Independent regulation of Hsp70 and Hsp90 chaperones by Hsp70/Hsp90-organizing protein Sti1 (Hop1)

Hsp70 and Hsp90 protein chaperones cooperate in a protein-folding pathway required by many "client" proteins. The co-chaperone Sti1p coordinates functions of Hsp70 and Hsp90 in this pathway. Sti1p has three tetratricopeptide repeat (TPR) domains. TPR1 binds Hsp70, TPR2a binds Hsp90, and the ligand for TPR2b is unknown. Although Sti1p is thought to be dedicated to the client folding pathway, we earlier showed that Sti1p regulated Hsp70, independently of Hsp90, in a way that impairs yeast [PSI+] prion propagation. Using this prion system to monitor Sti1p regulation of Hsp70 and an Hsp90-inhibiting compound to monitor Hsp90 regulation, we identified Sti1p mutations that separately affect Hsp70 and Hsp90. TPR1 mutations impaired Sti1p regulation of Hsp70, but deletion of TPR2a and TPR2b did not. Conversely, TPR2a and TPR2b mutations impaired Sti1p regulation of Hsp90, but deletion of TPR1 did not. All Sti1p mutations variously impaired the client folding pathway, which requires both Hsp70 and Hsp90. Thus, Sti1p regulated Hsp70 and Hsp90 separately, Hsp90 is implicated as a TPR2b ligand, and mutations separately affecting regulation of either chaperone impair a pathway that is dependent upon both. We further demonstrate that client folding depended upon bridging of Hsp70 and Hsp90 by Sti1p and find conservation of the independent regulation of Hsp70 and Hsp90 by human Hop1.     

Substrate transfer from the chaperone Hsp70 to Hsp90

Hsp90 is an essential chaperone protein in the cytosol of eukaryotic cells. It cooperates with the chaperone Hsp70 in defined complexes mediated by the adaptor protein Hop (Sti1 in yeast). These Hsp70/Hsp90 chaperone complexes play a major role in the folding and maturation of key regulatory proteins in eukaryotes. Understanding how non-native client proteins are transferred from one chaperone to the other in these complexes is of central importance. Here, we analyzed the molecular mechanism of this reaction using luciferase as a substrate protein. Our experiments define a pathway for luciferase folding in the Hsp70/Hsp90 chaperone system. They demonstrate that Hsp70 is a potent capture device for unfolded protein while Hsp90 is not very efficient in this reaction. When Hsp90 is absent, in contrast to the in vivo situation, Hsp70 together with the two effector proteins Ydj1 and Sti1 exhibits chaperone activity towards luciferase. In the presence of the complete chaperone system, Hsp90 exhibits a specific positive effect only in the presence of Ydj1. If this factor is absent, the transferred luciferase is trapped on Hsp90 in an inactive conformation. Interestingly, identical results were observed for the yeast and the human chaperone systems although the regulatory function of human Hop is completely different from that of yeast Sti1.     

Cns1 is an activator of the Ssa1 ATPase activity

Hsp90 is a key mediator in the folding process of a growing number of client proteins. The molecular chaperone cooperates with many co-chaperones and partner proteins to fulfill its task. In Saccharomyces cerevisiae, several co-chaperones of Hsp90 interact with Hsp90 via a tetratricopeptide repeat (TPR) domain. Here we show that one of these proteins, Cns1, binds both to Hsp90 and to the yeast Hsp70 protein Ssa1 with comparable affinities. This is reminiscent of Sti1, another TPR-containing co-chaperone. Unlike Sti1, Cns1 exhibits no influence on the ATPase of Hsp90. However, it activates the ATPase of Ssa1 up to 30-fold by accelerating the rate-limiting ATP hydrolysis step. This stimulating effect is mediated by the N-terminal TPR-containing part of Cns1, whereas the C-terminal part showed no effect. Competition experiments allow the conclusion that Hsp90 and Ssa1 compete for binding to the single TPR domain of Cns1. Taken together, Cns1 is a potent cochaperone of Ssa1. Our findings highlight the importance of the regulation of Hsp70 function in the context of the Hsp90 chaperone cycle.     

Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions

The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.     

Sti1 is a novel activator of the Ssa proteins

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.     

Sgt1p is a unique co-chaperone that acts as a client adaptor to link Hsp90 to Skp1p

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.     

Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.     

The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.     

Hsp104 interacts with Hsp90 cochaperones in respiring yeast

The highly abundant molecular chaperone Hsp90 functions with assistance from auxiliary factors, collectively referred to as Hsp90 cochaperones, and the Hsp70 system. Hsp104, a molecular chaperone required for stress tolerance and for maintenance of [psi(+)] prions in the budding yeast Saccharomyces cerevisiae, appears to collaborate only with the Hsp70 system. We now report that several cochaperones previously thought to be dedicated to Hsp90 are shared with Hsp104. We show that the Hsp90 cochaperones Sti1, Cpr7, and Cns1, which utilize tetratricopeptide repeat (TPR) domains to interact with a common surface on Hsp90, form complexes with Hsp104 in vivo and that Sti1 and Cpr7 interact with Hsp104 directly in vitro. The interaction is Hsp90 independent, as further emphasized by the fact that two distinct TPR domains of Sti1 are required for binding Hsp90 and Hsp104. In a striking parallel to the sequence requirements of Hsp90 for binding TPR proteins, binding of Sti1 to Hsp104 requires a related acidic sequence at the C-terminal tail of Hsp104. While Hsp90 efficiently sequesters the cochaperones during fermentative growth, respiratory conditions induce the interaction of a fraction of Hsp90 cochaperones with Hsp104. This suggests that cochaperone sharing may favor adaptation to altered metabolic conditions.     

Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones.

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co-chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90. These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP-dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.     

Nucleotide-dependent interaction of Saccharomyces cerevisiae Hsp90 with the cochaperone proteins Sti1, Cpr6, and Sba1

The ATP-dependent molecular chaperone Hsp90 and partner cochaperone proteins are required for the folding and activity of diverse cellular client proteins, including steroid hormone receptors and multiple oncogenic kinases. Hsp90 undergoes nucleotide-dependent conformational changes, but little is known about how these changes are coupled to client protein activation. In order to clarify how nucleotides affect Hsp90 interactions with cochaperone proteins, we monitored assembly of wild-type and mutant Hsp90 with Sti1, Sba1, and Cpr6 in Saccharomyces cerevisiae cell extracts. Wild-type Hsp90 bound Sti1 in a nucleotide-independent manner, while Sba1 and Cpr6 specifically and independently interacted with Hsp90 in the presence of the nonhydrolyzable analog of ATP, AMP-PNP. Alterations in Hsp90 residues that contribute to ATP binding or hydrolysis prevented or altered Sba1 and Cpr6 interaction; additional alterations affected the specificity of Cpr6 interaction. Some mutant forms of Hsp90 also displayed reduced Sti1 interaction in the presence of a nucleotide. These studies indicate that cycling of Hsp90 between the nucleotide-free, open conformation and the ATP-bound, closed conformation is influenced by residues both within and outside the N-terminal ATPase domain and that these conformational changes have dramatic effects on interaction with cochaperone proteins.     

Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37

In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a "scaffold," recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50(cdc37), whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50(cdc37), like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a "relaxed" ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50(cdc37) binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50(cdc37) is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50(cdc37) forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo.     

The Hsp90 co-chaperones Cdc37 and Sti1 interact physically and genetically

Cdc37 associates with the heat-shock protein 90 (Hsp90) molecular chaperone as one of several auxiliary proteins that are collectively referred to as Hsp90 co-chaperones. Cdc37 has been proposed to be a specificity factor for Hsp90, directing it notably towards kinases. It is not known whether Cdc37 is essential for viability in the budding yeast Saccharomyces cerevisiae because of Hsp90-dependent or -independent functions or both. Sti1 and Cpr7 are non-essential Hsp90 co-chaperones that bind to a common surface on Hsp90 through tetratricopeptide repeats (TPR). We have found that Sti1 is specifically retained from yeast extracts by immobilized Cdc37. Similarly, the endogenous proteins are also found in a complex. Moreover, purified recombinant Sti1 and Cdc37 interact in the complete absence of Hsp90. Complexes between Cdc37 and Sti1 are not unique to this TPR protein since endogenous Cdc37 can be co-purified with exogenously expressed Cpr7 fused to glutathione-S-transferase. The heterogeneity of Cdc37 complexes, both with and without Hsp90, may expand the functional diversity of Cdc37. Here we show that the combination of cdc37 and sti1 mutations is synthetically lethal, suggesting that direct contacts between Cdc37 and Sti1 may at least contribute to vital functions in yeast.     

Chaperone network in the yeast cytosol: Hsp110 is revealed as an Hsp70 nucleotide exchange factor

The Hsp110 proteins, exclusively found in the eukaryotic cytosol, have significant sequence homology to the Hsp70 molecular chaperone superfamily. Despite this homology and the cellular abundance of these proteins, the precise functional role has remained undefined. Here, we present the intriguing finding that the yeast homologue, Sse1p, acts as an efficient nucleotide exchange factor (NEF) for both yeast cytosolic Hsp70s, Ssa1p and Ssb1p. The mechanism involves formation of a stable nucleotide-sensitive complex, but does not require ATP hydrolysis by Sse1p. The NEF activity of Sse1p stimulates in vitro Ssa1p-mediated refolding of thermally denatured luciferase, and appears to have an essential role in vivo. Overexpression of the only other described cytosolic NEF, Fes1p, can partially compensate for a lethal sse1,2Delta phenotype, however, the cells are sensitive to stress conditions. Furthermore, in the absence of Sse, the in vivo refolding of thermally denatured model proteins is affected. This is the first report of a nucleotide exchange activity for the Hsp110 class of proteins, and provides a key piece in the puzzle of the cellular chaperone network.     

Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway

The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway.     

The yeast Hsp110 Sse1 functionally interacts with the Hsp70 chaperones Ssa and Ssb

There is growing evidence that members of the extended Hsp70 family of molecular chaperones, including the Hsp110 and Grp170 subgroups, collaborate in vivo to carry out essential cellular processes. However, relatively little is known regarding the interactions and cellular functions of Sse1, the yeast Hsp110 homolog. Through co-immunoprecipitation analysis, we found that Sse1 forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. Binding of Ssa or Ssb to Sse1 was mutually exclusive. The ATPase domain of Sse1 was found to be critical for interaction as inactivating point mutations severely reduced interaction with Ssa and Ssb. Sse1 stimulated Ssa1 ATPase activity synergistically with the co-chaperone Ydj1, and stimulation required complex formation. Ssa1 is required for post-translational translocation of the yeast mating pheromone alpha-factor into the endoplasmic reticulum. Like ssa mutants, we demonstrate that sse1delta cells accumulate prepro-alpha-factor, but not the co-translationally imported protein Kar2, indicating that interaction between Sse1 and Ssa is functionally significant in vivo. These data suggest that the Hsp110 chaperone operates in concert with Hsp70 in yeast and that this collaboration is required for cellular Hsp70 functions.