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The Yeast Hsp110 Family Member, Sse1, Is an Hsp90 Cochaperone

1999; Elsevier BV; Volume: 274; Issue: 38 Linguagem: Inglês

10.1074/jbc.274.38.26654

ISSN

1083-351X

Autores

Xiaodong Liu, Kevin A. Morano, Dennis J. Thiele,

Tópico(s)

Insect and Pesticide Research

Resumo

In eukaryotes, production of the diverse repertoire of molecular chaperones during normal growth and in response to stress is governed by the heat shock transcription factor HSF. The HSC82 and HSP82 genes, encoding isoforms of the yeast Hsp90 molecular chaperone, were recently identified as targets of the HSF carboxyl-terminal activation domain (CTA), whose expression is required for cell cycle progression during prolonged heat stress conditions. In the present study, we have identified additional target genes of the HSF CTA, which include nearly all of the heat shock-inducible members of the Hsp90 chaperone complex, demonstrating coordinate regulation of these components by HSF. Heat shock induction of SSE1, encoding a member of the Hsp110 family of heat shock proteins, was also dependent on the HSF CTA. Disruption of SSE1 along with STI1, encoding an established subunit of the Hsp90 chaperone complex, resulted in a severe synthetic growth phenotype. Sse1 associated with partially purified Hsp90 complexes and deletion of the SSE1 gene rendered cells susceptible to the Hsp90 inhibitors macbecin and geldanamycin, suggesting functional interaction between Sse1 and Hsp90. Sse1 is required for function of the glucocorticoid receptor, a model substrate of the Hsp90 chaperone machinery, and Hsp90-based repression of HSF under nonstress conditions. Taken together, these data establish Sse1 as an integral new component of the Hsp90 chaperone complex in yeast. In eukaryotes, production of the diverse repertoire of molecular chaperones during normal growth and in response to stress is governed by the heat shock transcription factor HSF. The HSC82 and HSP82 genes, encoding isoforms of the yeast Hsp90 molecular chaperone, were recently identified as targets of the HSF carboxyl-terminal activation domain (CTA), whose expression is required for cell cycle progression during prolonged heat stress conditions. In the present study, we have identified additional target genes of the HSF CTA, which include nearly all of the heat shock-inducible members of the Hsp90 chaperone complex, demonstrating coordinate regulation of these components by HSF. Heat shock induction of SSE1, encoding a member of the Hsp110 family of heat shock proteins, was also dependent on the HSF CTA. Disruption of SSE1 along with STI1, encoding an established subunit of the Hsp90 chaperone complex, resulted in a severe synthetic growth phenotype. Sse1 associated with partially purified Hsp90 complexes and deletion of the SSE1 gene rendered cells susceptible to the Hsp90 inhibitors macbecin and geldanamycin, suggesting functional interaction between Sse1 and Hsp90. Sse1 is required for function of the glucocorticoid receptor, a model substrate of the Hsp90 chaperone machinery, and Hsp90-based repression of HSF under nonstress conditions. Taken together, these data establish Sse1 as an integral new component of the Hsp90 chaperone complex in yeast. heat shock factor heat shock protein heat shock element carboxyl-terminal transcriptional activation domain hemagglutinin glucocorticoid receptor polymerase chain reaction synthetic complete polyacrylamide gel electrophoresis Cells respond to a variety of environmental stresses by inducing the heat shock response, the coordinated synthesis of a set of proteins that protect the cell from damage and facilitate recovery (1Feige, U., Morimoto, R. I., Yahara, I., and Polla, B. S. (eds) (1996) Stressinducible Cellular Responses, Vol. 77, Birkhauser Verlag, BaselGoogle Scholar, 2Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Plainview, Cold Spring Harbor, NY1994Google Scholar). In eukaryotes, this response is primarily governed at the transcriptional level by the heat shock transcription factor (HSF),1 a highly conserved protein that binds to heat shock elements (HSEs), specific cis-acting sequences upstream of genes encoding heat shock proteins (Hsps). HSF is a modular protein composed of a highly conserved amino-terminal DNA binding domain followed by a series of hydrophobic heptad repeats that comprise the trimerization interface for the active factor (3Wu C. Annu. Rev. Cell Dev. Biol. 1995; 11: 441-469Crossref PubMed Scopus (982) Google Scholar). In addition, all HSF molecules possess a transcriptional activation domain in the carboxyl terminus that shows low sequence conservation. In higher eukaryotes, multiple distinct HSF isoforms exist that display differential specificity for HSE binding and respond to diverse stimuli. In yeast, however, HSF is encoded by a single essential gene, HSF1, which possesses both an amino-terminal trans-activation domain and a carboxyl-terminal trans-activation domain (CTA) (4Sorger P.K. Cell. 1990; 62: 793-805Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 5Nieto-Sotelo J. Wiederrecht G. Okuda A. Parker C.S. Cell. 1990; 62: 807-817Abstract Full Text PDF PubMed Scopus (122) Google Scholar). The two domains appear to be differentially used for target gene activation, since expression of the copper metallothionein gene CUP1 in response to heat, oxidative stress, or glucose starvation is abolished when the CTA is deleted, while expression of at least three other heat shock genes, SSA1, SSA3, and SSA4, is largely unaffected (6Tamai K.T. Liu X. Silar P. Sosinowski T. Thiele D.J. Mol. Cell. Biol. 1994; 14: 8155-8165Crossref PubMed Scopus (123) Google Scholar, 7Santoro N. Johansson N. Thiele D.J. Mol. Cell. 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Genes Dev. 1997; 11: 1775-1785Crossref PubMed Scopus (181) Google Scholar), and the p23 ortholog Sba1 (30Fang Y. Fliss A.E. Rao J. Caplan A.J. Mol. Cell. Biol. 1998; 18: 3727-3734Crossref PubMed Scopus (133) Google Scholar). The Hsp90 chaperone complex is dynamic, with distinct subunits associating at various stages of the folding/chaperoning process for different client proteins (31Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1543) Google Scholar). For example, during maturation of the glucocorticoid receptor (GR), early complexes form, which include GR, Hsp90, Hsp70, Ydj1, and p60. After hormone binding, the Hsp70/Ydj1 pair dissociate, and p60 is replaced by the cyclophilin Cyp-40, probably through competition for the single tetratricopeptide repeat binding domain in Hsp90 (32Young J.C. Obermann W.M. Hartl F.U. J. Biol. Chem. 1998; 273: 18007-18010Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 33Prodromou C. Siligardi G. O'Brien R. 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The Hsp70 superfamily of molecular chaperones is divided into three subgroups based on sequence homology: the DnaK subfamily, the GRP170/Lhs1 subfamily, and the Hsp110/Sse1 subfamily (37Craven R.A. Tyson J.R. Stirling C.J. Trends Cell Biol. 1997; 7: 277-282Abstract Full Text PDF PubMed Scopus (48) Google Scholar). All share a high level of homology within their amino termini, which includes the ATP-binding domain essential for catalytic folding activity, but diverge within the carboxyl termini, thought to be responsible for interaction with substrate proteins. The founding members of the Hsp110/Sse1 subfamily are the SSE1 and SSE2 genes from yeast, which show 76% identity with each other but only 30–40% identity with the DnaK group of Hsp70s (38Mukai H. Kuno T. Tanaka H. Hirata D. Miyakawa T. Tanaka C. Gene (Amst.). 1993; 132: 57-66Crossref PubMed Scopus (104) Google Scholar). The SSE1 and SSE2 genes were first identified as calmodulin-binding proteins (38Mukai H. Kuno T. Tanaka H. Hirata D. Miyakawa T. Tanaka C. Gene (Amst.). 1993; 132: 57-66Crossref PubMed Scopus (104) Google Scholar), and SSE1 was also isolated independently in a genetic study as MSI3, a multicopy suppressor of the heat shock-sensitive phenotype of Ras-cAMP pathway hyperactivation (39Shirayama M. Kawakami K. Matsui Y. Tanaka K. Toh-e A. Mol. Gen. Genet. 1993; 240: 323-332Crossref PubMed Scopus (42) Google Scholar). However, the true functions of Sse1 and Sse2 in yeast are currently unknown. While heat shock-induced proteins of approximately 110 kDa have long been observed in higher eukaryotes, only recently have Hsp110 members from a variety of organisms including human, plant, nematode and fission yeast been cloned and found to share substantial homology (∼40%) to the SSE1/SSE2 genes (40Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 41Storozhenko S. De Pauw P. Kushnir S. Van Montagu M. Inze D. FEBS Lett. 1996; 390: 113-118Crossref PubMed Scopus (31) Google Scholar, 42Yasuda K. Nakai A. Hatayama T. Nagata K. J. Biol. Chem. 1995; 270: 29718-29723Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 43Chung K.S. Hoe K.L. Kim K.W. Yoo H.S. Gene (Amst.). 1998; 210: 143-150Crossref PubMed Scopus (16) Google Scholar, 44Plesofsky-Vig N. Brambl R. J. Biol. Chem. 1998; 273: 11335-11341Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 45Santos B.C. Chevaile A. Kojima R. Gullans S.R. Am. J. Physiol. 1998; 274: F1054-F1061PubMed Google Scholar). Interestingly, unlike Hsp70, the Hsp110 class of chaperones can maintain heat-denatured proteins such as firefly luciferase in a folding-competent state (46Oh H.J. Chen X. Subjeck J.R. J. Biol. Chem. 1997; 272: 31636-31640Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 47Brodsky J.L. Werner E.D. Dubas M.E. Goeckeler J.L. Kruse K.B. McCracken A.A. J. Biol. Chem. 1999; 274: 3453-3460Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). In order to more precisely determine the cause of cell cycle arrest in HSF (1–583) mutant cells, we sought to identify additional gene products whose heat shock-induced expression was abrogated relative to wild type cells. We demonstrate that expression of SSE1 is dependent upon the HSF CTA. In addition, most of the heat shock-inducible components of the Hsp90 chaperone complex were found to share this requirement, suggesting tight coordinate regulation of their expression. Moreover, SSE1 displays synthetic genetic interactions with chaperone genes, is physically associated with purified Hsp90 complexes, and is required for Hsp90 activity in vivo. Together these findings identify Sse1 as a novel member of the Hsp90 chaperone complex. In-frame fusion of the triple hemagglutinin (HA) tag to the amino terminus of the SSE1 gene was accomplished by insertion of annealed oligonucleotides encoding three repeats of the HA epitope 3′ of the initiator ATG codon. The fusion gene was cloned into the HIS3-based pRS313 vector to generate pRS313-HA-SSE1. For disruption of the STI1 gene, the oligonucleotides 5′-GAGCTCTTCACTAAAGCTATTGAAGTTTCTGAAACTCCAAACCATGTTTTATATTCTAACAGGTCCGCCTGTaagcttcgtacgctgcag-3′ and 5′-TTCATGGCCCTTTGATAGGTTTCTTCTGGGGTTTCGTTACTGGTACCAGGTTGGAATCTTTGTTGGCTTGCCggccactagtggatctga-3′ were used to amplify the KanMX2 cassette from the pFA-KanMX2 plasmid (48Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2241) Google Scholar) by PCR. Uppercase letters represent sequences native to the STI1 gene. The PCR-amplified fragment contains 72 base pairs of flanking sequences (both 5′ and 3′) that are homologous to the STI1 gene. Approximately 4 μg of the PCR product was used to transform Saccharomyces cerevisiae cells, and G418-resistant colonies were recovered. The SSE1 gene was similarly disrupted by a PCR product amplified from the pFA-KanMX2 cassette using oligonucleotides 5′-GATGAGTACTCCATTTGGTTTAGATTTAGGTAACAATAACTCTGTCCTTGCCGTTGCTAGAAACAGAGGTATaagcttcgtacgctgcag-3′ and 5′-GACCTTCTGGTAATTGAACACCAGTGATCTCCCAGTTAGCGATTTGTTCTGGAGTGTTTGGTGGTAACTGTGggccactagtggatctga-3′. The resulting strains carrying the appropriate disruptions were G418-resistant and displayed the expected phenotypes as described previously (48Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2241) Google Scholar), and the mutated alleles were verified by PCR. p413GPD-rGR and pYRP-GRElacZ (a kind gift from D. McDonnell, Duke University) expressing rat glucocorticoid receptor and a lacZ reporter construct containing GR-binding elements were as described (9Morano K.A. Santoro N. Koch K.A. Thiele D.J. Mol. Cell. Biol. 1999; 19: 402-411Crossref PubMed Scopus (72) Google Scholar). YEp-STI1 was a kind gift from Elizabeth Craig (University of Wisconsin) (49Nicolet C.M. Craig E.A. Mol. Cell. Biol. 1989; 9: 3638-3646Crossref PubMed Scopus (206) Google Scholar), and YEp-SSE1 was from Akio Toh-e (University of Tokyo) (39Shirayama M. Kawakami K. Matsui Y. Tanaka K. Toh-e A. Mol. Gen. Genet. 1993; 240: 323-332Crossref PubMed Scopus (42) Google Scholar). pCM64-SSA3-lacZ is as described (7Santoro N. Johansson N. Thiele D.J. Mol. Cell. Biol. 1998; 18: 6340-6352Crossref PubMed Scopus (90) Google Scholar). S. cerevisiae strains used in this study are listed in TableI. Cells were grown in synthetic complete (SC) medium minus the indicated nutrients (SC-x) at 30 °C to midlog phase (A650 = 0.7–1.4). For growth dilution series assays, saturated liquid cultures were adjusted to approximately 1 × 106 cells/ml, serially diluted, and plated on SC-x agar plates for incubation at 30 °C or 37 °C as indicated. Sensitivity to the benzoquinoid ansamycins macbecin and geldanamycin was assayed on YPD plates made by adding the drugs in Me2SO to melted YPD agar solution to a final concentration of 35 μm. Macbecin was obtained from the Drug Synthesis and Chemistry branch of NCI, National Institutes of Health. Geldanamycin was obtained from Dr. William Pratt (University of Michigan Medical School, Ann Arbor, MI). sse1Δ HSF (1–583) and sti1Δ HSF (1–583) cells were generated from XLY24 (sse1Δ, GAL1-HSF) and XLY25 (sti1Δ, GAL1-HSF) strains, respectively. XLY24 and XLY25 were transformed with HSF-(1–583)-expressing plasmids and grown to saturation in galactose-containing medium (to allow for expression of GAL1-HSF). The spot dilution assay was carried out as described above using SC agar plates with glucose as sole carbon source to repress GAL1-HSF production, causing HSF-(1–583) to be the only source of HSF. For heat shock treatment, cells were grown at 25 °C to midlog phase, and 5 ml of cells were either kept at 25 °C (control) or at 41 °C (heat shock) for 16 min or 1 h (Fig. 6). Cells were then washed with ice-cold sterile glass-distilled water and stored at −80 °C, prior to extraction of RNA for RNA blotting experiments or proteins for immunoblotting.Table IS. cerevisiae strains used in this studyStrainsGenotype (including plasmids)SourceParental strainPS145MAT a ade2–1 trp1 can1–100 leu2–3, -112 his3–11, -15 ura3 hsfΔ∷LEU2 Ycp50-GAL1-HSFRef. 59Sorger P.K. Pelham H.R.B. Cell. 1988; 54: 855-864Abstract Full Text PDF PubMed Scopus (570) Google ScholarW303NSY-AMAT a ade2–1 trp1 can1–100 leu2–3, -112 his3–11, -15 ura3 hsfΔ∷LEU2 pRS314-HSFRef.7Santoro N. Johansson N. Thiele D.J. Mol. Cell. Biol. 1998; 18: 6340-6352Crossref PubMed Scopus (90) Google ScholarPS145NSY-BMAT a ade2–1 trp1 can1–100 leu2–3, -112 his3–11, -15 ura3 hsfΔ∷LEU2 pRS314-HSF(1–583)Ref. 7Santoro N. Johansson N. Thiele D.J. Mol. Cell. Biol. 1998; 18: 6340-6352Crossref PubMed Scopus (90) Google ScholarPS145XLY18MAT a ade2–1 trp1 can1–100 leu2–3, -112 his3–11, -15 ura3 hsfΔ∷LEU2 sse1∷KANR pRS314-HSFThis studyNSY-AXLY19MAT a ade2–1 trp1 can1–100 leu2–3, -112 his3–11, -15 ura3 hsfΔ∷LEU2 sti1∷KANR pRS314-HSFThis studyNSY-AXLY24MAT a ade2–1 trp1 can1–100 leu2–3, -112 his3–11, -15 ura3 hsfΔ∷LEU2 sse1∷KANR Ycp50-GAL1-HSFThis studyPS145XLY25MAT a ade2–1 trp1 can1–100 leu2–3, -112 his3–11, -15 ura3 hsfΔ∷LEU2 sti1∷KANR Ycp50-GAL1-HSFThis studyPS145DS10MAT a GAL2 his3–11,15 leu2–3,112 lys1 lys2 trpΔ1 ura3–52Ref. 49Nicolet C.M. Craig E.A. Mol. Cell. Biol. 1989; 9: 3638-3646Crossref PubMed Scopus (206) Google ScholarCN11MAT a GAL2 his3–11,15 leu2–3,112 lys1 lys2 trpΔ1 ura3–52 sti1∷HIS3Ref. 49Nicolet C.M. Craig E.A. Mol. Cell. Biol. 1989; 9: 3638-3646Crossref PubMed Scopus (206) Google ScholarDS10XLY35MAT a GAL2 his3–11,15 leu2–3,112 lys1 lys2 trpΔ1 ura3–52 sse1∷KANRThis studyDS10XLY36MAT a GAL2 his3–11,15 leu2–3,112 lys1 lys2 trpΔ1 ura3–52 sti1∷HIS3 sse1∷KANRThis studyDS10XLY29MAT a his4–539 ura3–52 lys2–801 SUC2 sti1∷KANRThis studyDTY123XLY34MAT a ura3–52 lys2–801 ade2–101 trp1–63 his3–200 leu2–1 hsc82∷URA3 hsp82∷GAL1-HSP82∷LEU2 sse1∷KANR p-T-GPD-HSP82 (TRP+) pRS313-HA-SSE1This study5CG2 (60Kimura Y. Matsumoto S. Yahara I. Mol. Gen. Genet. 1994; 242: 517-527Crossref PubMed Scopus (42) Google Scholar)XLY30MAT a ura3–52 lys2–801 ade2–101 trp1–63 his3–200 leu2–1 hsc82∷URA3 hsp82∷GAL1-HSP82∷LEU2 sse1∷KANR p-T-HIS6-GPD-HSP82 (TRP+) pRS313-HA-SSE1This study5CG2 Open table in a new tab Five ml of cells were grown in SC-Met-Cys to early log phase, harvested, and resuspended in 1 ml of SC-Met-Cys. The cultures were incubated at 23 or 41 °C for 15 min with constant agitation and then transferred to prewarmed Eppendorf microcentrifuge tubes each containing 158 μCi of Tran35S-labelTM (ICN, CA). The tubes were further incubated for another 15 min at 23 or 41 °C. Cells were than washed once with ice-cold sterile glass-distilled H2O, and the pellet was frozen at −80 °C. Cell extracts were prepared as described below, and scintillation counting to determine total incorporation of label was carried out on a Beckman LS6500 scintillation counter (Beckman Instruments). Equivalent cpm of extracts were fractionated with SDS-PAGE (8%) using a Hoeffer gel electrophoresis system (SE400 model) at 4 °C. The gel was rinsed in glass-distilled H2O for 15 min, incubated in AutoFluorTM (National Diagnostics, Inc.) for 30 min at room temperature, vacuum-dried, and exposed to Kodak BioMax film at −80 °C. A Molecular Dynamics PhosphorImager system was used for quantitative assessment of labeled protein levels. Cell pellets containing between 5 and 15 A650 units of cells were resuspended in HEGN buffer (20 mm HEPES, pH 7.9, 1 mm EDTA, 10% glycerol, 50 mm NaCl) or SDS harvest buffer (0.5% SDS, 10 mm Tris-HCl at pH 7.4, 1 mm EDTA), with the following protease inhibitors added: 1 mm Pefabloc™ (Roche Molecular Biochemicals), 8 μg/ml aprotinin, 4 μg/ml pepstatin, 2 μg/ml leupeptin. After the addition of an equal volume of acid-washed sterile glass beads (450–600 μm), the samples were lysed by rapid agitation at 4 °C in a microtube mixer (model MT-360; Tomy Tech, Inc.) at top speed for 2 min for four cycles with 2-min intervals on ice between each round. Extracts were clarified by microcentrifugation at 2500 × g for 6 min at 4 °C, and protein concentration was determined by Bradford assay. The extracts were fractionated by SDS-PAGE (8%), and electroblotted to nitrocellulose. Standard immunoblot procedures were used, followed by protein detection using the ECL chemiluminescence system according to the manufacturer's instructions (Amersham Pharmacia Biotech). Monoclonal antibody recognizing phosphoglycerate kinase was purchased from Molecular Probes, Inc. (Eugene, OR), and the following antibodies were generous gifts from the following sources: Anti-Ssa3/Ssa4 from Elizabeth Craig (University of Wisconsin, Madison, WI); anti-hsp90 from Susan Lindquist (University of Chicago); anti-Ydj1 from Avrom Caplan (Mt. Sinai School of Medicine); anti-Sti1 from David Toft (Mayo Graduate School); anti-Cpr7 from Richard Gaber (Northwestern University); anti-Kar2 from James Gaut (University of Michigan Medical School). Lysates were prepared from cells (XLY34 and XLY30) grown to midlog phase in SC-His medium at 30 °C in LyB buffer (25Chang H.C. Lindquist S. J. Biol. Chem. 1994; 269: 24983-24988Abstract Full Text PDF PubMed Google Scholar), using similar procedures as described above for immunoblot analysis. Isolation of His6-Hsp82 and associated polypeptides with nickel affinity chromatography was carried out exactly as described (25Chang H.C. Lindquist S. J. Biol. Chem. 1994; 269: 24983-24988Abstract Full Text PDF PubMed Google Scholar). Five ml of cells transformed with p413GPD-rGR and pYRP-GRElacZ were grown to midlog phase in SC-Ura-His medium at 30 °C, and 5 μl of absolute ethanol (control) or 10 mm DOC (deoxycorticosterone; Sigma) dissolved in ethanol were added to the cell culture (final concentration of 10 μm). The cells were further incubated with shaking at 30 °C for 1 h, harvested, washed once with sterile glass-distilled H2O, and resuspended in 700 μl of Z buffer (100 mm sodium phosphate, pH 7.0, 10 mm KCl, 1 mm MgSO4, 50 mm β-mercaptoethanol). 50 μl of chloroform and 50 μl of 0.1% SDS were added, and the cell suspension was vortex-mixed at top speed for 10 s followed by incubation at 30 °C for 5 min. 200 μl of 4 mg/ml ONPG in Z buffer was added to each sample and incubated at 30 °C for 3–7 min. 350 μl of 1 mNa2CO3 was added to stop the reactions. After clarification by centrifugation, the supernatant was diluted, and A420 was measured within the linear response range. Specific β-galactosidase activity was calculated as follows: (dilution factor × 1000 ×A420)/(A650 × (volume of cells in ml) × (time of incubation in min)). Data shown are averages of three independent experiments with associated S.D. Previous work has demonstrated that expression of the two yeast Hsp90 genes HSC82 and HSP82 is markedly reduced in a strain expressing the HSF truncation mutant HSF (1–583), which displays a temperature-sensitive growth phenotype characterized by arrest in the G2/M phase of the cell cycle (9Morano K.A. Santoro N. Koch K.A. Thiele D.J. Mol. Cell. Biol. 1999; 19: 402-411Crossref PubMed Scopus (72) Google Scholar). Two points, however, suggest that loss of Hsp90 is not the sole determinant of this arrest. First, restoration of Hsp90 levels through introduction of the HSP82 gene on a high copy vector does not lead to growth rates at 37 °C comparable with wild type cells. Second, specific depletion of Hsp90 from cells wild type for HSF results in a mixed population of cells arrested in both the G1/S and G2/M phases of the cell cycle at 37 °C, in contrast to the fairly uniform G2/M arrest observed in HSF (1–583). Therefore, we reasoned that there must be additional genes under the specific control of the HSF CTA that are required for growth at higher temperatures and that are poorly expressed in HSF-(1–583) cells. To begin to identify these additional targets, SDS-PAGE protein profiles of 35S pulse-labeled HSF and HSF (1–583) cell extracts was conducted under control and heat shock conditions (Fig.1 A). Heat shock induces the synthesis of a number of known Hsps, whose SDS-PAGE migration patterns have been well documented (50Nicolet C.M. Craig E.A. Methods Enzymol. 1991; 194: 710-717Crossref PubMed Scopus (34) Google Scholar). We were able to faithfully recapitulate these patterns, demonstrating robust heat shock induction of a number of proteins, including Hsp104 and the Hsp70 isoforms Ssa1, Ssa3, and Ssa4. Three proteins whose heat shock inducibility was diminished in HSF (1–583) cells compared with HSF wild type were tentatively identified (Fig. 1 A, bands a–c) by comparison with published heat shock protein profiles (39Shirayama M. Kawakami K.

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