.

Substrates for Tra1 are unknown. Potentially these could include phospholipids; however, we have

detected only very weak binding of recombinant Tra1-PI3K domain to PIP strips (Echelon

Biosciences; not shown).

Eight alleles with mutations in the PI3K domain supported viability and displayed phenotypes, which

fell into two groups. The first group, tra1-P3446, -DKL3491, -P3612 and -N3617 were slightly resistant to

overexpression of VP16, although to a much lesser extent than the ada genes, and were partially

sensitive to hydroxyurea, characteristic of defects in DNA replication. This later phenotype has not

been previously ascribed to SAGA or NuA4 components and may reflect a novel function of one or

both of these complexes or perhaps defects in expression of genes involved in this process. The second

group of alleles containing mutations of P3408, SRR3413, S3463 and to a lesser extent EER3416, was more

marked. These alleles resulted in temperature sensitive growth, reduced growth in low phosphate

media and in media containing 6% ethanol. The latter has also been noted for deletions of ada2 and

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based on their reduced growth in the presence of Calcofluor white, this phenotype likely reflects

changes in cell wall properties (Takahashi et al., 2001) or the response to cell wall stress. Unlike

deletions of Ada components, the tra1-P3408A, -SRR3413 and -S3463A alleles did not result in resistance

to overexpression of VP16, nor is their temperature sensitive growth indicative of deletions of ada

genes in the KY320 strain background. tra1-P3408 and tra1-SRR3413, the alleles with the most

pronounced phenotypes, were partially sensitive to the microtubule destabilizing agent benomyl. This

is characteristic of deletions of NuA4 components, but again for the tra1 alleles the effect was not as

severe. The tra1-SRR3413 allele was the only allele showing sensitivity to MMS but dramatically less so

than for deletions of components of the NuA4 complex. Growth of tra1-P3408, SRR3413 and S3463 strains

was modestly reduced on media depleted of inositol, a characteristic of deletions of spt genes;

however, unlike deletion of spt7, the inositol auxotrophy of the tra1 alleles was not the result of

reduced expression of INO1. Cumulatively, these phenotypes suggest that Tra1 possesses functions

overlapping yet distinct from SAGA or NuA4 components.

The transcriptional effects of the tra1-P3408A, SRR3413 and S3463A alleles were compared to deletions of

spt7, gcn5 and yng2 using lacZ fusion reporters. The transcriptional change for the tra1 alleles was less

than that seen upon deletion of the NuA4 or SAGA components but most closely resembled that seen

upon deletion of gcn5. Microarray analyses confirmed the transcription defect arising from these

alleles and that they were acting by altering a common function. Cluster analysis versus the expression

profiles seen for deletions of SAGA and NuA4 components showed the greatest similarity though not

robust, to ada2. Comparison of the growth phenotypes of strains containing deletion of strains carrying

single and double mutations of ada2Δ0 and tra1-P3408A confirmed that the tra1 mutations were not

functioning exclusively through effects on the ADA genes (data not shown). This lack of similarity

suggests that the transcriptional effects of the tra1 alleles result from the integrative effect of

disturbing histone acetylation by both SAGA and NuA4 complexes and/or the possibility that the PI3K

domain of Tra1 has one or more roles distinct from other members of these complexes. The

mechanism by which the tra1 mutations affect histone acetylation is unclear; interestingly, in the three-

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dimensional structure of the SAGA complex revealed by electron microscopy, Tra1 and Gcn5 are

positioned on opposite sides of a nucleosome-sized cleft (Wu et al., 2004), suggesting that Tra1 may

play a role in orienting nucleosomes for acetylation.

The finding of a generation dependent telomere shortening in the tra1-SRR3413 strain points to a role of

Tra1 in telomere elongation or maintenance. The shortened telomeres may be the indirect result of

Tra1 regulating expression of genes required for these processes. In this regard eleven genes whose

deletion results in shortened telomeres (Askree et al, 2004) have decreased expression of 1.5-fold or

greater in the tra1-SRR3413 strain. Most notable of these is EST3 with decreased expression of 2.4-fold.

Alternatively, Tra1 may play a direct role in telomere elongation or maintenance. We do not believe

that the Tra1 effect is mediated solely through one of SAGA or NuA4-dependent histone acetylation,

as component genes were not found in the genome wide screen of Askree et al. (2004) nor in our

independent analyses of Spt7, Gcn5 and Eaf7 (data not shown). Additional studies will be required to

determine if Tra1 is recruited to telomeres and the role of the PI3K-domain in telomere elongation.

The ethanol and Calcofluor white sensitivity of tra1-SRR3413 as well as its genetic interactions with

genes involved in cell wall and membrane processes is consistent with the involvement of SAGA in

the response to stress (Huisinga and Pugh, 2004). Tra1 may be required to trigger transcriptional

changes necessary when membrane processes are disturbed during cellular stress. Associations

between SAGA components and membrane processes have previously been suggested by the finding

that Gcn5 and Spt20 are required for the unfolded protein response (Welihinda et al., 1997, 2000).

Interestingly, deletion of a number of genes involved in vesicular trafficking, including vps9, vps15,

vps28, vps34, vps36, vps39, bro1 and sur4, result in both telomere shortening and ethanol sensitivity

similar to tra1-SRR3413. The other two gene deletions with both of these phenotypes are bem2 and sit4,

each of which is involved in cytoskeletal and cell wall organization. (Expression of these genes is not

reduced in the tra1-SRR3413 background.) Other studies connect NuA4 components with membrane

processes; most notably, vid21/eaf1 (Vacuolar Import Degradation) was identified in a screen for

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defects in sorting of carboxypeptidase Y to the vacuole (Bonangelino et al., 2002), is sensitive to

ethanol (Fugita et al., 2006), and interacts with Vac8, which is required for several aspects of vacuolar

function (Tang et al., 2006).

Possible functions of the PI3K domain. Given the importance of PI3K domains in key signaling

proteins and the conservation of this domain in the Tra1/TRRAP family of molecules, it is reasonable

to consider possible activities of the PI3K domain. In this regard P3408, SRR3413 and S3463 map

proximally to what would be the cleft between N and C-terminal lobes of the PI3K-γ structure. P3408, in

fact, aligns with K833, which in PI3K-γ interacts with the α-phosphate of ATP and is covalently

modified by the kinase inhibitor wortmanin (Wymann et al., 1996). While clearly an approximation of

the Tra1 structure, S3463 may be located within the cleft at a position that putatively could be involved

with substrate interaction. Though speculative, it is attractive to propose that the putative cleft of Tra1

is a substrate-binding pocket, where the domain catalyzes the transfer of a modifying unit other than

phosphate moieties, transfers water as a hydrolase or perhaps has a role in the nuclear signaling by

inositol polyphosphates (for example; Steger et al., 2003).

ACKNOWLEDGEMENTS

We thank Drs.Brian Shilton, Catherine Bateman, David Litchfield and Greg Gloor for their comments

on the manuscript, Drs. Victoria Lundblad, Fred Winston, Kathy Gould, Joan Curcio, V. Zakian and

Raymund Wellinger for reagents, Shayna Oldegard, Dana Abrassart, Monica Piasecki, Courtney

Coschi and Kim Grant for assistance in preparation of DNA constructs and Dr. Joe Martens and

Melissa Bradford for assistance with the chromatin immunoprecipitations. This work was supported by

Canadian Institutes of Health Research grants to CJB. SMTH is a holder of a NSERC studentship. CH

was supported by an OGSST scholarship. AIM is a holder of a Western Graduate Research

Scholarship.

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FIGURE LEGENDS

Figure 1. Phenotypes of Tra1-SRR3413, P3408A and S3463A. A. Yeast strains containing the tra1 mutant

alleles and Tra1SB (parent molecule) expressed from plasmids or endogenous wild-type Tra1 (KY320)

were grown to saturation and ten-fold serial dilutions plated onto YP media containing 2% glucose and

grown at 30o and 37o, or at 30o in YPD containing 3% ethanol or 5 μg/ml Calcofluor white (lower

panels). Note that expression of Tra1SB on a plasmid results in slightly reduced growth as compared to

endogenous wild-type Tra1. B. Growth curves of Tra1 mutant strains after temperature shift to 37o.

The indicated tra1 mutant strains or the background wild-type strain (TRA1SB) were grown to

saturation and diluted into YPD media at 34o (time 0). Cells were grown for 4 hrs with the absorbance

at 600 nm determined on hourly intervals. One half of the culture volume was removed and shifted to

37o and absorbance determined for cultures at both temperatures. Note the scale differences for the

graphs. The arrow indicates 30 minutes after the temperature shift, the first time-point a reading at both

temperatures was made. C. Analyses of a strain containing an integrated version of the tra1-SRR3413

that lacks the BamHI site found in the plasmid copy. Yeast strains CY2437 (integrated wild-type

TRA1) and CY2438 (integrated tra1-SRR3413) were grown to saturation in YPD. Serial dilutions were

spotted onto YPD plates and grown at 30o and 37o, or at 30o in YPD containing 5 μg/ml Calcofluor

white.

Figure 2. Expression and interactions of the ethanol sensitive Tra1 mutants. A. Crude extracts were

prepared from yeast strain KY320 (- ve) or strains expressing TAP-Flag3x-tagged versions of Tra1SB,

Tra1-S3463A, Tra1-P4308A and Tra1-SRR3413. 25 μg of extract was separated by SDS PAGE (5%) and

Western blotted with anti-Flag antibody. The upper panel is the Western blot of the ~450 kDa band

corresponding to Tra1; the lower panel is a portion of an equivalent gel stained with Coomassie

Brilliant Blue to verify protein concentrations. B. tra1-SRR3413 associates with Spt7 and Yng2. Top

panel: Crude extracts were prepared from strains KY320 expressing wild-type Tra1 (-ve), TAP-Flag-

Tra1SB or TAP-Flag-Tra1-SRR3413 and with Flag-Yng2. 150 μg of crude extract was separated by

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electrophoresis on 5% SDS PAGE and visualized by Western blotting using anti-Flag antibody. Co-

purification: Extracts were subjected to tandem affinity purification. Equal volumes were separated by

electrophoresis on 5% and 8% SDS PAGE and Western blotted using anti-Flag antibody to detect Tra1

(upper panel) or Yng2 (middle panel). The presence of Spt7 (lower panel) was detected using anti-Spt7

antibody (kindly provided by Dr. Fred Winston). The SAGA-form and SLIK-form of Spt7 are marked

as SA and SL, respectively. C. Tra1-SRR3413 forms Ada2-containing complexes that elute from a

HiTrap Q Sepharose Fast Flow column with a similar profile to Tra1SB. Whole cell extract from strains

containing Tra1-SRR3413 or Tra1SB, each with HA-Ada2, were fractionated on a HiTrap Q Sepharose

Fast Flow column. Protein was eluted with a gradient of 0-1.0 M NaCl. Equal volumes of successive

fractions were separated on 8% SDS gels, Western blotted for HA-Ada2 and relative amounts

determined by densitometry. D. Tra1-SRR3413 forms Yng2-containing complexes that elute from a

HiTrap Q Sepharose Fast Flow column with a similar profile to Tra1SB. 25 mg of whole cell extract

from strains containing Tra1-SRR3413 or Tra1SB, each with Flag-Yng2, were fractionated on a HiTrap

Q Sepharose Fast Flow column as above. Equal volumes of successive fractions were separated on 8%

SDS gels and Western blotted with anti-Flag antibody.

Figure 3. Expression of β-galactosidase reporter constructs in tra1 mutant strains. A. Promoter

fragments from PHO5, INO1, HIS4 and ADH2 were cloned as his3-LACZ reporter fusions into the

LEU2 centromeric plasmid YCp87 and transformed into FY1093 (spt7Δ0), FY1370 (gcn5Δ0),

CY1514 (tra1-S3463A), CY1507 (tra1-P3408A) and CY1531 (tra1-SRR3413). β-galactosidase activity was

determined after growth in low phosphate media (PHO5), inositol depleted media (INO1), minimal

media (HIS4) or YP containing 3% ethanol plus 0.01% glucose (ADH2) at 30o. Activity was compared

to the appropriate wild-type strains, FY630 for spt7Δ, FY86 for gcn5Δ and CY1524 for the tra1 mutant

strains and are shown as percentages. Measurements were made in triplicate in two independent

experiments with the standard error indicated. B. Expression of INO1-lacZ was analyzed at 30 o and

35o for strains containing TRA1SB and tra1-SRR3413.

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Figure 4. Hierarchical cluster analysis of tra1-SRR3413 microarray expression data. Microarrary

analyses were performed for yeast strain CY1531 containing tra1-SRR3413 as the sole copy of Tra1

grown in YPD at 30oC using the Agilent Yeast Oligo Array Kit. The experiment was performed in

duplicate with dye-reversal using TRA1SB (yeast strain CY1524) as the control. Comparisons were

made to the expression profiles of strains with deletions of NuA4 (Krogan et al., 2004) and SAGA

(Ingvarsdottir et al., 2005) components. Also included were profiles of strains with deletions of cka2,

ckb2, gcn4, hat2, isw1, mbp1, rpd3, rtg1, sap30, sin3, ste12 and yap3 which were identified as being

most similar to tra1-SRR3413 in an initial clustering analysis using a subset of the compendium data

(Hughes et al., 2000). Gene families are indicated to the right.

Figure 5. Effects of Tra1-SRR3413 on interaction with the Gal4 activation domain and recruitment to

the PHO5 promoter. A. Interaction of Tra1-SRR3413 with the Gal4 activation domain. Yeast extract

was prepared from CY1021 (lane 1, negative control), CY1962 containing HA-tagged Ada2 (Tra1SB;

lane 2) and CY1963 containing HA-tagged Ada2 (Tra1-SRR3413; lane 3). Purified TAP-Tra1SB was

applied to the Glutathione Sepharose bound with GST-Gal4AD (lane 4) or control Glutathione

Sepharose bound with GST (lane 5). Similarly TAP-Tra1-SRR3413 was applied to GST-Gal4 AD beads

(lane 6) or GST beads (lane 7). Bound protein was eluted with reduced glutathione and the eluant

separated on a 10% SDS-PAGE gel. Interaction with Gal4 AD was monitored by Western blotting for

HA-Ada2. B . Recruitment of TAP-Tra1 to the PHO5 promoter was determined by chromatin

immunoprecipitation using strains BY4741 (lane 1) and BY4741 containing TAP-Tra13413 (lane 2) or

TAP-Tra1-SRR3413 (lane 3). The presence of PHO5 promoter DNA was determined by PCR and

separation on 5% native polyacrylamide gels stained with ethidium bromide. The lower panel shows

PCR products from the input DNA used for analysis. A serial dilution of the sample for Tra1-SRR3413

is shown in lanes 3 through 5. Lane 6 is a no template control.

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Figure 6. Histone acetylation in Tra1-SRR3413 containing strains. A. Acetylation of lysine 8 of histone

H4. Yeast strains CY1531 (tra1-SRR3413), CY1524 (TRA1SB), QY202 (yng2Δ0), QY204 (YNG2),

BY4916 (vid21Δ0) and BY4741 (VID21) were grown to A600=1.5 in YPD and ChIP analysis

performed using antibody directed against acetylated K8 of histone H4 and against histone H3. Levels

of immunoprecipitated ADE1 promoter, PHO5 promoter and the 3’ coding region of PHO5, were

determined after PCR and separation by electrophoresis and staining with ethidium bromide. Serial

dilutions were examined to ensure detection in a linear range. The histogram shows the ratio of

acetylated histone H4 to total histone H3 for each of the mutant strains as a percentage of that found in

the relevant wild-type strain for experiments performed in duplicate. B. Histone H3 acetylation. Yeast

strains CY1531 (tra1-SRR3413), CY1524 (TRA1SB), BY3281 (spt7Δ0), BY4282 (ada2Δ0) and BY4741

(wild-type) were grown in phosphate depleted media (PHO5) or YPD (PHO84 and PGK1) to an

A600=1.5. Equal amounts of chromatin were immunoprecipitated with anti acetylated (K9) histone H3

antibody or anti histone H3 antibody and levels of promoter determined after PCR. The ratio of

acetylated histone H3 to total histone H3 for each mutant is shown as a percentage of that found in the

relevant wild-type strain.

Figure 7. TRA1-SRR3413 results in a generation dependent reduction in telomere length. A. Plasmid

linearization assay. Yeast strains CY1524 (TRA1SB), CY1531 (tra1-SRR3413), and CY1507 (tra1-

P3408A) and CY1514 (tra1-S3463A) were transformed with pVL106 (kindly supplied by Dr. V.

Lundblad). Leu+ Ura+ transformants were grown sequentially in media lacking uracil then YPD,

washed, and spotted onto plates containing 5-FOA and lacking leucine. The number of 5-FOA resistant

Leu+ colonies were counted after 5 days of growth at 30o and normalized to the total number of cells

plated. The graph indicates the percentage of 5-FOA resistant Leu+ colonies in each of the tra1 mutant

strains as compared to that observed for TRA1SB for 5 experiments each performed in duplicate. B.

Southern blot of genomic DNA from TRA1SB (wt) and tra1-SRR3413 (SRR) containing strains cut with

XhoI and hybridized to a 32P-labeled TG1-3/C1-3A DNA probe. The terminal restriction fragment (TRF)

Page 40: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

40

is derived from a subset of yeast telomeres that have a Y’ telomere element. The Y’ element has a

conserved XhoI site that is located proximal to the terminal telomeric repeat tract. The larger DNA

fragments are derived from either telomeres lacking the Y’ element or subtelomeric DNA sequences

containing TG1-3 tracks. Cells were serially passaged for the indicated number of generations. After 10

generations the average TRF length for the two strains (~1110 basepairs) did not differ significantly

(left-hand panel) at either growth temperature. After 50 and 100 generations at 30˚ (right-hand panel)

the average TRF length for the tra1-SRR3413 strain was reduced by ~125 and 140 basepairs,

respectively.

Figure 8. H3530Y suppresses the ethanol sensitivity and transcription defects due to tra1-SRR3413. A.

Yeast strains containing the TRA1 alleles indicated in the legend to the right were plated onto YPD or

YPD containing 6% ethanol and grown at 30o. B. Delay in the induction of PHO5 resulting from

SRR3413 is suppressed by alteration of H3530Y. Yeast strains containing Tra1SB, Tra1-SRR3413, and Tra1-

SRR3413-H3530Y were transformed with PHO5-lacZ, grown to saturation in YPD, washed 4-times in

water, and diluted 1:12 into low phosphate media. Samples were taken at the indicated time points and

β-galactosidase activity determined.

Page 41: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

Tra1SB

Tra1WT

Tra1WT

S3463A

Tra1SB

P3408A

P3408A

S3463A

SRR3413

SRR3413

YPD 30o YPD 37o

YPD EtOH YPD CW

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12

Ab

sorb

ance

(60

0 n

M)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 2 4 6 8 10 12

Ab

sorb

ance

(60

0 n

M)

0

0.5

1

1.5

2

2.5

0 2 4 6 8 10 12

Ab

sorb

ance

(60

0 n

M)

34C37C

BTra1SB

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12

Ab

sorb

ance

(60

0 n

M)

P3408A

S3463A

SRR3413

Time (hr)

A

Figure 1

YPD 30o

YPD 37o

YPD CW

CSRR3413

SRR3413

SRR3413

Tra1WT

Tra1WT

Tra1WT

Page 42: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

0

50

100

150

200

250

20 30 40 50 60 70

P 3408

A

SRR 34

13

S 3463

A

TRA

1 SB

-ve

A

Figure 2

TAP

CrudeextractsTra1

Tra1

SBSR

R 3413

-ve

SASL

Tra1

Spt7

Yng2

B0

50

100

150

200

250

300

350

10 20 30 40 50

FractionD

ensi

tom

etry

units

Ada2 elution profile

Yng2 elution profile

C

Fraction

FractionD

ensi

tom

etry

Uni

tsD

ensi

tom

etry

Uni

tsD

Tra1SB

SRR3413

Tra1SB

SRR3413Tra1

Cont

Page 43: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

0

100

200

300

400

500

600

700

Rel

ativ

e IN

O1

exp

ress

ion 30

35

0

20

40

60

80

100

120

140

PHO5 INO1 HIS4 ADH2

Per

cen

t W

T e

xpre

ssio

nSRR3413P3408S3463yng2gcn5spt7

Tra1-SB Tra1-SRR

A B

o

o

Figure 3

Page 44: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

gcn4

cka2

ckb2

rtg1

isw

1∆

hat2

eaf5

yaf9

vid2

1∆yn

g2∆

ynl1

36w

sgf1

1∆

ubp8

yap3

ste1

2∆

mbp

1∆

rpd3

sap3

0∆

sin3

ada2

tra1

-SR

Rsp

t3

spt8

purine base metabolism (GO:0006144)

cell wall (GO:0005618)

carbohydrate catabolism (GO:0016052)

amino acid metabolism (GO:0006520)

phosphate metabolism (GO:0006796)

cell cycle (GO:0007049)

ion transporter (GO:0015075)

protein metabolism (GO:0019538)

fold repression fold induction1 ≥42≥4 323

Figure 4

Page 45: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

B

Figure 5

Tra1

SBSR

R

cont

rol

HA-Ada2

Tra1SB SRR3413

1 2 3 4 5 6 7inputs bound

A

Tra1 wt SB SRR3413

TAP - + +

1 2 3 4 5 6

IP

Input

Column Matrix

Gal4AD Gal4AD

-GST -GSTGST GST

Page 46: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

0

10

20

30

40

50

60

70

80

90

100

PHO84 PHO5 PGK1

Ac-

H3/

H3

(% m

uta

nt/

wt) tra1-SRR

spt7ada2

Figure 6

0

10

20

30

40

50

60

70

80

90

100

ADE1 PHO5

Ac-

H4/

H3

(% m

uta

nt/

wt) tra1-SRR

yng2vid21/eaf1

PHO5 (3’-coding)

A B

Page 47: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

0

20

40

60

80

100

120

140

Tra1SB SRR3413 P3408A S3463A

Per

cent

5-F

OA

res

ista

nt L

eu+

A

Tra1SB SRR3413 P3408 S3463

Figure 7

StrainGenerations

Temp ˚C 30 34 30 34

wt SRR

10 10

TRF

1 2 3 4

1.2

1.9

4.3

Size (kb)

M

wt SRR wt SRR50 50 100 100

30 34 30 34 30 34 30 34

1 2 3 4 5 6 7 8

TRF1.2

1.9

0.7

4.3

B

Page 48: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

H3530Y

P3408

SRRSRRH3530Y

P3408H3530YTra1SB

YPD

6%EtOH

A B

Figure 8

SRR3413

SRR3413H3530Y

Tra1SB

Time (hr)P

HO

5-Lac

ZE

xpre

ssio

n

Page 49: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

Table 1. Strains used in this study Strain Genotype TRA1 plasmid Reference KY320 MATa ura3-52 ade2-101 trp1-∆1 lys2-

801 his3-∆200 leu2::PET56 Chen and Struhl

(1988) CY1021 Isogenic to KY320 but tra1::Tn10LUK myc-TRA1 Saleh et al., 1998 CY1962 Isogenic to CY1021 TAP- TRA1SB This work CY1963 Isogenic to CY1021 TAP-tra1-

SRR3413 This work

CY1524 Isogenic to CY1021 Flag3- TRA1SB This work CY1507 Isogenic to CY1021 Flag3-tra1-

P3408A This work

CY1531 Isogenic to CY1021 Flag3-tra1-SRR3413

This work

CY1514 Isogenic to CY1021 Flag3-tra1-S3463A

This work

FY630 MATα his4-917 lys2-173R2 leu2∆1 ura3-52 trp1∆63

Gansheroff et al. (1995)

FY1093 Isogenic to FY630 but spt7::LEU2 CY1381 Isogenic to FY1093 but

leu2::HIS3 This work

FY86 MATα his3∆200 leu2∆1 ura3-52 Roberts and Winston (1997)

FY1370 isogenic to FY86 but gcn5::HIS3 Y5565 MATα can1∆::MFA1pr-HIS3

mfα1∆::MFα1pr-LEU2 lyp1∆ ura3∆ his3∆ met15∆

Tong and Boone (2006)

BY4741 MATa his∆0 leu2∆0 met1∆0 ura3∆0 Winzeler and Davis (1997)

BY3281 Isogenic to BY4741 except spt7::Kanr BY4196 Isogenic to BY4741 except vid21::Kanr BY4282 Isogenic to BY4741 except ada2::Kanr QY204 MAT his3∆200 trp1∆ 63 ura3-52

leu2∆1 lys2-128δ Nourani et al.

(2001) QY202 Isogenic to QY204 except yng2::Kanr

Page 50: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

Table 2. Growth of strains containing mutations throughout the length of Tra1. Mutation1 Reason for analysis Viability2

30o 37o DSP171 Potential phosphorylation Yes Yes EFS184 Potential phosphorylation Yes Yes KEL345 Conserved3 Yes Yes VLL837 LXXLL sequence Yes Yes VRL867 Conserved Yes Yes KKK1686 Found in chromatin associated Sth1 Yes Yes GLW2677 Conserved in yeast4 Yes Yes GYH2939 Conserved in yeast Yes Yes TLP2972 Conserved in yeast Yes Yes HFQ3168 Conserved in FAT domain5 Yes Yes EER3416 Conserved Yes Reduced PFR3621 Conserved in PI3K domain No No 1 Alleles were constructed by placing a NotI linker encoding a triple alanine tag in place of the indicated three amino acids. The number refers to the amino acid position of the first of the three amino acids. 2 Growth on YPD media at 30o or 37o 3 Conserved indicates identity in S. pombe and human Tra1 molecules. 4 Sequence similarity or identity in yeast homologs 5 See Bosotti et al., 2000

Page 51: Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29  · 1 Structure Function Analysis of the Phosphatidylinositol-3-kinase Domain of Yeast Tra1 A. Irina Mutiu*,

Table 3. Phenotypes of tra1 mutations. Mutation1 30o 2 37 o 3 Phos4 Ino5 EtOH6 VP167 Ben8 K3571 ++++ ++++ ++++ ++++ ++++ - ++++ P3655 ++++ ++++ ++++ ++++ +++ - ++++ P3408 +++ + + +++ + - ++ SRR3413 ++ - + ++ - - + EER3416 ++++ ++ ++++ ++++ ++++ - ++++ S3463 ++++ + ++ +++ + - +++ F3443 + - - - - - - P3446 ++++ ++++ ++++ ++++ +++ +/- ++++ DKL3491 ++++ ++++ ++++ ++++ ++++ +/- ++++ P3612 ++++ ++++ ++++ ++++ +++ +/- ++++ N3617 ++++ ++++ ++++ ++++ +++ +/- ++++ 1 The indicated mutations were engineered into TRA1SB which contains a BamHI restriction site that results in N3580A and a silent SalI restrictions site that converts nucleotides A9714G and T9717G. All amino acid changes were to alanine. 2 Alleles were transformed into CY1021 and tested for viability by growth on mimimal plates containing 5-FOA. Growth at 30o was determined on YPD. Comparisons were made to a strain containing TRA1SB 3 Growth at 37 o on YPD plates 4 Growth on YPD plates deficient for phosphate 5 Growth on complete minimal media lacking inositol 6 Growth on YPD plates containing 6% ethanol 7 Strains were transformed with padhGV16-gal4-VP16 (Berger et al., 1992) and growth examined on minimal media lacking leucine. 8 Growth on synthetic complete media containing 20 µg/ml benomyl

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Table 4: Gene expression profile in tra1-P3408A, -SRR3413, and -S3463A tra1-P3408A

GENE1 Fold2 tra1-SRR3413

GENE Fold tra1-S3463A

GENE Fold 1 FET3 3.2 TKL2 4.5 YGL262W 6.0 2 PCL1 3.2 YHR033W 3.9 AFT2 3.9 3 ATF2 3.2 SCW10 3.5 COS12 3.5 4 RNR1 3.1 YLR327C 3.4 SCW10 3.0 5 SCW10 3.1 YER053C 3.4 FET3 3.0 6 HXT4 2.9 PDR12 3.3 RNR1 3.0 7 SVS1 2.8 YLR312C 3.1 PDR12 2.9 8 YKL097W-A 2.7 SVS1 3.1 PCL1 2.8 9 PDR12 2.7 MNN1 3.1 PUS2 2.7 10 DBP2 2.6 RNR1 3.0 KAP122 2.7 11 YHR033W 2.6 ATF2 3.0 SVS1 2.7 12 RMD6 2.5 PCL1 3.0 RRN3 2.7 13 MNN1 2.5 RRN3 2.9 MNN1 2.7 14 NCE4 2.5 MND1 2.8 HXT4 2.6 15 RRN3 2.4 ECM11 2.8 DBP2 2.6 16 APG10 2.4 KCC4 2.7 AGP10 2.5 17 YER053C 2.4 TRA1 2.7 YHR033W 2.5 18 HO 2.4 APG14 2.7 HO 2.5 19 RPL7B 2.3 PUS2 2.7 TRA1 2.4 20 PLB3 2.3 INO1 2.6 YPL207W 2.3 21 RNA14 2.3 FET3 2.6 FDH2 2.3 22 MIR1 2.3 BDF2 2.6 DHR2 2.3 23 YLR312C 2.3 HRP1 2.6 YER053C 2.3 24 MEP3 2.3 SIT1 2.6 MDV1 2.3 25 PUS2 2.3 ATP7 2.5 TOP3 2.3 TOTAL#3 85 152 97 1 IMD2 -14.7 IMD2 -19.3 IMD2 -18.3 2 ADE17 -14.2 ADE17 -13.5 SPL2 -10.1 3 SPL2 -10.9 SPL2 -11.4 YBR285W -7.9 4 HIS4 -6.9 AAD4 -11.3 ADE17 -7.2 5 YBR285W -6.9 YLR053C -8.7 AAD4 -6.3 6 AAD4 -6.8 YBR285W -8.5 HIS4 -5.5 7 ADE1 -6.5 ADE1 -8.5 YAL061W -5.2 8 LAP4 -6.3 PHO84 -7.9 TSA2 -5.2 9 VPS73 -6.1 HIS4 -7.4 YMR090W -5.1 10 KHS1 -5.8 VPS73 -7.3 ZTA1 -5.1 11 YAL061W -5.8 GTT2 -7.2 EMI2 -5.0 12 SNZ1 -5.8 YMR090W -7.2 SNZ1 -4.9 13 YLR053C -5.5 YAL061W -6.5 YBR047W -4.9 14 SHM2 -5.2 TSA2 -6.5 YLR460C -4.8 15 PH05 -5.1 BNA1 -6.1 YLR053C -4.7 16 ZTA1 -5.1 LYS20 -6.1 YBR147W -4.6 17 PHO84 -5.0 ADE5,7 -6.1 YPS73 -4.5 18 TSA2 -5.0 LAP4 -5.8 YJL038C -4.3 19 EMI2 -4.9 IMD1 -5.8 SNO1 -4.3 20 IMD1 -4.8 ZTA1 -5.8 YMR118C -4.2 21 ADE5,7 -4.7 LYS9 -5.7 YKL107W -4.2 22 YJL038C -4.7 LYS7 -5.5 LAP4 -4.2 23 YBR147W -4.7 GCV2 -5.4 ECM12 -4.2 24 YNL194C -4.6 YPS6 -5.3 PHO84 -4.1 25 GCV2 -4.6 YKL107W -5.3 IMD1 -4.1 TOTAL#3 258 294 261

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1 Genes in bold are found either a minimum of 2-fold elevated or 4-fold decreased in one of the other strains. Those italicized are 3-fold decreased in one of the other strains. Dubious ORFs are not listed. HIS4 was down regulated in the gene array data more so than in the β-galactosidase assays, likely reflecting different levels of Tra1 contribution in different media. 2 Values are the fold-change in expression relative to that in CY1524. Cells were grown in YPD media to an A600 nm ~1.5. Values are the average of two experiments with dye reversal. 3 Total number of genes affected 2-fold or greater.