Sti1 Is a Novel Activator of the Ssa Proteins
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m301548200
ISSN1083-351X
AutoresHarald Wegele, Martin Haslbeck, Jochen Reinstein, Johannes Büchner,
Tópico(s)thermodynamics and calorimetric analyses
ResumoThe molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins in eukaryotes. Of specific importance in this context is a ternary multichaperone complex in which Hsp70 and Hsp90 are connected by Hop. In Saccharomyces cerevisiae two components of the complex, yeast Hsp90 (yHsp90) and Sti1, the yeast homologue of Hop, had already been identified, but it remained to be shown which of the 14 different yeast Hsp70s are part of the Sti1 complex and what were the functional consequences resulting from this interaction. With a two-hybrid approach and co-immunoprecipitations, we show here that Sti1 specifically interacts with the Ssa group of the cytosolic yeast Hsp70 proteins. Using purified components, we reconstituted the dimeric Ssa1-Sti1 complex and the ternary Ssa1-Sti1-yHsp90 complex in vitro. The dissociation constant between Sti1 and Ssa1 was determined to be 2 orders of magnitude weaker than the affinity of Sti1 for yHsp90. Surprisingly, binding of Sti1 activates the ATPase of Ssa1 by a factor of about 200, which is in contrast to the behavior of Hop in the mammalian Hsp70 system. Analysis of the underlying activation mechanism revealed that ATP hydrolysis is rate-limiting in the Ssa1 ATPase cycle and that this step is accelerated by Sti1. Thus, Sti1 is a potent novel effector for the Hsp70 ATPase. The molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins in eukaryotes. Of specific importance in this context is a ternary multichaperone complex in which Hsp70 and Hsp90 are connected by Hop. In Saccharomyces cerevisiae two components of the complex, yeast Hsp90 (yHsp90) and Sti1, the yeast homologue of Hop, had already been identified, but it remained to be shown which of the 14 different yeast Hsp70s are part of the Sti1 complex and what were the functional consequences resulting from this interaction. With a two-hybrid approach and co-immunoprecipitations, we show here that Sti1 specifically interacts with the Ssa group of the cytosolic yeast Hsp70 proteins. Using purified components, we reconstituted the dimeric Ssa1-Sti1 complex and the ternary Ssa1-Sti1-yHsp90 complex in vitro. The dissociation constant between Sti1 and Ssa1 was determined to be 2 orders of magnitude weaker than the affinity of Sti1 for yHsp90. Surprisingly, binding of Sti1 activates the ATPase of Ssa1 by a factor of about 200, which is in contrast to the behavior of Hop in the mammalian Hsp70 system. Analysis of the underlying activation mechanism revealed that ATP hydrolysis is rate-limiting in the Ssa1 ATPase cycle and that this step is accelerated by Sti1. Thus, Sti1 is a potent novel effector for the Hsp70 ATPase. Molecular chaperones are a diverse set of functionally related proteins that are involved in protein folding in the cell. An important feature in this context are dynamic interactions of various components of the chaperone machinery. Two of the most abundant chaperones found in the eukaryotic cell are Hsp70 and Hsp90. The Hsp70 and Hsp90 chaperones cooperate in the formation and maintenance of protein structures of a set of target proteins in vivo (1Smith D.F. Toft D.O. Mol. Endocrinol. 1993; 7: 4-11Crossref PubMed Scopus (57) Google Scholar, 2Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1543) Google Scholar, 3Buchner J. Trends Biochem. Sci. 1999; 24: 136-141Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 4Jolly C. Morimoto R.I. J. Natl. Cancer Inst. 2000; 92: 1564-1572Crossref PubMed Scopus (879) Google Scholar, 5Richter K. Buchner J. J. Cell. Physiol. 2001; 188: 281-290Crossref PubMed Scopus (509) Google Scholar, 6Zylicz M. King F.W. Wawrzynow A. EMBO J. 2001; 20: 4634-4638Crossref PubMed Scopus (187) Google Scholar, 7Walter S. Buchner J. Angew. Chem. 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Hop/Sti1 is a unique co-chaperone consisting mainly of three tetratricopeptide repeat (TPR) 1The abbreviations used are: TPR, tetratricopeptide repeat; HPLC, high pressure liquid chromatography; yHsp90, yeast Hsp90; hHsp90, human Hsp90. domains. TPRs are helical modules that mediate interactions between proteins (24Honore B. Leffers H. Madsen P. Rasmussen H.H. Vandekerckhove J. Celis J.E. J. Biol. Chem. 1992; 267: 8485-8491Abstract Full Text PDF PubMed Google Scholar). In the case of Hop, its TPR domains can bind to the C-terminal ends of both Hsp70 and Hsp90 (25Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar), thus providing a physical link between the Hsp70 and Hsp90 chaperone machinery (26Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 27Chen S. Smith D.F. J. Biol. Chem. 1998; 273: 35194-35200Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Despite its importance for the Hsp90 cycle, the composition and function of this complex are still ill-defined. Using purified components, it had been shown previously that yHsp90 and Sti1 form a binary complex in which Sti1 inhibits the ATPase of yHsp90 (28Prodromou 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 (356) Google Scholar). However, neither the question of whether in addition to the Ssa class (29Chang H.C. Lindquist S. J. Biol. Chem. 1994; 269: 24983-24988Abstract Full Text PDF PubMed Google Scholar) any of the nine cytosolic Hsp70 proteins in yeast is incorporated into the Sti1-yHsp90 complex nor the functional consequences of this interaction had been addressed. These nine Hsp70s can be divided into the four subfamilies: Ssa, Ssb, Sse, and Ssz (Ss denotes stress seventy-related; a, b, e, and z indicate the subfamily) (8Craig E.A. Boorstein W.R. Park H.O. Stone D.N.C.M. UCLA Symp. Mol. Cell. Biol. 1989; 96: 51-61Google Scholar). The Ssa subfamily, which is the only essential Hsp70 family in the cytosol, comprises four members (Ssa1–Ssa4). The expression of at least one of the four Ssa proteins is essential for viability (30Werner-Washburne M. Stone D.E. Craig E.A. Mol. Cell. Biol. 1987; 7: 2568-2577Crossref PubMed Scopus (294) Google Scholar). The Ssa1 gene is expressed at high levels at physiological temperature; in addition, its expression is stimulated ∼10-fold after a temperature shift to 37 °C, whereas Ssa2 is expressed at the same level at all temperatures (30Werner-Washburne M. Stone D.E. Craig E.A. Mol. Cell. Biol. 1987; 7: 2568-2577Crossref PubMed Scopus (294) Google Scholar). Ssa3 and Ssa4 are expressed at extremely low levels at physiological temperature, but after a temperature upshift, the amounts of Ssa3 and Ssa4 mRNAs increase severalfold (8Craig E.A. Boorstein W.R. Park H.O. Stone D.N.C.M. UCLA Symp. Mol. Cell. Biol. 1989; 96: 51-61Google Scholar). In contrast to the Ssa proteins, which are soluble in the cytosol, Ssb1 and Ssb2 are associated with nascent polypeptides of translating ribosomes (31Nelson R.J. Ziegelhoffer T. Nicolet C. Werner-Washburne M. Craig E.A. Cell. 1992; 71: 97-105Abstract Full Text PDF PubMed Scopus (427) Google Scholar, 32Pfund C. Lopez-Hoyo N. Ziegelhoffer T. 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Finally, Sse1 and Sse2 are up-regulated severalfold under heat shock conditions. Under normal growth conditions, Sse1 is present at moderate levels, and Sse2 is present at very low levels (37Mukai H. Kuno T. Tanaka H. Hirata D. Miyakawa T. Tanaka C. Gene (Amst.). 1993; 132: 57-66Crossref PubMed Scopus (104) Google Scholar). To analyze the function of the Hsp70·Sti1·Hsp90 complex from yeast, we first set out to identify the Hsp70 components. We show that the Ssa proteins are the only Hsp70 component of the complex and that Sti1, unlike Hop in the mammalian Hsp90 multichaperone complex, is a novel regulator for their ATPase activity. Yeast Strains, Culture Media, and General Methods—All strains except for GS 115, which is a Pichia pastoris strain, are Saccharomyces cerevisiae strains. Strains used in this study have the following geno-types: Y190, MATa ura3–52 his3–200 ade2–101 lys2–801 trp1–901 leu2–3,112 gal4Δ gal80Δ cyhr2 LYS2::GAL1 UAS -HIS3 TATA -HIS3 MEL1 URA3:: GAL1 UAS -GAL1 TATA -lacZ; W303, MATa ade2–1, leu2–3, 112 his3–11, and 15 trp1–1 ura3–1 can1–100; CN11a, MAT a Δtrp1 lys1 lys2 ura3–52 leu2–3,112 his3–11,15 sti1–1::HIS3; and GS 115, his4 (Invitrogen). All S. cerevisiae strains were grown in YPD medium (1% yeast extract, 2% bacto peptone, 2% glucose). The P. pastoris strain GS 115 containing the pPICZB-Ssa1 plasmid was grown in dextrose histidine medium (1.34% (w/v) yeast nitrogen base, 0.4% (w/v) l-histidine, 2% (w/v) glucose, 4 × 10–5% biotin), and the expression of Ssa1 was induced in methanol histidine medium (1.34% (w/v) yeast nitrogen base, 0.4% (w/v) l-histidine, 4 × 10–5% biotin, and 0.5% (v/v) methanol). The lysates for immunoprecipitations were prepared as follows. The strains W303 and CN11a were grown in 100 ml of YPD medium to a A 600 of ∼1.0, harvested, and resuspended in 5 ml of 40 mm Hepes, pH 7.5, 150 mm KCl, 5 mm MgCl2. The cells were lysed using a cell disruption system (Constant Systems, Warwick, UK), shock frozen with liquid nitrogen, and stored in 100 μl portions at –80 °C. Construction of pAS and pACT Clones—Ssa1–4, Ssb1–2, Sse1–2, Ssz1, and Sti1 were amplified by PCR using yeast genomic DNA (strain W303) and ligated in the vectors pAS1-CYH2 and/or pACTII (38Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar) as fusion proteins with the GAL4 DNA-binding domain (pAS1-CYH2) and the GAL4 activation domain (pACTII) (Clontech, Palo Alto, CA). The identity of the clones was confirmed by DNA sequencing. Two-hybrid Screening—The yeast strain Y190 was transformed with either Sti1 in pAS1-CYH2 or pACTII or the nine different yeast Hsp70s in pAS1-CYH2 or pACTII as described in the manufacturer's instructions (Clontech). Single transformants were confirmed as His– and LacZ– to ensure that the fusion proteins alone do not exhibit transcriptional activity in Y190. Double transformants were incubated for 3 days at 30 °C before positive clones were picked, restreaked onto His– Leu– Trp– plates and assayed for the lacZ phenotype (39Breeden L. Nasmyth K. Cold Spring Harbor Symp. Quant. Biol. 1985; 50: 643-650Crossref PubMed Scopus (470) Google Scholar). Construction of the Mutants his-Ssa1 (K69Q) and his-yHsp90 (E33A)—The design of the Ssa1 (K69Q) mutant is described elsewhere (40McClellan A.J. Brodsky J.L. Genetics. 2000; 156: 501-512Crossref PubMed Google Scholar). The yHsp90 (E33A) point mutation was generated in vitro using overlap PCR technique according to Mikaelian and Sergeant (41Mikaelian I. Sergeant A. Nucleic Acids Res. 1992; 20: 376Crossref PubMed Scopus (211) Google Scholar). The identity of this construct was confirmed by DNA sequencing. Protein Purification—S. cerevisiae Ssa1 was expressed using the P. pastoris expression system (42Wegele H. Haslbeck M. Buchner J. J. of Chromatogr. B. 2003; 786: 109-115Crossref PubMed Scopus (18) Google Scholar). The mutant his-Ssa1 (K69Q) was expressed and purified as described previously (40McClellan A.J. Brodsky J.L. Genetics. 2000; 156: 501-512Crossref PubMed Google Scholar). Sti1 was expressed in the Escherichia coli strain BL21-CodonPlus (DE3) and purified as described elsewhere (28Prodromou 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 (356) Google Scholar, 43Mayr C. Richter K. Lilie H. Buchner J. J. Biol. Chem. 2000; 275: 34140-34146Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Wild type and mutant yHsp90 (E33A) were purified as described (44Buchner J. Bose S. Mayr C. Jakob U. Methods Enzymol. 1998; 290: 409-418Crossref PubMed Scopus (21) Google Scholar, 45Richter K. Muschler P. Hainzl O. Buchner J. J. Biol. Chem. 2001; 276: 33689-33696Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Human Hsp70 was expressed in a DnaK– variant of the E. coli strain BL21-CodonPlus (DE3) and purified as described elsewhere (46Schumacher R.J. Hansen W.J. Freeman B.C. Alnemri E. Litwack G. Toft D.O. Biochemistry. 1996; 35: 14889-14898Crossref PubMed Scopus (144) Google Scholar). Human Hop and human Hsp90 were purified as described (44Buchner J. Bose S. Mayr C. Jakob U. Methods Enzymol. 1998; 290: 409-418Crossref PubMed Scopus (21) Google Scholar, 47Buchner J. Weikl T. Bugl H. Pirkl F. Bose S. Methods Enzymol. 1998; 290: 418-429Crossref PubMed Scopus (22) Google Scholar). Shock frozen proteins were stored in 40 mm Hepes, pH 7.5, 150 mm KCl, 5 mm MgCl2, and 1 mm dithiothreitol (standard buffer) at –80 °C. Immunoprecipitations—The immunoprecipitations were performed with lysates from the S. cerevisiae wild type strain W303 or the Sti1 knock-out strain CN11a and with the purified proteins Ssa1, Sti1, and yHsp90 in a volume of 100 μl. The immunoprecipitations were incubated at 25 °C for 1 h in standard buffer with or without 2 mm ATP, using the indicated antibodies. Immune complexes were recovered by binding to protein A/G-Sepharose (Sigma). After three washes with 5 volumes of 1× phosphate-buffered saline (4 mm KH2PO4, 16 mm Na2HPO4, 0.12 m NaCl, pH 7.4), the immunocomplexes were eluted with 1× Laemmli SDS loading buffer. For immunodetection, the proteins were separated by SDS-PAGE and transferred to Immobilon-nitrocellulose (Millipore, Bedford, MA) or Immobilon-polyvinylidene difluoride (Millipore) membranes and incubated with polyclonal anti-sera specific for either Ssa (C-terminal 80 amino acids), Ssb (C-terminal 80 amino acids) (48Lopez-Buesa P. Pfund C. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15253-15258Crossref PubMed Scopus (67) Google Scholar), Sti1 (this study), or yHsp90 (this study). As a secondary antibody, anti-rabbit IgG conjugated with peroxidase for ECL detection (Amersham Biosciences) was used. ATPase Activity (Radioactive Assay)—ATPase assays were performed according to Kornberg et al. (49Kornberg A. Scott J.F. Bertsch L.L. J. Biol. Chem. 1978; 253: 3298-3304Abstract Full Text PDF PubMed Google Scholar). The indicated amounts of Ssa1 ± Sti1 ± yHsp90 ± Ydj1 ± Hop, Hsp70 ± Hop ± Sti1, and Hsp90 ± Hop ± Sti1 (±, with or without) were incubated in standard buffer at 37 °C. The ATPase reaction was initiated by the addition of the respective ATP concentrations containing [α-32P]ATP (Hartmann Analytic, Braunschweig, Germany). A sample contained 0.1 μCi of [α-32P]ATP. As a control for unspecific protein effects, Sti1 was added after incubation at 70 °C for 30 min. For steady state hydrolysis measurements, a final ATP concentration of 15 mm was used. In the case of single turnover experiments, the ATP to protein ratio was kept constant at 0.8:1. The ATP to ADP ratio was quantified with a Typhoon 9200 PhosphorImager (Amersham Biosciences). The hydrolysis rates were corrected for uncatalyzed, spontaneous ATP hydrolysis. ATPase Activity of Ssa1 (HPLC)—The formation of ADP in steady state and single turnover experiments was measured at 37 °C by incubating Ssa1 (Ssa1 K69Q) ± Sti1 and/or yHsp90 (yHsp90 E33A) with ATP for various times and subsequent nucleotide analysis by HPLC as described (50Theyssen H. Schuster H.P. Packschies L. Bukau B. Reinstein J. J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (199) Google Scholar). Biacore Measurements—Surface plasmon resonance (51Schuck P. Curr. Opin. Biotechnol. 1997; 8: 498-502Crossref PubMed Scopus (142) Google Scholar, 52Nice E.C. Catimel B. Bioessays. 1999; 21: 339-352Crossref PubMed Scopus (184) Google Scholar) data were collected on a Biacore X Instrument (Biacore, Uppsala, Sweden). For experiments in the yeast system, either Ssa1 or yHsp90 were covalently linked to the CM5 chip via amine residues according to the supplier's instructions; for determination of binding constants in the human system, Hop was linked the same way to the CM5 chip. The measurements were performed at a flow rate of 5 μl/min at 25 °C. To determine the binding constant, we directly measured the change in response units, when different amounts of Sti1 were injected onto a Ssa1- or yHsp90-coated chip in standard buffer with or without 2 mm ATP or different amounts of Hsp70 and Hsp90 were injected onto the Hop-coated chip in standard buffer with or without 2 mm ATP. Data analysis for direct binding curves used the linear relationship between the resonance signal (RU) and the amount of protein (c) bound to the chip. RU max (Equation 1) is the maximum signal at which all of the Ssa1 molecules on the chip are saturated with Sti1. RU=RUmax×cc+KD(Eq. 1) The Hsp70 Component of the Sti1 Complex Is the Ssa Proteins—An important step in characterizing the Hsp90 multichaperone complex in S. cerevisiae is to elucidate which of the 14 Hsp70 proteins present in yeast interacts with cytosolic Sti1. Nine of the 14 different yeast Hsp70 proteins are cytosolic and therefore potential partners of the co-chaperone Sti1. It had been shown previously that at least one member of the Ssa class interacts with Sti1 in yeast (29Chang H.C. Lindquist S. J. Biol. Chem. 1994; 269: 24983-24988Abstract Full Text PDF PubMed Google Scholar), but it was not clear whether other yeast Hsp70s also interact with Sti1 in the Hsp90 system. To determine which of these Hsp70 proteins interacts with Sti1, we tested Ssa1–4, Ssb1–2, Sse1–2, and Ssz1 in a yeast two-hybrid reporter system for interaction with Sti1 (Fig. 1 A). Only for the Ssa class (Ssa1–4) positive results were obtained suggesting that the Ssa proteins are the only Hsp70 component in the yHsp90 complex. As an independent proof for this interaction, we immunoprecipitated Sti1 from a wild type yeast lysate (strain W303) and tested the precipitate by Western blotting for the presence of Ssa proteins. Fig. 1B (lane 1) shows that the Ssa proteins were precipitated together with Sti1. When the cell lysates were supplemented with purified Sti1, the amount of Ssa1 co-precipitated increased with increasing amounts of purified Sti1 added (Fig. 1B, lanes 2 and 3). As a control for the specificity of the Sti1 antibody, we repeated the experiment using lysate from the Sti1 knock-out strain CN11a (Fig. 1B, lane 4). No Ssa1 could be detected in this case. To test whether the Sti1 complex contained only Ssa proteins and not Ssb proteins, we probed the blots with an antibody specific for Ssb proteins. This experiment showed that Ssb proteins could not be detected in the Sti1 complex (Fig. 1B, lane 5). Ssb proteins were, however, present in the lysate as shown in Fig. 1B (lane 6). This result is in agreement with previous studies in the mammalian system (25Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar), because only the Ssa class of Hsp70s share the C-terminal sequence motif (I/L/M)EEVD-COOH. Because Ssa3 and Ssa4 are only expressed under heat shock, these proteins are not relevant for the function of the yHsp90 multichaperone complex under normal growth conditions. Therefore, we focused in the following on the Ssa1 protein, because Ssa1 and Ssa2 are 97% identical and expressed in equal amounts under these conditions (8Craig E.A. Boorstein W.R. Park H.O. Stone D.N.C.M. UCLA Symp. Mol. Cell. Biol. 1989; 96: 51-61Google Scholar). Sti1 Binds to Ssa1 with Low Affinity—To further characterize the interaction between Ssa1 and Sti1, we purified both proteins and studied complex formation under physiological conditions concerning salt concentration and temperature. First, we incubated the proteins and performed immunoprecipitations with anti-Sti1 or anti-Ssa antibodies and separated the precipitate by SDS-PAGE. Fig. 2A indicates that the Sti1-Ssa1 complex was formed efficiently under these conditions. We found that the amount of Ssa1 in the complex was not significantly affected by ATP (Fig. 2A, lanes 2 and 4). To gain quantitative data on the interaction between Ssa1 and Sti1, surface plasmon resonance spectroscopy was employed. Binding of different Sti1 concentrations with or without ATP to immobilized Ssa1 was analyzed. The data from the resulting sensorgrams were used to determine the dissociation constant of the Ssa1-Sti1 interaction (Fig. 2B). We found that in the absence of ATP, Sti1 binds to Ssa1 with an affinity of 7.5 ± 2 μm, whereas in the presence of ATP, the affinity is 3 ± 1 μm (Fig. 2B and Table I). Compared with the affinity of Sti1 for yHsp90 (28Prodromou 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 (356) Google Scholar, 43Mayr C. Richter K. Lilie H. Buchner J. J. Biol. Chem. 2000; 275: 34140-34146Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) (Table I), Sti1 binding to Ssa1 is about 2 orders of magnitude weaker.Table ISummary of KD valuesProteinKDμMSsa1-Sti17.5 ± 2Ssa1-Sti1-ATP3 ± 1Ssa1-HopNDyHsp90-Sti10.1 ± 0.02yHsp90-HopNDHsp70-Hop1.5 ± 0.2Hsp70-Hop-ATP1.5 ± 0.2Hsp70-Sti1NDhHsp90-Hop0.1 ± 0.02hHsp90-Sti1ND Open table in a new tab Sti1 Is a Potent Activator of the ATPase Activity of Ssa1—It has been described previously that Sti1 inhibits the ATPase activity of yHsp90 (28Prodromou 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 (356) Google Scholar). We were interested in determining whether Sti1 influences the ATPase activity of Ssa1 as well. Sti1 itself does not exhibit ATPase activity (data not shown). The ATPase activity of Ssa1 is similar to that of other Hsp70s studied, with a K m for ATP of 0.2 μm and a k cat for ATP of 0.04 min–1 at physiological potassium concentrations (48Lopez-Buesa P. Pfund C. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15253-15258Crossref PubMed Scopus (67) Google Scholar). When Sti1 was added to Ssa1, we found that, unexpectedly, the ATPase activity of Ssa1 increased with increasing amounts of Sti1 (Fig. 3A). A stimulation by a factor of 200 was reached at a Sti1:Ssa1 ratio of 1:1 (Fig. 3B). Half-maximal activation was reached at a ratio of Ssa1:Sti1 = 1:0.5. When thermally unfolded Sti1 was added in a control experiment, no significant increase in the ATPase activity of Ssa1 was detected (Table II).Table IISummary of kcat valuesProteinkcatmin-1Ssa10.04 ± 0.02Ssa1-Sti1 (1:1)7.1 ± 0.2Ssa1-Sti1 nonnative (1:1)0.04 ± 0.02Ssa1-Ydj1 (1:4)0.4 ± 0.05Ssa1-Ydj1-Sti1 (1:4:1)7.1 ± 0.2Ssa1-Hop (1:5)0.04 ± 0.02yHsp900.4 ± 0.05yHsp90-Sti1 (1:5)0.05 ± 0.02yHsp90-Hop (1:5)0.4 ± 0.05Hsp700.4 ± 0.05Hsp70-Hop (1:5)0.4 ± 0.05Hsp70-Sti1 (1:5)0.4 ± 0.05hHsp900.04 ± 0.01hHsp90-Hop (1:5)0.04 ± 0.01hHsp90-Sti1 (1:5)0.04 ± 0.01 Open table in a new tab To determine which step in the Ssa1 ATPase cycle is accelerated by Sti1, we compared steady state measurements with single turnover experiments in the presence and absence of Sti1 (Fig. 3). It was not known whether ATP hydrolysis or nucleotide exchange represents the rate-limiting step in the ATPase cycle of Ssa1. To discriminate between the two possibilities, we analyzed the steady state k cat and the single turnover k cat. In steady state measurements, excess of ATP is used, and therefore Ssa1 hydrolyzes ATP with maximum velocity, whereas in single turnover assays, about 80% of Ssa1 is saturated with ATP. Thus, a single hydrolysis step could be monitored, which allows, in comparison with the steady state measurement, determination of the rate-limiting step of the ATPase cycle. We found that the k cat values (0.04 min–1) were identical in the absence of Sti1. Thus, we conclude that ATP hydrolysis, or a conformational change preceding it, is rate-limiting for the Ssa1 ATPase cycle. Increasing amounts of Sti1 resulted in an acceleration of the single turnover ATPase activity of Ssa1 (Fig. 3C). A stimulation factor of 200 could also be seen in the single turnover experiments at a Sti1 to Ssa1 ratio of 1:1. In the presence of Sti1, both the steady state k cat and the single turnover k cat were accelerated (Fig. 3). This demonstrates that Sti1 stimulates ATP hydrolysis and confirms that this step is rate-limiting in the Ssa1 ATPase cycle. As previously described (53Cyr D.M. Lu X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar), Ydj1 is able to accelerate the ATPase of Ssa1 by a factor of 10. When Ydj1 was added to a preformed Ssa1-Sti1 complex at a 1:1 ratio or at high excess, the activation resulting from Sti1 was not influenced (data not shown). Further, if Sti1 was added to an Ssa1-Ydj1 complex, a 200-fold activation was observed. Influence of Sti1 on Ssa1 and yHsp90 in the Ternary Multichaperone Complex—To study the ternary multichaperone complex consisting of Ssa1, Sti1, and yHsp90, we incubated the purified Ssa1, Sti1, and yHsp90, precipitated with anti-Ssa1, anti-Sti1 or anti-yHsp90 antibodies and separated the precipitate by SDS-PAGE (Fig. 4A). The three proteins could be detected in equal amounts no matter which antibody was used. This complex is also present in the absence of ATP (Fig. 4A, lane 4). To study the influence of Sti1 on the ATPase activities of yHsp90 and Ssa1 in the ternary complex, it was necessary to incorporate either a Ssa1 mutant (K69Q) (40McClellan A.J. Brodsky J.L. Genetics. 2000; 156: 501-512Crossref PubMed Google Scholar) or a yHsp90 mutant (E33A) (54Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (494) Google Scholar, 55Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref
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