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• W2088377890 abstract "Sgt1p is a conserved, essential protein required for kinetochore assembly in both yeast and animal cells. Sgt1p has homology to both TPR and p23 domains, sequences often found in proteins that interact with and regulate the molecular chaperone, Hsp90. The presence of these domains and the recent findings that Sgt1p interacts with Hsp90 has led to the speculation that Sgt1p and Hsp90 form a co-chaperone complex. To test this possibility, we have used purified recombinant proteins to characterize the in vitro interactions between yeast Sgt1p and Hsp82p (an Hsp90 homologue in yeast). We show that Sgt1p interacts directly with Hsp82p via its p23 homology region in a nucleotide-dependent manner. However, Sgt1p binding does not alter the enzymatic activity of Hsp82p, suggesting that it is distinct from other co-chaperones. We find that Sgt1p can form a ternary chaperone complex with Hsp82p and Sti1p, a well characterized Hsp90 co-chaperone. Sgt1p interacts with its binding partner Skp1p through its TPR domains and links Skp1p to the core Hsp82p-Sti1p co-chaperone complex. The multidomain nature of Sgt1p and its ability to bridge the interaction between Skp1p and Hsp82p argue that Sgt1p acts as a “client adaptor” recruiting specific clients to Hsp82p co-chaperone complexes. Sgt1p is a conserved, essential protein required for kinetochore assembly in both yeast and animal cells. Sgt1p has homology to both TPR and p23 domains, sequences often found in proteins that interact with and regulate the molecular chaperone, Hsp90. The presence of these domains and the recent findings that Sgt1p interacts with Hsp90 has led to the speculation that Sgt1p and Hsp90 form a co-chaperone complex. To test this possibility, we have used purified recombinant proteins to characterize the in vitro interactions between yeast Sgt1p and Hsp82p (an Hsp90 homologue in yeast). We show that Sgt1p interacts directly with Hsp82p via its p23 homology region in a nucleotide-dependent manner. However, Sgt1p binding does not alter the enzymatic activity of Hsp82p, suggesting that it is distinct from other co-chaperones. We find that Sgt1p can form a ternary chaperone complex with Hsp82p and Sti1p, a well characterized Hsp90 co-chaperone. Sgt1p interacts with its binding partner Skp1p through its TPR domains and links Skp1p to the core Hsp82p-Sti1p co-chaperone complex. The multidomain nature of Sgt1p and its ability to bridge the interaction between Skp1p and Hsp82p argue that Sgt1p acts as a “client adaptor” recruiting specific clients to Hsp82p co-chaperone complexes. Hsp90 is a highly conserved molecular chaperone that has been linked to maintaining the activity of a number of cellular proteins involved in signal transduction and cell division. This role has been linked to the normal cycle of protein activation and inactivation associated with the highly dynamic pathways that control cell division (1Whitesell L. Lindquist S.L. Nat. Rev. Cancer. 2005; 5: 761-772Crossref PubMed Scopus (1975) Google Scholar). In addition, Hsp90 has been proposed to play a more general role in “buffering” the proteome against the genetic changes associated with the rapid accumulation of mutations found in cancers or in slow accumulation of changes that contribute to gene evolution (2Sangster T.A. Lindquist S. Queitsch C. BioEssays. 2004; 26: 348-362Crossref PubMed Scopus (195) Google Scholar). How Hsp90 is targeted to its substrates, or clients, remains a major unresolved question. A large group of Hps90-associated proteins have been proposed to assist Hsp90 in client recognition or in the transition of client to its final active state. One class of Hsp90-associated proteins includes “co-chaperones,” proteins that interact with Hsp90 and frequently are found to modulate its ATPase activity. Co-chaperones typically interact with Hsp90 through two conserved domains: (i) a tetracopeptide repeat (TPR) 2The abbreviations used are: TPR, tetracopeptide repeat; CBF3, centromere binding factor-3; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate). domain or an Hsp20/α-crystallin domain, also known as a p23-domain, after the founding member of the family (3Lamb J.R. Tugendreich S. Hieter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (552) Google Scholar, 4Blatch G.L. Lassle M. BioEssays. 1999; 21: 932-939Crossref PubMed Scopus (963) Google Scholar). SGT1 encodes a protein that has both a putative TPR and p23 homology domains and was originally identified in the budding yeast Saccharomyces cerevisiae as a high copy suppressor of skp1-4 (5Kitagawa K. Skowyra D. Elledge S.J. Harper J.W. Hieter P. Mol Cell. 1999; 4: 21-33Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), a temperature-sensitive allele of SKP1. The relevance of this genetic interaction lies in the fact that Skp1p is a Sgt1p-associated protein. The Skp1-4p mutant fails to interact with Sgt1p, and this failure compromises the assembly of the budding yeast centromere-DNA binding complex, CBF3 (6Rodrigo-Brenni M.C. Thomas S. Bouck D.C. Kaplan K.B. Mol. Biol. Cell. 2004; 15: 3366-3378Crossref PubMed Scopus (37) Google Scholar, 7Bansal P.K. Abdulle R. Kitagawa K. Mol. Cell. Biol. 2004; 24: 8069-8079Crossref PubMed Scopus (66) Google Scholar). A major advance in the understanding of Sgt1p function came from evidence found in multiple systems for an interaction between Sgt1p and Hsp90 (7Bansal P.K. Abdulle R. Kitagawa K. Mol. Cell. Biol. 2004; 24: 8069-8079Crossref PubMed Scopus (66) Google Scholar, 8Hubert D.A. Tornero P. Belkhadir Y. Krishna P. Takahashi A. Shirasu K. Dangl J.L. EMBO J. 2003; 22: 5679-5689Crossref PubMed Scopus (321) Google Scholar, 9Takahashi A. Casais C. Ichimura K. Shirasu K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11777-11782Crossref PubMed Scopus (393) Google Scholar, 10Lee Y.T. Jacob J. Michowski W. Nowotny M. Kuznicki J. Chazin W.J. J. Biol. Chem. 2004; 279: 16511-16517Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 11Lingelbach L.B. Kaplan K.B. Mol. Cell. Biol. 2004; 24: 8938-8950Crossref PubMed Scopus (60) Google Scholar). Although the biochemical details remain unclear, it has been proposed that Sgt1p may link Hsp90 to Skp1p and the core CBF3 subunit, Ctf13p, thus allowing Ctf13p activation and the assembly of the CBF3 complex (6Rodrigo-Brenni M.C. Thomas S. Bouck D.C. Kaplan K.B. Mol. Biol. Cell. 2004; 15: 3366-3378Crossref PubMed Scopus (37) Google Scholar). Consistent with this possibility, CBF3 assembly in rabbit reticulocyte lysates is sensitive to inhibition of Hsp90 (7Bansal P.K. Abdulle R. Kitagawa K. Mol. Cell. Biol. 2004; 24: 8069-8079Crossref PubMed Scopus (66) Google Scholar, 12Stemmann O. Neidig A. Kocher T. Wilm M. Lechner J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8585-8590Crossref PubMed Scopus (56) Google Scholar). Although Skp1p has also been implicated in function of the E3 ubiquitin ligase SCF and a vacuolar [H+] ATPase, the importance of Sgt1p in these complexes is less clear (13Feldman R.M.R. Correll C.C. Kaplan K.B. Deshaies R.J. Cell. 1997; 91: 221-230Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar, 14Li F.N. Johnston M. EMBO J. 1997; 16: 5629-5638Crossref PubMed Scopus (182) Google Scholar, 15Verma R. Feldman R.M. Deshaies R.J. Mol. Biol. Cell. 1997; 8: 1427-1437Crossref PubMed Scopus (138) Google Scholar, 16Skowyra D. Craig K.L. Tyers M. Elledge S.J. Harper J.W. Cell. 1997; 91: 209-219Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar, 17Seol J.H. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (207) Google Scholar). So while it may be that Sgt1p and Skp1p function together in some contexts, it is likely that Sgt1p is important for a broad set of cellular functions, including regulation of protein kinase A signaling through control of adenylyl cyclase function and in disease resistance in plants (18Schadick K. Fourcade H.M. Boumenot P. Seitz J.J. Morrell J.L. Chang L. Gould K.L. Partridge J.F. Allshire R.C. Kitagawa K. Hieter P. Hoffman C.S. Eukaryot. Cell. 2002; 1: 558-567Crossref PubMed Scopus (32) Google Scholar, 19Dubacq C. Guerois R. Courbeyrette R. Kitagawa K. Mann C. Eukaryot. Cell. 2002; 1: 568-582Crossref PubMed Scopus (88) Google Scholar, 20Azevedo C. Sadanandom A. Kitagawa K. Freialdenhoven A. Shirasu K. Schulze-Lefert P. Science. 2002; 295: 2073-2076Crossref PubMed Scopus (486) Google Scholar, 21Austin M.J. Muskett P. Kahn K. Feys B.J. Jones J.D. Parker J.E. Science. 2002; 295: 2077-2080Crossref PubMed Scopus (348) Google Scholar, 22Muskett P. Parker J. Microbes Infect. 2003; 5: 969-976Crossref PubMed Scopus (59) Google Scholar, 23Liu Y. Burch-Smith T. Schiff M. Feng S. Dinesh-Kumar S.P. J. Biol. Chem. 2004; 279: 2101-2108Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 24Bieri S. Mauch S. Shen Q.H. Peart J. Devoto A. Casais C. Ceron F. Schulze S. Steinbiss H.H. Shirasu K. Schulze-Lefert P. Plant Cell. 2004; 16: 3480-3495Crossref PubMed Scopus (216) Google Scholar, 25Holt III B.F. Belkhadir Y. Dangl J.L. Science. 2005; 309: 929-932Crossref PubMed Scopus (200) Google Scholar). Recent work in human tumor cells further argues that essential mitotic functions of Sgt1p overlap with those of Hsp90 in these cells (26Niikura Y. Ohta S. Vandenbeldt K.J. Abdulle R. McEwen B.F. Kitagawa K. Oncogene. 2006; 25: 4133-4146Crossref PubMed Scopus (56) Google Scholar). Interpreting these genetic findings requires some basic understanding of the biochemical role of Sgt1p in the context of Hsp90 function. Recent reports have shown that Sgt1p co-immunoprecipitates with the molecular chaperone Hsp90 in yeast, plants, and humans (7Bansal P.K. Abdulle R. Kitagawa K. Mol. Cell. Biol. 2004; 24: 8069-8079Crossref PubMed Scopus (66) Google Scholar, 9Takahashi A. Casais C. Ichimura K. Shirasu K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11777-11782Crossref PubMed Scopus (393) Google Scholar, 10Lee Y.T. Jacob J. Michowski W. Nowotny M. Kuznicki J. Chazin W.J. J. Biol. Chem. 2004; 279: 16511-16517Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 11Lingelbach L.B. Kaplan K.B. Mol. Cell. Biol. 2004; 24: 8938-8950Crossref PubMed Scopus (60) Google Scholar). Alone, this observation does not distinguish between Sgt1p being an Hsp90 client or co-chaperone. Co-precipitation data shows only a small fraction of total Sgt1p in a complex with Hsp90 and does not address whether additional factors are required to mediate the Sgt1p-Hsp90 interaction (7Bansal P.K. Abdulle R. Kitagawa K. Mol. Cell. Biol. 2004; 24: 8069-8079Crossref PubMed Scopus (66) Google Scholar, 11Lingelbach L.B. Kaplan K.B. Mol. Cell. Biol. 2004; 24: 8938-8950Crossref PubMed Scopus (60) Google Scholar). Furthermore, work with known co-chaperones has suggested that they may bind with much higher affinity than seen for Sgt1p and Hsp90 in extracts; the yeast co-chaperones Sti1p and Cpr6p have dissociation constants that suggest co-chaperone-Hsp90 complexes should predominate in vivo (27Prodromou C. Siligardi G. O'Brien R. Woolfson D.N. Regan L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; 18: 754-762Crossref PubMed Scopus (355) Google Scholar). One possible interpretation is that Sgt1p forms a transient interaction with Hsp90, one more reminiscent of the essential Hsp90-targeting subunit, Cdc37p (28Pearl L.H. Curr. Opin. Genet. Dev. 2005; 15: 55-61Crossref PubMed Scopus (202) Google Scholar). To explore the relationship between Sgt1p and Hsp90 in more detail, we have used purified recombinant proteins to reconstitute Hsp90-Sgt1p complexes in vitro. We have found that the p23 homology domain of Sgt1p is required for the direct interaction between Sgt1p and Hsp90. Sgt1p binds preferentially to non-ATP bound forms of Hsp90 and does not alter the intrinsic ATPase activity of the chaperone. Interestingly, Sgt1p can form at least two distinct ternary complexes; in the first, Hsp90 interacts directly with Sgt1p and Sti1p and in the second, Sgt1p interacts with Skp1p through its TPR domain and Hsp90 through its p23 homology domain. Importantly, we demonstrate that Sgt1p can recruit Skp1p to the core co-chaperone complex containing Hps90 and Sti1p. Together, these findings argue that Sgt1p functions as a client adaptor, linking Hsp90 to a specific set of clients. Plasmids and Cloning—Construction of His-Sgt1p for baculoviral expression has been previously described (6Rodrigo-Brenni M.C. Thomas S. Bouck D.C. Kaplan K.B. Mol. Biol. Cell. 2004; 15: 3366-3378Crossref PubMed Scopus (37) Google Scholar) as has His-Skp1p (29Russell I.D. Grancell A.S. Sorger P.K. J. Cell Biol. 1999; 145: 933-950Crossref PubMed Scopus (84) Google Scholar). Yeast HSP82, STI1, and CPR6 were PCR-amplified with the addition of appropriate cloning sites from yeast genomic DNA and cloned into a modified pFASTBAC™ vector (Invitrogen), pFBNHis10HA (6Rodrigo-Brenni M.C. Thomas S. Bouck D.C. Kaplan K.B. Mol. Biol. Cell. 2004; 15: 3366-3378Crossref PubMed Scopus (37) Google Scholar), or pMIT-77 (pFASTBACGST followed by PreScission Protease cleavage site; details provided upon request) to generate His-Hsp82p, GST-Hsp82p, GST-Sti1p, or GST-Cpr6. Inserts were confirmed by DNA sequencing. p23ET, a plasmid for bacterial expression of Histagged yeast Sba1, was a kind gift of Avrom Caplan. Site-directed mutagenesis using PCR was performed to generate mutant versions of Sgt1p and Hsp82p. Coding sequences were confirmed by DNA sequencing. Details of cloning techniques or primers used are available upon request. Protein Expression and Purification—His-Sba1p was expressed and purified from bacterial lysates as previously described (30Fang Y. Fliss A.E. Rao J. Caplan A.J. Mol. Cell. Biol. 1998; 18: 3727-3734Crossref PubMed Scopus (133) Google Scholar). All other proteins used in this paper were expressed and purified using the baculoviral expression system. High Five™ insect cells (Invitrogen) were infected with the appropriate virus for 48 h, chilled on ice 15 min, then washed in phosphate-buffered saline. Cells were lysed on ice 15 min using 1% Triton buffer (10 mm HEPES, pH 8.0, 150 mm NaCl, 50 mm β-glycerophosphate, 0.1 mm EDTA, 1% Triton X-100, 1 mm dithiothreitol, 10% glycerol) plus protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 mm N-tosyl-l-phenylalanine chloromethyl ketone, and 10 μg/ml leupeptin, pepstatin, and chymostatin) then centrifuged at 4 °C for 15 min at 21,000 × g. The soluble fraction was removed, aliquoted, flash frozen, and stored at -80 °C for subsequent purification. To purify GST- or His-tagged fusion proteins, 100 μl of glutathione-Sepharose 4B beads (GE Healthcare, Piscataway, NJ) or 100 μl of nickel-nitrilotriacetic acid beads (Qiagen, Valencia CA), as appropriate, were added to soluble baculoviral extract containing the desired fusion protein diluted 1:10 in 1% Triton buffer and rocked at 4 °C for 2 h. Generally, extract containing 40 μg of fusion protein was used, although the protocol could be scaled up or down without altering purity or percent yield. After rocking, beads were washed three times in 500 μl of 1% Triton buffer, followed by three washes in 500 μl of SHB-Tris (25 mm Tris, pH 8.0, 150 mm KCl, 0.05% Triton X-100, 10% glycerol, 1 mm dithiothreitol, with protease inhibitors as described for 1% Triton buffer). GST fusion protein to be used as bait for binding assays was stored as purified at 4 °C for up to 1 week. To produce cleaved Sti1p or Cpr6p, GST fusion protein-bound beads were further washed three times in PreScission protease buffer followed by addition of 2 units of PreScission protease and overnight cleavage at 4 °C as described by the manufacturer (GE Healthcare). Eluted protein was aliquoted and flash frozen. His fusion proteins bound to nickel-nitrilotriacetic acid beads were washed three times in five bead volumes of SHB-Tris plus 50 mm imidazole, and protein was then eluted three times in one bead volume of SHB-Tris plus 250 mm imidazole. Eluted protein was combined, aliquoted, and flash frozen. Contaminating chaperones were released from Sti1p and Sgt1p protein fusions by including a final wash step with SHB-Tris buffer containing 5 mm ATP for 30 min at 4 °C, followed by three washes in SHB-Tris buffer with no nucleotide. Although this step reduced co-purifying proteins, it had no effect on the binding assays. Because the ATPase assays described below require large amounts of highly concentrated protein, the above protein purification protocol was scaled up, and eluted proteins were concentrated using Microcon YM-30 centrifugal filter devices (Millipore, Billerica, MA). Protein Binding Assays—For the quantitative binding assays shown in Fig. 1 (A and B) proteins to be assayed were quantified using Bradford reagent (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. 0.025, 0.050, or 0.100 nmol of Sgt1p, Sti1p, or Cpr6p was added to 0.100 nmol of GSTp or GST-Hsp82p bound to glutathione beads in 200 μl of SHB-Tris and processed as described below. For the other binding reactions shown, 10 μl of GST, GST-Hsp82p, or GST-Skp1p (∼2 μg of protein), bound to glutathione-Sepharose 4B beads (Amersham Biosciences) and purified as described above, was added to 200 μl of SHB-Tris followed by addition of ∼2 μg of additional protein(s) to be assayed. Binding reactions were rocked at room temperature 1 h and then centrifuged at 1000 × g. Depleted supernatants were removed, and proteins were concentrated after adding 20% w/v trichloroacetic acid. Beads were washed three times in 200 μl of cold SHB-Tris. Bound and unbound fractions were resolved by SDS-PAGE and Coomassie-stained. For GST-Skp1p binding experiments, PreScission Protease was used (as described above) to cleave the fusion protein prior to SDS-PAGE because GST-Skp1p and His-Sgt1p co-migrate. To quantify bound Sgt1p, Sti1p, and Cpr6 bound to GST-Hsp82p (Fig. 1B), the intensity of the band (or doublet of bands for Sgt1p) was determined from the Coomassie-stained gel using ImageQuaNT (GE Healthcare), converted to micrograms of bound using the molecular weight ladder as an internal control, then converted to nanomoles of bound using the molecular weight of the protein. Nucleotide Dependence of Binding—Binding reactions of Sgt1p to GST-Hsp82p with or without various nucleotides were conducted as above with the following modifications. SHB-Tris binding buffer was modified via addition of 6 mm MgOAc and 5 mm ADP, ATP, AMP-PNP (Sigma), or ATPγS (Roche Applied Science) as indicated. Geldanamycin (a kind gift of the Drug Synthesis and Chemistry Branch, Developmental Therapeutics, Division of Cancer Treatment and Diagnosis, NCI, National Institutes of Health) was diluted from a 10 mg/ml (17.8 mm) stock in Me2SO to a final concentration of 60 μm. This concentration has previously been shown to completely inhibit the ATPase activity of purified Hsp90 (31McLaughlin S.H. Smith H.W. Jackson S.E. J. Mol. Biol. 2002; 315: 787-798Crossref PubMed Scopus (214) Google Scholar, 32Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (625) Google Scholar). GST-Hsp82 and indicated nucleotide or geldanamycin were mixed on ice and then preincubated at 30 °C for 30 min followed by the addition of potential binding partner to be assayed, and assays were completed as described above. Reactions were prepared on ice, then GST-Hsp82, and indicated amounts of nucleotide or geldanamycin were preincubated at 30 °C for 30 min, potential binding partner to be assayed was added, and assays were completed as described above. For Sba1p binding (Fig. 1D), binding was performed as described (33Weikl T. Abelmann K. Buchner J. J. Mol. Biol. 1999; 293: 685-691Crossref PubMed Scopus (86) Google Scholar), but no cross-linking reagent was used. ATPase Assays—Pyruvate kinase/lactate dehydrogenase-coupled ATPase assays were conducted essentially as described (32Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (625) Google Scholar, 34Ali J.A. Jackson A.P. Howells A.J. Maxwell A. Biochemistry. 1993; 32: 2717-2724Crossref PubMed Scopus (310) Google Scholar), except that buffer SHB-Tris (25 mm Tris, pH 8.0, 150 mm KCl, 0.05% Triton X-100, 10% glycerol) was modified with 13 mm MgOAc and 5 mm ATP. Reactions were carried out at 37 °C in 200 μl of reaction volume with 0.4 nmol of Hsp82p and/or 1.2 nmol of co-chaperone (Sgt1p, Sti1p, Cpr6p, and Sba1p) unless otherwise noted. Addition of 60 μm geldanamycin completely inhibited the ATPase activity of our His-Hsp82p preparation (data not shown), ruling out the possibility of contaminating ATPases. Sgt1p Directly Interacts with Yeast Hsp90—To characterize Sgt1p-Hsp90 complexes in more detail, we expressed and purified yeast Sgt1p and Hsp82p using baculoviral expression in insect cells (see “Experimental Procedures”). To serve as positive controls and to allow comparison of Sgt1p to known HSP90 co-chaperones, the well characterized co-chaperones Cpr6p, Sti1p, and Sba1p were also purified as either GST fusions, or multihistidine fusions (supplemental Fig. S1). Sti1p is the yeast homolog of human HOP (35Nicolet C.M. Craig E.A. Mol. Cell. Biol. 1989; 9: 3638-3646Crossref PubMed Scopus (206) Google Scholar) and is thought to mediate transfer of client proteins from Hsp70p to Hsp90 (36Smith D.F. Sullivan W.P. Marion T.N. Zaitsu K. Madden B. McCormick D.J. Toft D.O. Mol. Cell. Biol. 1993; 13: 869-876Crossref PubMed Scopus (247) Google Scholar, 37Hernandez M.P. Sullivan W.P. Toft D.O. J. Biol. Chem. 2002; 277: 38294-38304Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Cpr6p is a yeast cyclophilin-like molecule (38Duina A.A. Chang H.C. Marsh J.A. Lindquist S. Gaber R.F. Science. 1996; 274: 1713-1715Crossref PubMed Scopus (186) Google Scholar), and Sba1p is the homolog of human p23 (30Fang Y. Fliss A.E. Rao J. Caplan A.J. Mol. Cell. Biol. 1998; 18: 3727-3734Crossref PubMed Scopus (133) Google Scholar). Both Cpr6p and Sba1p are believed to act at a late step of Hsp90 client activation (39Wegele H. Muller L. Buchner J. Rev. Physiol. Biochem. Pharmacol. 2004; 151: 1-44Crossref PubMed Scopus (518) Google Scholar). Previous efforts have co-purified only small amounts of Sgt1p associated with Hsp90 under conditions where the presence of other potential bridging proteins could not be ruled out (7Bansal P.K. Abdulle R. Kitagawa K. Mol. Cell. Biol. 2004; 24: 8069-8079Crossref PubMed Scopus (66) Google Scholar, 11Lingelbach L.B. Kaplan K.B. Mol. Cell. Biol. 2004; 24: 8938-8950Crossref PubMed Scopus (60) Google Scholar). To test if the interaction between Sgt1p and Hps90p is direct, we purified recombinant, His-tagged versions of yeast Sgt1p (His-Sgt1p) and a GST fusion of the yeast Hsp90 protein, Hsp82p (GST-Hsp82). When added at equimolar ratios, His-Sgt1p specifically interacted with GST-Hsp82, but not GST, bound to glutathione-Sepharose beads (Fig. 1A). His-Sgt1p purified from insect cells migrates as a doublet due to protein phosphorylation (see arrows, Fig. 1A) 3B. A. Macher and K. B. Kaplan, unpublished observations. ; interestingly, both forms of His-Sgt1p bind equally well to GST-Hsp82 (compare with load gel, supplemental Fig. S1). To assess the relative affinity of Sgt1p for GST-Hsp82p, we compared its ability to bind with two known co-chaperones, Sti1p and Cpr6p (Fig. 1A). We assayed the binding of Sgt1p, Sti1p, and Cpr6p when added at 1:4, 1:2, or 1:1 molar ratios to GST-Hsp82p. Taking into account the two forms of Sgt1p, quantitative analysis of digitized gel images suggest Sgt1p binds to GST-Hsp82p with similar affinity to other known co-chaperones (Fig. 1B). Multiple modes of interactions link Hsp90 to its co-chaperone and clients. Some interactions are sensitive to conformational changes in Hsp90 mediated by nucleotide binding and hydrolysis. For example, it has been reported that Sba1p/p23 interacts preferentially with the ATP-bound form of Hsp90; Sti1p/Hop1 interacts preferentially with the ADP-bound form of Hsp90; and Cpr6p binds to Hsp90 independent of nucleotide (27Prodromou C. Siligardi G. O'Brien R. Woolfson D.N. Regan L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; 18: 754-762Crossref PubMed Scopus (355) Google Scholar, 30Fang Y. Fliss A.E. Rao J. Caplan A.J. Mol. Cell. Biol. 1998; 18: 3727-3734Crossref PubMed Scopus (133) Google Scholar, 40Sullivan W. Stensgard B. Caucutt G. Bartha B. McMahon N. Alnemri E.S. Litwack G. Toft D. J. Biol. Chem. 1997; 272: 8007-8012Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 41Johnson J.L. Toft D.O. Mol. Endocrinol. 1995; 9: 670-678Crossref PubMed Scopus (211) Google Scholar). To test the effect of nucleotide on the interaction between Sgt1p and Hsp90, we incubated purified His-Sgt1p with GST-Hsp82p in the absence of nucleotide, in the presence of ADP, ATP as well as the non-hydrolyzable ATP analogues, ATP-γ-S and AMP-PNP (see “Experimental Procedures”). His-Sgt1p bound to GST-Hsp82p in the absence of nucleotide, suggesting that binding does not require Hsp82p to be bound to nucleotide. Consistent with this possibility, geldanamycin, a highly specific Hsp90 inhibitor that displaces ATP from the active site (42Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1332) Google Scholar, 43Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1121) Google Scholar, 44Stebbins C.E. Russo A.A. Schneider C. Rosen N. Hartl F.U. Pavletich N.P. Cell. 1997; 89: 239-250Abstract Full Text Full Text PDF PubMed Scopus (1249) Google Scholar), has no effect on the interaction of His-Sgt1p with GST-Hsp82p (Fig. 1C). Although incubation with ADP resulted in similar amounts of His-Sgt1p bound to GST-Hsp82p compared with the no nucleotide condition, ATP, and to a greater extent ATPγS and AMP-PNP had an inhibitory effect on the binding of His-Sgt1p to GST-Hsp82p. As anticipated from the participation of Sti1p in the HSP90 “intermediate” complex and Cpr6p in the HSP90 “mature complex” (27Prodromou C. Siligardi G. O'Brien R. Woolfson D.N. Regan L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; 18: 754-762Crossref PubMed Scopus (355) Google Scholar), the binding of Cpr6p to GST-Hsp82p increased when GST-Hsp82 was ATP-bound, whereas Sti1p failed to interact with GST-Hsp82p in the presence of nonhydrolyzable ATP analogues (Fig. 1C). Although a weaker interaction, Sba1p bound to Hsp82p best in the presence of non-hydrolyzable nucleotide analogues and was further enhanced with the addition of sodium molybdate under conditions previously described (Fig. 1D) (33Weikl T. Abelmann K. Buchner J. J. Mol. Biol. 1999; 293: 685-691Crossref PubMed Scopus (86) Google Scholar). Skp1p did not bind directly to GST-Hsp82p under any of the nucleotide conditions tested (Fig. 1B). These controls argue that the preparation of GST-Hsp82p bound to glutathione Sepharose is behaving as predicted, allowing us to conclude that, like Sti1p, Sgt1p has a lower affinity for Hsp90 in its ATP-bound conformation. Sgt1p Does Not Alter the Enzymatic Activity of Hsp82p—The binding of Sti1p and Sgt1p to the ATP-bound form of Hsp82p raised the possibility that these two proteins have a similar effect on the enzymatic activity of Hsp82p. Previous reports have shown that Sti1p/HOP blocks access of ATP to Hsp90 (27Prodromou C. Siligardi G. O'Brien R. Woolfson D.N. Regan L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; 18: 754-762Crossref PubMed Scopus (355) Google Scholar). We therefore asked if Sgt1p could alter the hydrolysis rate of Hsp82p. For these experiments, we used a His-tagged version of yeast Hsp82p (see “Experimental Procedures”). Because the enzymatic activity of yeast His-Hsp82p produced in insect cells using baculovirus had not been previously determined, we first established conditions where reliable kinetics could be measured. Using conditions similar to previously published work, we observed His-Hsp82p to have a hydrolysis rate of 0.285 mol/min/mol at 37 °C (Table 1), a rate comparable although slightly lower than previously reported (0.4 mol/min/mol (32Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (625) Google Scholar)). Adding 60 μm of the ATP binding site competitor geldanamycin completely blocked ATP hydrolysis in our assay, arguing that there are no nonspecific ATPases present in the His-Hsp82p preparation. Importantly, ATP hydrolysis was almost completely inhibited when we added 3-fold molar excess of Sti1p or Sba1p; in contrast, addition of 3-fold excess of Cpr6p had no effect on ATP hydrolysis by His-Hsp82p (Table 1). These results are consistent with previously published reports and further validate the integrity of the purified system (27Prodromou C. Siligardi G. O'Brien R. Woolfson D.N. Regan L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; 18: 754-762Crossref PubMed Scopus (355) Google Scholar, 45Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (121) Google Scholar). To determine if Sgt1p could alter the rate of ATP hydrolysis, it was added at a 3:1 ratio to Hsp82p. We observed no affect on ATP hydrolysis by His-Hsp82p (Table 1). An increase in the molar ratio of Sgt1p:His-Hsp82p also had no effect on the rate of ATP hydrolysis (>10-fold Sgt1p:His-Hsp82p; data not shown). This is consistent with the preference of Sgt1p for the non-ATP bound form of Hsp82p. We conclude that, although Sgt1p and Sti1p both bind to Hsp82p with sim" @default.
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• W2088377890 title "Sgt1p Is a Unique Co-chaperone That Acts as a Client Adaptor to Link Hsp90 to Skp1p" @default.
• W2088377890 cites W1607384859 @default.
• W2088377890 cites W1966034049 @default.
• W2088377890 cites W1967036913 @default.
• W2088377890 cites W1968592557 @default.
• W2088377890 cites W1971707631 @default.
• W2088377890 cites W1972299037 @default.
• W2088377890 cites W1975331444 @default.
• W2088377890 cites W1980119734 @default.
• W2088377890 cites W1982177408 @default.
• W2088377890 cites W1985212818 @default.
• W2088377890 cites W1989074864 @default.
• W2088377890 cites W1992690825 @default.
• W2088377890 cites W1993525477 @default.
• W2088377890 cites W1995143644 @default.
• W2088377890 cites W1995369191 @default.
• W2088377890 cites W1997173534 @default.
• W2088377890 cites W2001462925 @default.
• W2088377890 cites W2002315746 @default.
• W2088377890 cites W2004893394 @default.
• W2088377890 cites W2007996847 @default.
• W2088377890 cites W2013947447 @default.
• W2088377890 cites W2022531767 @default.
• W2088377890 cites W2028966970 @default.
• W2088377890 cites W2029663913 @default.
• W2088377890 cites W2031556797 @default.
• W2088377890 cites W2033228162 @default.
• W2088377890 cites W2038186997 @default.
• W2088377890 cites W2042042682 @default.
• W2088377890 cites W2046699174 @default.
• W2088377890 cites W2057668430 @default.
• W2088377890 cites W2059406989 @default.
• W2088377890 cites W2068435780 @default.
• W2088377890 cites W2073886258 @default.
• W2088377890 cites W2082390299 @default.
• W2088377890 cites W2087427383 @default.
• W2088377890 cites W2093444601 @default.
• W2088377890 cites W2095686344 @default.
• W2088377890 cites W2104615400 @default.
• W2088377890 cites W2104956294 @default.
• W2088377890 cites W2107466812 @default.
• W2088377890 cites W2112133866 @default.
• W2088377890 cites W2113659644 @default.
• W2088377890 cites W2116659929 @default.
• W2088377890 cites W2120316538 @default.
• W2088377890 cites W2126632528 @default.
• W2088377890 cites W2127692414 @default.
• W2088377890 cites W2131581447 @default.
• W2088377890 cites W2132387250 @default.
• W2088377890 cites W2138767066 @default.
• W2088377890 cites W2138821198 @default.
• W2088377890 cites W2139306838 @default.
• W2088377890 cites W2140937615 @default.
• W2088377890 cites W2141850284 @default.
• W2088377890 cites W2144950416 @default.
• W2088377890 cites W2144993348 @default.
• W2088377890 cites W2147056656 @default.
• W2088377890 cites W2160114316 @default.
• W2088377890 cites W2161069579 @default.
• W2088377890 cites W2165360238 @default.
• W2088377890 cites W2166252272 @default.
• W2088377890 cites W2168705084 @default.
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