Genetics, Vol. 177, 151-166, September 2007, Copyright © 2007
doi:10.1534/genetics.107.074476

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Structure/Function Analysis of the Phosphatidylinositol-3-Kinase Domain of Yeast Tra1

A. Irina Mutiu^*,1, Stephen M. T. Hoke^*,1, Julie Genereaux^*, Carol Hannam^*,2, Katherine MacKenzie^*, Olivier Jobin-Robitaille, Julie Guzzo, Jacques Côté, Brenda Andrews, David B. Haniford^* and Christopher J. Brandl^*,3

^* Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A5C1, Canada, Laval University Cancer Research Center, Hotel-Dieu de Quebec (CHUQ), Quebec City, Quebec G1R-2J6, Canada and Department of Medical Genetics and Microbiology and the Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada

³ Corresponding author: Department of Biochemistry, University of Western Ontario, London, ON N6A5C1, Canada.
E-mail: cbrandl{at}uwo.ca

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
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 SRR_3413-3415AAA included temperature sensitivity and reduced growth in media containing 6% ethanol or calcofluor white or depleted of phosphate. These alleles resulted in a twofold or greater 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.

The Saccharomyces cerevisiae Spt-Ada-Gcn5-Acetyltransferase (SAGA) 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 (GRANT et al. 1998b; SALEH et al. 1998) 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 (MCMAHON et al. 1998; BOUCHARD et al. 2001; DELEU et al. 2001; ARD et al. 2002; KULESZA et al. 2002). Its deletion results in defects in cell cycle progression and early embryonic lethality (HERCEG et al. 2001).

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 (SMITH et al. 1998; CLARKE et al. 1999). 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 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 FRAP-ATM-TRRAP (FAT) 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; although 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.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
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 was 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 BstBI 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.

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 at http://www.genetics.org/supplemental/ 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, 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 TRA1_SB. 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.

Tandem affinity purification (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–SacI fragment into this molecule after PCR using oligonucleotides 4149-4 and 4168-1 and pFA6a as the template (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–SacI fragment.

TAP:
TAP 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 phenylmethylsulfonylfluoride, 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 x g and incubated with 600 µl of IgG-Agarose (Sigma-Aldrich Canada, Oakville, Ontario) for 2 hr. 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 with 100 units TEV protease for 2 hr. Three microliters of 1 M CaCl₂ were added to the eluent and then diluted with 3 ml of calmodulin binding buffer. The protein was incubated with 600 µl of calmodulin–sepharose (Stratagene, La Jolla, CA) for 1 hr, washed with 30 ml of calmodulin binding buffer, and protein eluted.

ß-Galactosidase assays:
HIS4-lacZ cells grown in minimal media to an A₆₀₀ of 1.5 were pelleted, washed in LacZ buffer, and concentrated approximately fivefold. ß-Galactosidase was determined using o-nitrophenol-ß-D-galactosidase as substrate, standardizing to cell density (AUSUBEL et al. 1998). Analysis of PHO5-lacZ, INO1-lacZ, and ADH2-lacZ was performed similarly except that overnight cultures were washed three times in water and then grown to an A₆₀₀ 1.5 in YPD depleted of phosphate (HAN et al. 1988)or depleted of inositol or in minimal media containing 3% ethanol (v/v) and 0.1% glucose.

Gal4 affinity chromatography:
Amino acid residues 764–882 of Gal4 were expressed as a GST fusion in the vector pGEX1. One liter of Escherichia coli cells was induced with 0.6 mM isopropyl ß-D-thiogalactopyranoside for 6 hr at 37°. 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 hr, and broken by sonication. Cleared extract was incubated on ice with 1 ml glutathione sepharose 4B (GE Healthcare, Waukesha, WI) and 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, rotated at 4° for 60 min, and washed with lysis buffer, and bound protein was eluted with 10 mM reduced glutathione. Thirty microliters of the eluent were separated on a 10% SDS–PAGE gel, transferred to PVDF membrane, and blotted with anti-HA antibody.

Genetic analyses—identification of an intragenic suppressor of tra1-SRR₃₄₁₃:
Approximately 2 x 10⁶ 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 were recovered (HOFFMAN and WINSTON 1987). The SalI–SacI fragment was recloned into TRA1_SB 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 A_600nm = 2.0 at 30°. 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 (Mogene, LC, St. Louis). 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 were log transformed and averages obtained. Genes displaying an expression change >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 and 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 were visualized using MapleTree (http://rana.lbl.gov/EisenSoftware.htm). Gene ontology terms associated with gene clusters were determined using FunSpec (ROBINSON et al. 2002).

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 A_600nm 2.0. To equalize amounts of input DNA, cell extract was reverse crosslinked 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, Piscataway, NJ), transferred to clean tubes, and rotated overnight with 1 µl of antibody (anti-H3, ab1791, Abcam, Cambridge, MA; anti-AcH3, Lys9, 07-352, Upstate Biotechnology). 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 (1035–1322). PCR conditions were 5 min at 95°, followed by 25 cycles of 30 sec at 95°, 52°, and 72°, and then 5 min at 72°. Assays of acetylated histone H4 were performed with 250-ml cultures grown to A_600nm 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 (ab1760, Abcam). 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 and transferred to PVDF membranes (Roche Applied Science, Indianapolis) using a wet transfer system in 48 mM Tris, 40 mM glycine, 0.0375% SDS, and 20% methanol for 1 hr at 100 V. Anti-Flag antibody (M2, Sigma-Aldrich Canada ) 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, Madison, WI) used at a ratio of 1:10,000 was detected using a 20% solution of Immobilon Western (Millipore). Densitometric scanning of films was performed using AlphaImager 3400 software (Alpha Innotech, San Leandro, CA).

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 and then YPD. Ten microliters were spotted onto leucine-depleted plates containing 5-fluoroorotic acid (5-FOA). Values reported are the percentage 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 was performed (generation zero). Ten-milliliter saturated cultures were grown from these colonies at 30° or 34° and DNA was prepared (AUSUBEL et al. 1998). Each culture was also diluted 1:1000 into YPD and grown again to saturation, and then the process was 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:
Eighteen milligrams of whole-cell extract containing HA-Ada2p and Tra1_SB 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 sulfate (0.2%), rotated at 4° for 30 min, cleared by centrifugation at 147,000 x 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. Aliquots (20 µl) of 200-µl fractions were separated by SDS–PAGE and analyzed by Western blotting with anti-HA antibody. Twenty-five milligrams of whole-cell extract containing TAP-Flag-Yng2 and Tra1_SB or Tra1-SRR₃₄₁₃ were fractionated and blotted using anti-Flag antibody.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
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-FOA. Phenotypes examined included mating efficiency; temperature sensitivity (37°); growth on media depleted for inositol; amino acids and phosphate; fermentation of galactose and raffinose; and sensitivity to hydroxyurea, methylmethanesulfonate (MMS), ultraviolet irradiation, -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 do 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 2 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.

The phenotypes of strains containing the mutagenized tra1 alleles are shown in Table 3. Of the 19 alleles with targeted mutations in the PI3K domain, 8 did not support viability. Two of the 8, 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 3–ß6 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- (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-, which is found within ß7 and conserved in many of the lipid kinases (WALKER et al. 1999). FRK₃₅₄₄ and RDE₃₆₆₈ align with 6 and 9, respectively, and are uniquely conserved in the Tra1/TRRAP molecules.

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 37°, 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 with 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 does deletion of ada2 (not shown), and like ada2 they 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 34°, the tra1-SRR₃₄₁₃, tra1-P₃₄₀₈A, and tra1-S₃₄₆₃A alleles resulted in a reduced growth rate in rich media relative to the TRA1_SB allele. Upon heat shock the mutant strains showed an immediate growth arrest but did recover; however, 4 hr after the shift to 37°, growth stopped. To ensure that the effects of these mutations were not due to expression on a plasmid or the background N₃₅₈₀A mutation from the BamHI site within TRA1_SB, 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.

View larger version (71K): [in this window] [in a new window] [Download PPT slide]   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 10-fold serial dilutions plated onto YP media containing 2% glucose and grown at 30° and 37° or at 30° in YPD containing 3% ethanol or 5 µg/ml calcofluor white (bottom). 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 37°. The indicated tra1 mutant strains or the background wild-type strain (TRA1SB) were grown to saturation and diluted into YPD media at 34° (time 0). Cells were grown for 4 hr with the absorbance at 600 nm determined on hourly intervals. One-half of the culture volume was removed and shifted to 37° and absorbance determined for cultures at both temperatures. Note the scale differences for the graphs. The arrow indicates 30 min 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 30° and 37° or at 30° in YPD containing 5 µg/ml calcofluor white.

To evaluate whether the growth phenotypes might result from reduced expression of the altered forms of Tra1, the levels of Flag_3x-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 Tra1_SB, although 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 was compared to that found in a strain containing Tra1_SB (Figure 2C and 2D). The elution profiles for Tra1-SRR₃₄₁₃ varied little with Tra1_SB, suggesting that it associates in the appropriate high molecular mass complexes.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   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. Twenty-five micrograms of extract were separated by SDS–PAGE (5%) and Western blotted with anti-Flag antibody. (Top) Western blot of the 450-kDa band corresponding to Tra1; (bottom) a portion of an equivalent gel stained with Coomassie Brilliant Blue to verify protein concentrations. (B) tra1-SRR3413 associates with Spt7 and Yng2. Top section: Crude extracts were prepared from strains KY320 expressing wild-type Tra1 (–ve), TAP-Flag-Tra1SB, or TAP-Flag-Tra1-SRR3413 and with Flag-Yng2. One hundred fifty micrograms of crude extract were separated by electrophoresis on 5% SDS–PAGE and visualized by Western blotting using anti-Flag antibody. Copurification: 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 (top) or Yng2 (middle). The presence of Spt7 (bottom) was detected using anti-Spt7 antibody (provided by 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 and 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. Twenty-five milligrams 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.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   FIGURE 3.— Expression of ß-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 (spt70), FY1370 (gcn50), CY1514 (tra1-S3463A), CY1507 (tra1-P3408A), and CY1531 (tra1-SRR3413). ß-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 30°. Activity was compared to that in the appropriate wild-type strains—FY630 for spt7, FY86 for gcn5, and CY1524 for the tra1 mutant strains—and is 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° and 35° for strains containing TRA1SB and tra1-SRR3413.

View larger version (73K): [in this window] [in a new window] [Download PPT slide]   FIGURE 4.— Hierarchical cluster analysis of tra1-SRR3413 microarray expression data. Microarray analyses were performed for yeast strain CY1531 containing tra1-SRR3413 as the sole copy of Tra1 grown in YPD at 30° 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.

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 Tra1_SB to associate with the Gal4 activation domain (Gal4_AD). Tandem- affinity-purified Tra1_SB and Tra1-SRR₃₄₁₃, prepared from strains containing HA-tagged Ada2, were chromatographed on GST-Gal4_AD columns. Association of SAGA with Gal4 was detected by Western blotting for HA-Ada2 (Figure 5A). SAGA containing Tra1-SRR₃₄₁₃ bound Gal4_AD to the same extent as SAGA-containing Tra1_SB (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 2233–2836 (BROWN et al. 2001). 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 Tra1_SB (compare lanes 2 and 3, top; input DNAs are shown in the bottom).

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   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-Gal4AD 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 Gal4AD 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. PCR products from the input DNA used for analysis are shown in the bottom. A serial dilution of the sample for Tra1-SRR3413 is shown in lanes 3–5. Lane 6 is a no-template control.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   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 (yng20), QY204 (YNG2), BY4916 (vid210), and BY4741 (VID21) were grown to A600 = 1.5 in YPD and ChIP analysis was 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 (spt70), BY4282 (ada20), 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.

View larger version (49K): [in this window] [in a new window] [Download PPT slide]   FIGURE 7.— TRA1-SRR3413 results in a generation-dependent reduction in telomere length. (A) Plasmid linearization assay. Yeast strains CY1524 (TRA1SB), CY1531 (tra1-SRR3413), CY1507 (tra1-P3408A), and CY1514 (tra1-S3463A) were transformed with pVL106 (supplied by V. Lundblad). Leu+ Ura+ transformants were grown sequentially in media lacking uracil and then in YPD, washed, and spotted onto plates containing 5-FOA and lacking leucine. The number of 5-FOA-resistant Leu+ colonies was counted after 5 days of growth at 30° 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 five 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) 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 bp) did not differ significantly (left) at either growth temperature. After 50 and 100 generations at 30° (right) the average TRF length for the tra1-SRR3413 strain was reduced by 125 and 140 bp, respectively.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   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 30°. (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 four times in water, and diluted 1:12 into low-phosphate media. Samples were taken at the indicated time points and ß-galactosidase activity was determined.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
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. On the basis of 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- 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, Salt Lake City; 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, was slightly resistant to overexpression of VP16, although to a much lesser extent than the ada genes, and was partially sensitive to hydroxyurea, characteristic of defects in DNA replication. This latter 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 and reduced growth in low-phosphate media and in media containing 6% ethanol. The latter result has also been noted for deletions of ada2 and on the basis of 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 vs. 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 ada20 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-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 11 genes whose deletion results in shortened telomeres (ASKREE et al. 2004) have decreased expression of 1.5-fold 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 genomewide screen of ASKREE et al. (2004) or 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, results in both telomere shortening and ethanol sensitivity similar to that in 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 (vacuole import and degradation) was identified in a screen for defects in sorting of carboxypeptidase Y to the vacuole (BONANGELINO et al. 2002), is sensitive to ethanol (FUJITA et al. 2006), and interacts with Vac8, which is required for several aspects of vacuolar function (TANG et al. 2006).

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- structure. P₃₄₀₈, in fact, aligns with K₈₃₃, which in PI3K- interacts with the -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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
We thank Brian Shilton, Catherine Bateman, David Litchfield, and Greg Gloor for their comments on the manuscript; 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 Joe Martens and Melissa Bradford for assistance with the chromatin immunoprecipitations. This work was supported by Canadian Institutes of Health Research grant to C.J.B. S.M.T.H. is a holder of a Natural Sciences and Engineering Research Council studentship. C.H. was supported by an Ontario Graduate Scholarship in Science and Technology scholarship. A.I.M. is a holder of a Western Graduate Research Scholarship.

	FOOTNOTES

 
Microarray data from this article have been submitted to the GEO database at NCBI under accession no. GSE6847.

¹ These authors contributed equally to this work.

² Present address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.

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