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The Function of the Yeast Molecular Chaperone Sse1 Is Mechanistically Distinct from the Closely Related Hsp70 Family

2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês

10.1074/jbc.m313739200

ISSN

1083-351X

Autores

Lance Shaner, Amy Trott, Jennifer L. Goeckeler-Fried, Jeffrey L. Brodsky, Kevin A. Morano,

Tópico(s)

Protein Structure and Dynamics

Resumo

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Δ and lethality of sse1Δsse2Δ cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70. The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Δ and lethality of sse1Δsse2Δ cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70. Cells respond to heat shock by induction of a specific set of genes that allow them to cope with and recover from stress. Many of these heat shock proteins (HSPs) 1The abbreviations used are: HSP, heat shock protein; PBD, peptide binding domain; HA, hemagglutinin. 1The abbreviations used are: HSP, heat shock protein; PBD, peptide binding domain; HA, hemagglutinin. function as molecular chaperones, binding unfolded proteins and preventing aggregation or facilitating their refolding (1Craig E.A. Baxter B.K. Becker J. Halladay J. Ziegelhoffer T. Morimoto R.I. Tissieres A. Georgopolos C. The Biology of Heat Shock Proteins and Molecular Chaperones. 26. Cold Spring Harbor Laboratory Press, New York1994: 31-52Google Scholar). One of the most well studied classes of chaperones is the Hsp70 family. Saccharomyces cerevisiae possesses 14 Hsp70 homologs, distributed in the cytosol, mitochondria, and endoplasmic reticulum (2Craven R.A. Egerton M. Stirling C.J. EMBO J. 1996; 15: 2640-2650Crossref PubMed Scopus (141) Google Scholar). Hsp70s promote folding of nascent polypeptides, facilitate translocation of proteins across membranes, and protect the cell from protein-denaturing stress. All Hsp70s share a common domain architecture consisting of an N-terminal ATPase domain and a C-terminal peptide binding domain (PBD). The PBD is responsible for binding unfolded peptide substrates and is regulated by the nucleotide binding status of the ATPase domain (3Liberek K. Skowyra D. Zylicz M. Johnson C. Georgopoulos C. J. Biol. Chem. 1991; 266: 14491-14496Abstract Full Text PDF PubMed Google Scholar). The PBD of the Escherichia coli Hsp70 homolog DnaK is composed of a series of eight β-strands (β-subdomain) that form a peptide binding cleft, followed by a series of α-helices that form the "lid" (α-subdomain) thought to regulate entry and exit of the substrate (4Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1048) Google Scholar). In E. coli, the DnaK folding cycle is regulated by DnaJ and GrpE, which stimulate ATPase activity and act as a nucleotide exchange factor, respectively (5Schroder H. Langer T. Hartl F.U. Bukau B. EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (497) Google Scholar). DnaJ homologs are found in all cellular compartments of eukaryotes while GrpE homologs have only been found in mitochondria (6Bolliger L. Deloche O. Glick B.S. Georgopoulos C. Jeno P. Kronidou N. Horst M. Morishima N. Schatz G. EMBO J. 1994; 13: 1998-2006Crossref PubMed Scopus (141) Google Scholar).The eukaryotic cytoplasmic Hsp110/Sse1 and ER-resident Grp170/Lhs1 proteins are divergent members of the Hsp70 chaperone family (7Easton D.P. Kaneko Y. Subjeck J.R. Cell Stress Chaperones. 2000; 5: 276-290Crossref PubMed Scopus (243) Google Scholar, 8Craven R.A. Tyson J.R. Stirling C.J. Trends Cell Biol. 1997; 7: 277-282Abstract Full Text PDF PubMed Scopus (48) Google Scholar). Hsp110 and Grp170 proteins are significantly larger than Hsp70, possessing a loop region between the β- and α-subdomains, as well as an extended tail. Hsp110 has been shown to bind denatured proteins in vitro but is unable to actively refold them and instead acts as a "holdase," maintaining substrate polypeptides in a folding-competent state (9Oh H.J. Chen X. Subjeck J.R. J. Biol. Chem. 1997; 272: 31636-31640Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 10Oh H.J. Easton D. Murawski M. Kaneko Y. Subjeck J.R. J. Biol. Chem. 1999; 274: 15712-15718Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). No endogenous substrates have been identified for the Hsp110s to date; however overexpression of Hsp110 increases thermotolerance in Chinese hamster ovary (CHO) cells (9Oh H.J. Chen X. Subjeck J.R. J. Biol. Chem. 1997; 272: 31636-31640Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). More recently overexpression of the Hsp110 family member Hsp105α was shown to suppress protein aggregation and subsequent apoptosis in COS-7 cells expressing the polyglutamine tract-containing truncated androgen receptor (tAR), suggesting a potential role for Hsp110 in the prevention of protein plaque-associated pathologies (11Ishihara K. Yamagishi N. Saito Y. Adachi H. Kobayashi Y. Sobue G. Ohtsuka K. Hatayama T. J. Biol. Chem. 2003; 278: 25143-25150Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar).SSE1 and its close paralog SSE2 are the S. cerevisiae members of the Hsp110 subfamily. SSE1 was identified biochemically as a calmodulin-binding protein and genetically as a high copy suppressor of the hyperactive PKA mutant ira1Δ (12Mukai H. Kuno T. Tanaka H. Hirata D. Miyakawa T. Tanaka C. Gene (Amst.). 1993; 132: 57-66Crossref PubMed Scopus (101) Google Scholar, 13Shirayama M. Kawakami K. Matsui Y. Tanaka K. Toh-e A. Mol. Gen. Genet. 1993; 240: 323-332Crossref PubMed Scopus (42) Google Scholar). Yeast lacking SSE1 are slow growing and slightly temperature sensitive. Deletion of SSE2 results in no observable growth defects, and deletion of both SSE genes was reported to be equivalent to deletion of SSE1 alone (12Mukai H. Kuno T. Tanaka H. Hirata D. Miyakawa T. Tanaka C. Gene (Amst.). 1993; 132: 57-66Crossref PubMed Scopus (101) Google Scholar, 13Shirayama M. Kawakami K. Matsui Y. Tanaka K. Toh-e A. Mol. Gen. Genet. 1993; 240: 323-332Crossref PubMed Scopus (42) Google Scholar). Mammalian Hsp110 does not complement the deletion of SSE1 in yeast (data not shown), 2J. Subjeck, personal communication. 2J. Subjeck, personal communication. suggesting distinct cellular substrate or cofactor specificities. In vitro, Sse1 binds denatured luciferase and accelerates its refolding upon addition of yeast cytosol (14Brodsky 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 (231) Google Scholar, 15Goeckeler J.L. Stephens A. Lee P. Caplan A.J. Brodsky J.L. Mol. Biol. Cell. 2002; 13: 2760-2770Crossref PubMed Scopus (69) Google Scholar). Sse1 also participates in Hsp90 signal transduction; its absence leads to the derepression of the yeast heat shock transcription factor, Hsf1, and loss of glucocorticoid receptor (GR) signaling, two established roles for Hsp90 (16Liu X.D. Morano K.A. Thiele D.J. J. Biol. Chem. 1999; 274: 26654-26660Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Recently, SSE1 was isolated as a high copy suppressor of a mutated form of the cytosolic Hsp40, Ydj1, possibly because of the participation of both proteins in Hsp90-dependent functions (15Goeckeler J.L. Stephens A. Lee P. Caplan A.J. Brodsky J.L. Mol. Biol. Cell. 2002; 13: 2760-2770Crossref PubMed Scopus (69) Google Scholar).The apparent lack of folding activity for Sse1/Hsp110 calls into question the role of nucleotide cycling in the ATPase domain. Several Hsp70 ATPase domain residues have been demonstrated to be vital for ATP binding and hydrolysis in multiple mammalian and yeast homologs (17Wei J. Gaut J.R. Hendershot L.M. J. Biol. Chem. 1995; 270: 26677-26682Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 18McClellan A.J. Brodsky J.L. Genetics. 2000; 156: 501-512Crossref PubMed Google Scholar, 19McClellan A.J. Endres J.B. Vogel J.P. Palazzi D. Rose M.D. Brodsky J.L. Mol. Biol. Cell. 1998; 9: 3533-3545Crossref PubMed Scopus (73) Google Scholar, 20Flaherty K.M. Wilbanks S.M. DeLuca-Flaherty C. McKay D.B. J. Biol. Chem. 1994; 269: 12899-12907Abstract Full Text PDF PubMed Google Scholar, 21Wilbanks S.M. DeLuca-Flaherty C. McKay D.B. J. Biol. Chem. 1994; 269: 12893-12898Abstract Full Text PDF PubMed Google Scholar). The Sse1 ATPase domain is 53% similar to Hsc70, whereas Ssa1 and Hsc70 share 87% similarity; many of the substitutions between the chaperones are within the ATP binding pocket. For example, a glutamate residue at position 175 in Hsc70 is replaced by aspartate in Sse1, whereas aspartate 206 of Hsc70, proposed to be a proton acceptor during the hydrolysis cycle, is replaced by threonine in Sse1 and glutamine in CHO Hsp110 (20Flaherty K.M. Wilbanks S.M. DeLuca-Flaherty C. McKay D.B. J. Biol. Chem. 1994; 269: 12899-12907Abstract Full Text PDF PubMed Google Scholar, 22Lee-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 (128) Google Scholar). Recombinant mammalian Hsp110 expressed in E. coli was shown to bind ATP only in the absence of the PBD, and furthermore neither Hsp110 nor Sse1 have been reported to hydrolyze ATP (10Oh H.J. Easton D. Murawski M. Kaneko Y. Subjeck J.R. J. Biol. Chem. 1999; 274: 15712-15718Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar).To learn more about this abundant yet ill defined chaperone, we have undertaken a molecular genetic analysis of Sse1 in baker's yeast. Residues known to be involved in ATP binding and hydrolysis in the Hsp70s were mutagenized in Sse1 and assayed for complementation of sse1Δ phenotypes. Mutants that were able to bind ATP affinity resin also complemented sse1Δ, whereas mutants that did not bind were non-functional in vivo. Deletion analysis of the C terminus revealed that Sse1 can tolerate removal of 44 residues with no effect on function, suggesting that this region is not involved in chaperone-chaperone communication as it is in Hsp70. Mutations abrogating the β-strand or α-helical subdomains of the PBD substantially destabilized the protein. Remarkably, the ATPase domain and PBD were found to function and interact when expressed in trans, indicating direct interdomain communication between the two domains. These results demonstrate significant differences in the cellular function of Sse1 versus the classical Hsp70s.EXPERIMENTAL PROCEDURESStrains and Plasmids—All strains are isogenic derivatives of S. cerevisiae strain W303 (MATa ade2-1 trp1 can1-100 leu2-3,-112 his3-11,-15 ura3). The sse1Δ::kanR strain was previously described (16Liu X.D. Morano K.A. Thiele D.J. J. Biol. Chem. 1999; 274: 26654-26660Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Construction details of strain sse1Δ::kanRsse2Δ::LEU2 will be described elsewhere. 3A. Trott, L. Shaner, and K. A. Morano, manuscript in preparation. Synthetic complete (SC) media lacking the appropriate nutrient for plasmid selection was purchased from BIO101 (Carlsbad, CA). Standard yeast propagation and transformation procedures were employed (23Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, New York1994Google Scholar). The SSE1 open reading frame was amplified by PCR from pYEp24SSE1 and cloned into p414TEF, p416TEF, and p426GPD yeast expression vectors as listed in Table I (16Liu X.D. Morano K.A. Thiele D.J. J. Biol. Chem. 1999; 274: 26654-26660Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 24Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1571) Google Scholar). SpeI (5′) and XhoI (3′) restriction sites were incorporated by PCR into all constructs to facilitate subcloning. N-terminal FLAG and HA epitope-tagged SSE1 clones were described previously (16Liu X.D. Morano K.A. Thiele D.J. J. Biol. Chem. 1999; 274: 26654-26660Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). SSE1K69Q and SSE1G233D point mutants were described previously (15Goeckeler J.L. Stephens A. Lee P. Caplan A.J. Brodsky J.L. Mol. Biol. Cell. 2002; 13: 2760-2770Crossref PubMed Scopus (69) Google Scholar). SSE1D8N, SSE1D174N, SSE1D203N, and SSE1G205D point mutants and the HA-SSE1Δ394–419 deletion mutant were made by the PCR overlap extension method using primers incorporating the appropriate mutation or deletion (25Aiyar A. Xiang Y. Leis J. Methods Mol. Biol. 1996; 57: 177-191PubMed Google Scholar). ATPase domain fragments were made by PCR incorporating a stop codon after residue 393 using p416TEF HA-SSE1 or p416TEF FLAG-SSE1 as template. N-terminal peptide binding domain truncations were constructed by PCR incorporating a start codon in front of residue 394, 419, 444, or 469. C-terminal truncations were constructed by PCR, incorporating a stop codon after residue 683, 649, 590, or 505 using p416TEF HA-SSE1 as template. To facilitate subcloning, an SpeI site was added to pFLAG-MAC (Sigma) in-frame and upstream of the XhoI site by PCR, creating pFLAG-Spe. Wild-type and ATPase point mutant SSE1 alleles were then subcloned by ligation of a SpeI/XhoI fragment into similarly digested pFLAG-Spe. Sequences for all oligonucleotides used in this report are available upon request.Table IPlasmidsPlasmidRef.pYep24SSE1p414TEFSSE1This studyp414TEFSSE1D8NThis studyp414TEFSSE1K69QThis studyp414TEFSSE1D174NThis studyp414TEFSSE1D203NThis studyp414TEFSSE1G205DThis studyp414TEFSSE1G233DThis studyp416TEFSSE1This studyp416TEFSSE1D8NThis studyp416TEFSSE1K69QThis studyp416TEFSSE1D174NThis studyp416TEFSSE1D203NThis studyp416TEFSSE1G205DThis studyp416TEFSSE1G233DThis studyp426GPDSSE1This studyp426GPDSSE1D8NThis studyp426GPDSSE1K69QThis studyp426GPDSSE1D174NThis studyp426GPDSSE1D203NThis studyp426GPDSSE1G205DThis studyp426GPDSSE1G233DThis studyp414TEFSSE1-CTD-(394-693)This studyp414TEFSSE1-CTD-(419-693)This studyp414TEFSSE1-CTD-(444-693)This studyp414TEFSSE1-CTD-(469-693)This studyp416TEF HA-SSE1This studyp416TEF HA-SSE1-ATPase-(1-393)This studyp416TEF FLAG-SSE1This studyp416TEF FLAG-SSE1-ATPase-(1-393)This studyp416TEF HA-SSE1-(1-683)This studyp416TEF HA-SSE1-(1-649)This studyp416TEF HA-SSE1-(1-590)This studyp416TEF HA-SSE1-(1-505)This studyp416TEF HA-SSE1Δ394-419This studypCM64-SSA3-lacZpFLAG-MACSigmapFLAG-SpeThis studypFLAG-Spe-SSA1This studypFLAG-Spe-SSE1This studypFLAG-Spe-SSE1D8NThis studypFLAG-Spe-SSE1K69QThis studypFLAG-Spe-SSE1D174NThis studypFLAG-Spe-SSE1D203NThis studypFLAG-Spe-SSE1G205DThis studypFLAG-Spe-SSE1G233DThis study Open table in a new tab Immunoblot Analysis—Cells were pelleted and resuspended in TEGN buffer (20 mm Tris, pH 7.9, 0.5 mm EDTA, 10% glycerol, 50 mm NaCl) + protease inhibitors (aprotinin, 2 μg/ml; pepstatin A, 2 μg/ml; leupeptin, 1 μg/ml; phenylmethylsulfonyl fluoride, 1 mm; chymostatin, 2 μg/ml) (Roche Diagnostics) followed by addition of glass beads. The samples were then agitated using a vortex mixer in four rounds of 1.5 min each followed by 1.5 min on ice. The lysate was then cleared by centrifugation at 4,500 × g. Proteins were separated by SDS-PAGE and transferred to nitrocellulose for immunodetection. 12CA5 monoclonal antibody recognizing the HA epitope was purchased from Roche Diagnostics. Anti-phosphoglycerate kinase antibody was purchased from Molecular Probes, Inc. (Eugene, OR). M2 anti-FLAG antibody was purchased from Sigma. Anti-Sse1 polyclonal antibody was previously described (15Goeckeler J.L. Stephens A. Lee P. Caplan A.J. Brodsky J.L. Mol. Biol. Cell. 2002; 13: 2760-2770Crossref PubMed Scopus (69) Google Scholar). Proteins were detected by addition of appropriate horseradish peroxidase-conjugated goat anti-rabbit or goat-anti-mouse secondary antibodies (Bio-Rad) followed by ECL chemiluminescence. An Alpha Innotech Corp. (San Leandro, CA) FluorChem 8800 Imaging System and AlphaEaseFC software were used for image capture and analysis.ATP Binding Assay—E. coli strain BL21 (B F–, ompT, hsdS (r –B, m –B), gal, dcm) was transformed with pFLAG constructs for overexpression of wild-type and mutant proteins. Strains were grown overnight in LB with ampicillin (100 μg/ml) at 37 °C, subcultured to an OD600 of 0.1 in 150 ml, and grown to an OD600 of 0.5, at which time cells were induced by addition of isopropyl-β-d-thiogalactoside to 0.5 mm and shifted to 30 °C for 4 h. Cells were then pelleted by centrifugation and resuspended in 15 ml of TEGN and protease inhibitors (aprotinin, 2 μg/ml; pepstatin A, 2 μg/ml; leupeptin, 1 μg/ml; phenylmethylsulfonyl fluoride, 1 mm; chymostatin, 2 μg/ml). The cell suspension was passed through a French pressure cell twice at 1,200 psi (SLM Instruments, Urbana, IL). Unbroken cells and debris were pelleted by centrifugation at 7,500 × g for 10 min. The medium speed supernatant was then centrifuged at 100,000 × g for 30 min, glycerol was added to the soluble fraction to a final concentration of 25%, and the samples were aliquoted and stored at –80 °C.ATP-agarose binding was performed essentially as described (10Oh H.J. Easton D. Murawski M. Kaneko Y. Subjeck J.R. J. Biol. Chem. 1999; 274: 15712-15718Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Briefly, ATP-agarose (Sigma, A2767) and nucleotide-free agarose were stored as a 1:1 slurry in buffer B (20 mm Tris-HCl, 20 mm NaCl, 0.1 mm EDTA, 2 mm dithiothreitol). A total of 1 mg of total protein was added to 200 μl of ATP-agarose or nucleotide-free agarose slurry and incubated at 4 °C overnight with mixing. The beads were then washed six times with Buffer B, eluted with SDS-PAGE sample buffer, and proteins were separated by SDS-PAGE and transferred to nitrocellulose for immunoblot analysis.Immunoprecipitation Analysis—sse1Δ cells harboring p416TEF FLAG-SSE1 ATPase-(1–393) and p414TEF SSE1-CTD-(394–693) or p416TEF and p414TEFSSE1-CTD-(394–693) were grown in SC-Ura-Trp to mid-log phase at 30 °C. Total soluble protein was then obtained by glass bead lysis. Protein extracts were incubated with 40 μl of anti-FLAG M2 resin for 2 h at 4 °C. Resin was then pelleted and washed five times with TEGN buffer followed by elution with SDS-PAGE sample buffer at 65 °C for 10 min. Proteins were then separated by SDS-PAGE followed by immunoblot analysis.Cycloheximide Chase Analysis—sse1Δ cells harboring either p416TEF HA-SSE1 or p416TEF HA-SSE1Δ394–419) were grown in SC-Ura medium to mid-log phase at which time cycloheximide was added to a final concentration of 200 μg/ml (t = 0). Samples were taken at 0, 2, 4, and 6 h, pelleted, and stored at –80 °C. Soluble protein was obtained by glass bead lysis and separated by SDS-PAGE followed by transfer to nitrocellulose and immunoblotting. Quantitation was performed using real time 16-bit CCD image acquisition and AlphaEaseFC image analysis software. Half-life was determined by normalization of the Sse1 signal to PGK at each time point and fitting the data to a single exponential curve. The experiment was performed twice with similar results.β-Galactosidase Assay—Liquid β-galactosidase assays for Hsf1 activity were performed exactly as described (16Liu X.D. Morano K.A. Thiele D.J. J. Biol. Chem. 1999; 274: 26654-26660Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).RESULTSSse1 ATPase Domain Mutants Are Functional—Previous research has shown that the Hsp70 ATPase activity is essential for function, both in vivo and in vitro. Extensive biochemical and genetic studies have been carried out using point mutations in the ATPase domain to determine which residues are important in the ATP binding and hydrolysis cycle of Hsp70s (17Wei J. Gaut J.R. Hendershot L.M. J. Biol. Chem. 1995; 270: 26677-26682Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 18McClellan A.J. Brodsky J.L. Genetics. 2000; 156: 501-512Crossref PubMed Google Scholar, 19McClellan A.J. Endres J.B. Vogel J.P. Palazzi D. Rose M.D. Brodsky J.L. Mol. Biol. Cell. 1998; 9: 3533-3545Crossref PubMed Scopus (73) Google Scholar, 20Flaherty K.M. Wilbanks S.M. DeLuca-Flaherty C. McKay D.B. J. Biol. Chem. 1994; 269: 12899-12907Abstract Full Text PDF PubMed Google Scholar, 21Wilbanks S.M. DeLuca-Flaherty C. McKay D.B. J. Biol. Chem. 1994; 269: 12893-12898Abstract Full Text PDF PubMed Google Scholar). For example, several residues in bovine Hsc70 are important for ATP hydrolysis, and mutations caused dramatic decreases in kcat and moderate increases in Km values (20Flaherty K.M. Wilbanks S.M. DeLuca-Flaherty C. McKay D.B. J. Biol. Chem. 1994; 269: 12899-12907Abstract Full Text PDF PubMed Google Scholar, 21Wilbanks S.M. DeLuca-Flaherty C. McKay D.B. J. Biol. Chem. 1994; 269: 12893-12898Abstract Full Text PDF PubMed Google Scholar). Equivalent mutations in the yeast cytosolic Hsp70, Ssa1, and endoplasmic reticulum luminal Hsp70 Kar2/BiP, were shown genetically and biochemically to render the protein non-functional (18McClellan A.J. Brodsky J.L. Genetics. 2000; 156: 501-512Crossref PubMed Google Scholar, 19McClellan A.J. Endres J.B. Vogel J.P. Palazzi D. Rose M.D. Brodsky J.L. Mol. Biol. Cell. 1998; 9: 3533-3545Crossref PubMed Scopus (73) Google Scholar). Notably, the ATPase domain of Sse1 shares significant sequence homology with other Hsp70s (36% identity with the Ssa1 ATPase domain). In order to study the role of the N-terminal ATPase domain in Sse1 function, we mutagenized residues known to be involved in the ATP binding and hydrolysis cycle of these other Hsp70s. The residues we targeted for mutation were D8N (Asp-10 in bHsc70), K69Q (Lys-71), D174N (Glu-175), D203N (Asp-199), G205D (Gly-201), and G233D (Gly-229). It was reported previously that Hsf1-regulated genes are derepressed in sse1Δ (16Liu X.D. Morano K.A. Thiele D.J. J. Biol. Chem. 1999; 274: 26654-26660Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Because SSE1 is a known target gene of Hsf1 we decided to place wild-type and mutant SSE1 constructs under the control of the heterologous TEF1 promoter (12Mukai H. Kuno T. Tanaka H. Hirata D. Miyakawa T. Tanaka C. Gene (Amst.). 1993; 132: 57-66Crossref PubMed Scopus (101) Google Scholar, 24Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1571) Google Scholar). Immunoblotting experiments showed that expression levels of these proteins were ∼2-fold higher than that expressed from the native SSE1 promoter (data not shown).Two different complementation assays were employed to assess function of the Sse1 mutants. In the first, the mutants were assayed for the ability to rescue the lethality of an sse1Δsse2Δ strain upon loss of a Yep24SSE1 plasmid on 5-FOA, shown in Fig. 1A. It was previously reported that deletion of SSE2 in a sse1Δ strain did not show any additional phenotypes (12Mukai H. Kuno T. Tanaka H. Hirata D. Miyakawa T. Tanaka C. Gene (Amst.). 1993; 132: 57-66Crossref PubMed Scopus (101) Google Scholar). However, our laboratory recently found that SSE1 and SSE2 constitute an essential gene pair.3 Surprisingly, we found that the D8N, K69Q, D174N, D203N, and G205D mutants all allowed for the efficient loss of pYep24SSE1. G233D was the only point mutant unable to permit loss of wild-type SSE1 in the sse1Δsse2Δ strain background. A construct consisting of the C-terminal PBD lacking the ATPase domain was also unable to complement sse1Δsse2Δ (data not shown). We then assayed for the ability of the mutants to complement the temperature-sensitive phenotype of the sse1Δ mutant. Fig. 1B shows that the D8N, K69Q, D174N, and D203N mutants complemented this phenotype while the G205D and G233D mutants did not. All of the mutants are expressed at or near the levels of wild-type Sse1 under control of the TEF1 promoter (Fig. 1C).The ability of the G205D mutant to function as the sole source of SSE1 in sse1Δsse2Δ at 30 °C and the failure of this mutant to complement the temperature sensitivity of sse1Δ suggested that this was a temperature-sensitive allele. This hypothesis was tested by assaying the growth of sse1Δsse2Δ mutants harboring a plasmid expressing either wild-type SSE1 or sse1-G205D at 30 °C and 37 °C. As shown in Fig. 1D, the inability of sse1Δsse2Δ-expressing G205D to grow at 37 °C illustrates that sse1-G205D is a temperature-sensitive allele. Comparative Western blots of samples grown at 30 and 37 °C showed that the mutant protein is stably expressed in both cases, indicating that G205D renders the Sse1 protein temperature sensitive for function, but not for stability (data not shown).Sse1, Hsp90, and the cyclophilin Cpr7 function similarly to repress Hsf1 under non-stress conditions (26Duina A.A. Kalton H.M. Gaber R.F. J. Biol. Chem. 1998; 273: 18974-18978Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Using a reporter containing the lacZ gene under control of the SSA3 promoter, which contains the Hsf1-responsive heat shock element (HSE), it was shown that sse1Δ cells derepress Hsf1 under steady-state conditions (16Liu X.D. Morano K.A. Thiele D.J. J. Biol. Chem. 1999; 274: 26654-26660Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 27Santoro N. Johansson N. Thiele D.J. Mol. Cell. Biol. 1998; 18: 6340-6352Crossref PubMed Scopus (88) Google Scholar). Utilizing this reporter, we assayed the SSE1 ATPase mutants for the ability to repress expression of Hsf1 target genes under non-stress conditions (30 °C, mid-log phase). Fig. 2 illustrates the results of this experiment. The D8N, K69Q, D174, and D203N mutants were able to repress expression of the reporter to near wild-type levels. In contrast, G205D, G233D, and the PBD all exhibited β-galactosidase activity ranging from ∼2.5–3.5 times higher than wild type, equivalent to the empty vector control. Taken together, these genetic data suggest that ATP binding is required for Sse1 function in vivo.Fig. 2Repression of Hsf1 transcriptional activity under nonstress conditions by SSE1 mutant alleles.sse1Δ cells expressing the indicated mutant alleles of SSE1 expressed from p414TEF were transformed with pCM64-SSA3-lacZ (URA3). The strains were grown to mid-log phase in SC-Ura-Trp media followed by determination of β-galactosidase activity as described under "Experimental Procedures."View Large Image Figure ViewerDownload (PPT)Sse1 and Complementing ATPase Mutants Bind ATP in Vitro—Although the ATPase domain shares significant homology with other Hsp70s, Sse1 has not been reported to bind or hydrolyze ATP. Mammalian Hsp110 binds ATP only in the absence of the peptide binding domain, but has not been reported to actively hydrolyze ATP, indicating that this subfamily of chaperones might function differently than Hsp70s (10Oh H.J. Easton D. Murawski M. Kaneko Y. Subjeck J.R. J. Biol. Chem. 1999; 274: 15712-15718Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). To establish the nucleotide binding capabilities of wild-type Sse1, we incubated total yeast protein extracts with ATP immobilized on agarose followed by elution of all proteins bound to the resin. Using this technique we were unable to detect Sse1 binding to the resin, while Ssa1 bound efficiently, demonstrating the efficacy of the procedure (data not shown). To test the po

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