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.     

Sti1 Regulation of Hsp70 and Hsp90 Is Critical for Curing of Saccharomyces cerevisiae [PSI+] Prions by Hsp104

Although propagation of Saccharomyces cerevisiae prions requires Hsp104 protein disaggregating activity, overproducing Hsp104 “cures” cells of [PSI⁺] prions. Earlier evidence suggests that the Hsp70 mutant Ssa1-21 impairs [PSI⁺] by a related mechanism. Here, we confirm this link by finding that deletion of STI1 both suppresses Ssa1-21 impairment of [PSI⁺] and blocks Hsp104 curing of [PSI⁺]. Hsp104''s tetratricopeptide repeat (TPR) interaction motif was dispensable for curing; however, cells expressing Sti1 defective in Hsp70 or Hsp90 interaction cured less efficiently, and the Hsp90 inhibitor radicicol abolished curing, implying that Sti1 acts in curing through Hsp70 and Hsp90 interactions. Accordingly, strains lacking constitutive or inducible Hsp90 isoforms cured at reduced rates. We confirm an earlier finding that elevating free ubiquitin levels enhances curing, but it did not overcome inhibition of curing caused by Hsp90 defects, suggesting that Hsp90 machinery is important for the contribution of ubiquitin to curing. We also find curing associated with cell division. Our findings point to crucial roles of Hsp70, Sti1, and Hsp90 for efficient curing by overexpressed Hsp104 and provide evidence supporting the earlier suggestion that destruction of prions by protein disaggregation does not adequately explain the curing.Saccharomyces cerevisiae prions are self-replicating misfolded forms of normal cellular proteins. They are believed to propagate as amyloid, which is a highly ordered fibrous aggregate. What triggers prion formation is uncertain, but in order to be maintained in an expanding yeast population, prions must grow, replicate, and be transmitted to daughter cells during cell division. Growth occurs when soluble protein joins the fiber ends and is converted into the prion form (30, 52, 58). Replication is associated with fragmentation of prion polymers, which generates new prions from preexisting material (37, 50). Transmission is believed to occur by passive diffusion of prions with cytoplasm (57).Although it is uncertain to what extent cellular factors influence growth or transmission of prions, it is clear that the Hsp104 disaggregation machinery is necessary for prion replication (10, 17, 55, 70). Hsp104 is a hexameric AAA+ chaperone that protects cells from a variety of stresses by resolubilizing proteins from aggregates (24, 25, 53). With help from Hsp70 and Hsp40, it extracts monomers from aggregates and extrudes them through its central pore (24, 41, 68). This machinery could act in prion replication by extracting monomers from amyloid fibers (29, 68), which would destabilize the fibers, causing them to break into more numerous pieces that each can continue to propagate the prion.Paradoxically, overexpressing Hsp104 very efficiently “cures” cells of the [PSI⁺] prion, which is composed of the translation termination factor Sup35 (10). A widely held view of this curing is that elevating the cellular protein disaggregation activity causes complete destruction of prions. However, elevating Hsp104 has little or no effect on most other amyloidogenic prions (15, 16, 38, 47, 54, 66), although it can be inferred to cure [MCA] prions in cells also propagating a prion of an Mca1-Sup35 fusion (49). Together, these results suggest that prions of Sup35, and perhaps those of Mca1, are particularly sensitive to Hsp104 disaggregation activity. Alternatively, something in addition to or other than a simple increase in protein disaggregation is involved in the curing.Although protein disaggregation activity of Hsp104 is required for both thermotolerance and prion propagation, we and others have identified mutations in Hsp104 that affect these processes separately (27, 32, 39, 60). The ability of Hsp104 to thread proteins through its central pore, however, is required for both processes (29, 41, 68), so this distinction in Hsp104 function could be due to differences in how Hsp104 interacts with amorphous aggregates of thermally denatured proteins and highly ordered prion aggregates or with cofactors that interact with the different prions as substrates. In any scenario, efficiency and specificity of Hsp104 function are affected by interactions with other components of the disaggregation machinery, in particular the Hsp70s and Hsp40s, which are believed to interact first with substrates to facilitate action of Hsp100 family disaggregases (2, 71, 72).Increasing expression of either ubiquitin (Ub) or Ssb, an Hsp70 that has roles in protein translation and proteasome degradation, enhances Hsp104 curing of [PSI⁺] (3, 11, 12). Predictably, reducing expression of either of them reduces curing efficiency. The mechanisms underlying these effects are unknown, but the combined effects of Ssb and Ub are additive, suggesting that they act in different pathways. The role of Ub is indirect, as Sup35 is neither ubiquitylated nor degraded during curing. Whether other chaperones are involved in the effects of Ub on curing has not been investigated.Earlier we isolated a mutant of the Hsp70 Ssa1, designated Ssa1-21, that weakens and destabilizes [PSI⁺] propagation (33). We later isolated several Hsp104 mutants that suppress this antiprion effect (29). The Hsp104 mutants retain normal functions in thermotolerance, protein disaggregation, and prion propagation, but when overexpressed, they are unable to cure [PSI⁺], even in wild-type cells. These findings argue against a specific hypersensitivity of [PSI⁺] to disaggregation and support the notion that something distinct from or in addition to complete destruction of prions is involved in the curing. They also imply that Ssa1-21 and elevated Hsp104 inhibit [PSI⁺] prions by similar mechanisms. A prediction from this conclusion is that other suppressors of Ssa1-21 will also inhibit curing of [PSI⁺] by overexpressed Hsp104. Indeed, we find here that alterations that suppress Ssa1-21 inhibition of [PSI⁺] do interfere with curing of [PSI⁺] by overexpressed Hsp104. We also provide evidence that Hsp90 has a critical role in this curing and that the ability of Ub to enhance curing depends on proper function of Hsp90 machinery.     

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.     

Hsp70 and Hsp40 Functionally Interact with Soluble Mutant Huntingtin Oligomers in a Classic ATP-dependent Reaction Cycle

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.     

Cooperative interaction of Hsp40 and TPR1 with Hsp70 reverses Hsp70-HspBp1 complex formation

Hsp40 and TPR1 are chaperone adaptors that regulate Hsp70-dependent folding processes by interacting with the amino terminal and carboxy terminal domains of Hsp70, respectively. In this study, we report cooperative interactions involving Hsp70, Hsp40, and TPR1 that enhance Hsp70-dependent folding of chemically denatured substrates. Hsp40 and Hsp70 dependent folding of chemically denatured luciferase was enhanced by up to 80% when TPR1 was also present. HspBp1, a negative modulator of Hsp70, completely inhibited Hsp70-dependent folding in the presence of Hsp40. However, when TPR1 was included in the reaction, the inhibitory effect of HspBp1 was reversed. To analyze the interactions, Kd analysis and competition assays were carried out. The Kds of the interactions of Hsp40, TRP1, and HspBp1 with Hsp70 were 0.5, 0.6, and 0.04 mM, respectively. Interestingly, the Hsp70/HspBp1 complex could only be dissociated in the presence of both Hsp40 and TPR1, suggesting cooperative interaction between Hsp70, Hsp40 and TPR1. To examine these interactions in vivo, we established a tetracycline-regulatable Hela cell line that expresses Hsp70 in the absence of doxycycline. Expression of HspBp1 inhibited Hsp70-dependent folding of heat-denatured luciferase, and this effect was only reversed in the presence of Hsp40 and TPR1. Our findings reveal a novel mechanism of positive regulation of Hsp70-dependent folding.     

The Hsp70/90 cochaperone,Sti1, suppresses proteotoxicity by regulating spatial quality control of amyloid-like proteins

Conformational diseases are associated with the conversion of normal proteins into aggregation-prone toxic conformers with structures similar to that of β-amyloid. Spatial distribution of amyloid-like proteins into intracellular quality control centers can be beneficial, but cellular mechanisms for protective aggregation remain unclear. We used a high-copy suppressor screen in yeast to identify roles for the Hsp70 system in spatial organization of toxic polyglutamine-expanded Huntingtin (Huntingtin with 103Q glutamine stretch [Htt103Q]) into benign assemblies. Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci. Subtle modulation of Sti1 activity reciprocally affects Htt toxicity and the packaging of Htt103Q into foci. Loss of Sti1 exacerbates Htt toxicity and hinders foci formation, whereas elevation of Sti1 suppresses Htt toxicity while organizing small Htt103Q foci into larger assemblies. Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1–monomeric red fluorescent protein into distinct foci. Sti1-inducible foci are perinuclear and contain proteins that are bound by the amyloid indicator dye thioflavin-T. Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.     

Hsp70 Targets a Cytoplasmic Quality Control Substrate to the San1p Ubiquitin Ligase

Accumulation of misfolded proteins in cellular compartments can result in stress-induced cell death. In the endoplasmic reticulum (ER), ER-associated degradation clears aberrant proteins from the secretory pathway. In the cytoplasm and nucleus, this job is left to the cytoplasmic quality control (CytoQC) machinery. Both processes utilize chaperones and the ubiquitin-proteasome system to aid in protein elimination. Previous studies in yeast have drawn comparisons between these processes using data from structurally and topologically different substrates. We sought to draw a direct comparison between ERAD and CytoQC by studying the elimination of a single misfolded domain that, depending on its residence, is disposed by either of these pathways. The truncated, second nucleotide binding domain (NBD2*) from a yeast ERAD substrate, Ste6p*, resides at the cytoplasmic face of the ER. We show that a soluble form of NBD2* is cytoplasmic and unlike wild-type NBD2 is targeted for proteasome-mediated degradation. In contrast to Ste6p*, which employs the ER-localized Doa10p ubiquitin ligase, NBD2* is ubiquitinated by a nuclear E3 ligase San1p, a factor that is also required for its degradation. Although the yeast cytoplasmic Hsp70 chaperone, Ssa1p, has been thought to facilitate the nuclear import or to maintain the solubility of most CytoQC substrates, we discovered that Ssa1p facilitates the interaction between San1p and NBD2*, demonstrating that chaperones can aid in substrate recognition and San1p-dependent protein degradation. These results emphasize the diverse action of molecular chaperones during CytoQC.     

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.     

Characterization and regulation of the major histocompatibility complex-encoded proteins Hsp70-Hom and Hsp70-1/2

Vertebrate cells contain at least 12 different genes for Hsp70 proteins, 3 of which are encoded in the major histocompatibility complex (MHC) class III region. In the human MHC, these are named Hsp70-1, -2, and -Hom. To characterize these proteins, we have determined their substrate binding specificity, their cellular and tissue distribution, and the regulation of their expression. We show for the first time (1) peptide binding specificity of Hsp70-Hom; (2) endogenous expression of Hsp70-Hom in human cell lines; (3) cytoplasmic location of Hsp70-Hom protein under basal conditions and concentration in the nucleus after heat shock; (4) unique RNA expression profiles in human tissues for each of the MHC-encoded Hsp70s, significantly different from that for the constitutive Hsc70; (5) a relative increase in levels of Hsp70-Hom protein, compared with other Hsp70s, in response to interferon gamma; and (6) a specific increase on lipopolysaccharide (LPS) treatment of in vivo messenger RNA levels for the MHC-encoded Hsp70s and the DnaJ homologue, hdj2, relative to other chaperones. The unique tissue distributions and specific up-regulation by LPS of the MHC-encoded Hsp70s suggest some specialization of functions for these members of the Hsp70 family, possibly in the inflammatory response.     

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.     

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca²⁺/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca²⁺/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca²⁺/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca²⁺/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca²⁺-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.     

In vivo bipartite interaction between the Hsp40 Sis1 and Hsp70 in Saccharomyces cerevisiae

The essential Hsp40, Sis1, is a J-protein cochaperone for the Ssa class of Hsp70's of Saccharomyces cerevisiae. Sis1 is required for the maintenance of the prion [RNQ(+)], as Sis1 lacking its 55-amino-acid glycine-rich region (G/F) does not maintain [RNQ(+)]. We report that overexpression of Sis1DeltaG/F in an otherwise wild-type strain had a negative effect on both cell growth and [RNQ(+)] maintenance, while overexpression of wild-type Sis1 did not. Overexpression of the related Hsp40 Ydj1 lacking its G/F region did not cause inhibition of growth, indicating that this dominant effect of Sis1DeltaG/F is not a characteristic shared by all Hsp40's. Analysis of small deletions within the SIS1 G/F region indicated that the observed dominant effects were caused by the absence of sequences known to be important for Sis1's unique cellular functions. These inhibitory effects of Sis1DeltaG/F were obviated by alterations in the N-terminal J-domain of Sis1 that affect interaction with Ssa's ATPase domain. In addition, a genetic screen designed to isolate additional mutations that relieved these inhibitory effects identified two residues in Sis1's carboxy-terminal domain. These alterations disrupted the interaction of Sis1 with the 10-kD carboxy-terminal regulatory domain of Ssa1, indicating that Sis1 has a bipartite interaction with Ssa in vivo.     

Characterization of Hsp70 binding and nucleotide exchange by the yeast Hsp110 chaperone Sse1

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors. We demonstrate here that Sse1 forms high-affinity (Kd approximately 10-8 M) heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones and that binding of ATP to Sse1 is required for binding to Hsp70s. Sse1.Hsp70 heterodimerization confers resistance to exogenously added protease, indicative of conformational changes in Sse1 resulting in a more compact structure. The nucleotide binding domains of both Sse1/2 and the Hsp70s dictate interaction specificity and are sufficient for mediating heterodimerization with no discernible contribution from the peptide binding domains. In support of a strongly conserved functional interaction between Hsp110 and Hsp70, Sse1 is shown to associate with and promote nucleotide exchange on human Hsp70. Nucleotide exchange activity by Sse1 is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. The Hsp110 family must therefore be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.     

Heat shock proteins Hsp70-1 and Hsp70-3 Are necessary and sufficient to prevent arsenite-induced dysmorphology in mouse embryos.

Heat Shock Proteins (HSPs) represent a variety of protein families that are induced by stressors such as heat and toxicants, and the induction of HSPs in the organogenesis stage rodent embryo is well established. It has been proposed that thermotolerance and chemotolerance result from expression of the HSPs. However, whether these proteins function to prevent dysmorphogenesis and which family members serve this function are unknown. Therefore, we evaluated the specific ability of stress-inducible Hsp70-1 and Hsp70-3 to prevent arsenite-induced dysmorphology in the cultured mouse embryo using gain- and loss-of-function models. Loss of HSP function was accomplished by injecting antisense oligonucleotides directed against hsp70-1 and hsp 70-3 mRNAs into the amniotic cavity of cultured Day 9 mouse embryos. Suppression of hsp70-1 and hsp70-3 expression resulted in an up to six-fold increase in the incidence of arsenite-induced neural tube defects. Gain of HSP function was accomplished by microinjecting a transgene with a constitutive promotor driving expression of the hsp70-1 coding region, and resulted in a decreased incidence of arsenite-induced neural tube defects. These results indicate that Hsp70-1 and Hsp70-3 are both necessary and sufficient for preventing arsenite-induced dysmorphology in early-somite staged mouse embryos. Mol. Reprod. Dev. 59:285-293, 2001.     

Binding of the maize cytosolic Hsp70 to calmodulin, and identification of calmodulin-binding site in Hsp70

Using a gel-overlay technique of biotinylated calmodulin (CaM), we showed that maize cytosolic Hsp70 protein could bind to CaM in the presence of 1 mM CaCl2. The purified maize cytosolic Hsp70 inhibited the activity of CaM-dependent NADK in a concentration-dependent manner. A synthetic peptide, which possesses the 21 amino acid sequence, PRALRRLRTACERAKRTLSST, at positions 261-281 in maize cytosolic Hsp70, could associate with CaM in the presence of 1 mM calcium. The synthetic peptide inhibited CaM-dependent NADK activity and PDE activity. This indicates that the 21-amino acid sequence at positions 261-281 is the CaM-binding site. The binding of CaM to Hsp70 inhibited the ATPase activity of Hsp70. The possible regulator function of Hsp70 in cell signaling events in response to heat stress is discussed.     

CNS1 Encodes an Essential p60/Sti1 Homolog in Saccharomyces cerevisiae That Suppresses Cyclophilin 40 Mutations and Interacts with Hsp90

Cyclophilins are cis-trans-peptidyl-prolyl isomerases that bind to and are inhibited by the immunosuppressant cyclosporin A (CsA). The toxic effects of CsA are mediated by the 18-kDa cyclophilin A protein. A larger cyclophilin of 40 kDa, cyclophilin 40, is a component of Hsp90-steroid receptor complexes and contains two domains, an amino-terminal prolyl isomerase domain and a carboxy-terminal tetratricopeptide repeat (TPR) domain. There are two cyclophilin 40 homologs in the yeast Saccharomyces cerevisiae, encoded by the CPR6 and CPR7 genes. Yeast strains lacking the Cpr7 enzyme are viable but exhibit a slow-growth phenotype. In addition, we show here that cpr7 mutant strains are hypersensitive to the Hsp90 inhibitor geldanamycin. When overexpressed, the TPR domain of Cpr7 alone complements both cpr7 mutant phenotypes, while overexpression of the cyclophilin domain of Cpr7, full-length Cpr6, or human cyclophilin 40 does not. The open reading frame YBR155w, which has moderate identity to the yeast p60 homolog STI1, was isolated as a high-copy-number suppressor of the cpr7 slow-growth phenotype. We show that this Sti1 homolog Cns1 (cyclophilin seven suppressor) is constitutively expressed, essential, and found in protein complexes with both yeast Hsp90 and Cpr7 but not with Cpr6. Cyclosporin A inhibited Cpr7 interactions with Cns1 but not with Hsp90. In summary, our findings identify a novel component of the Hsp90 chaperone complex that shares function with cyclophilin 40 and provide evidence that there are functional differences between two conserved sets of Hsp90 binding proteins in yeast.     

Effect of mutation of the tetratricopeptide repeat and asparatate-proline 2 domains of Sti1 on Hsp90 signaling and interaction in Saccharomyces cerevisiae

Through simultaneous interactions with Hsp70 and Hsp90 via separate tetratricopeptide repeat (TPR) domains, the cochaperone protein Hop/Sti1 has been proposed to play a critical role in the transfer of client proteins from Hsp70 to Hsp90. However, no prior mutational analysis demonstrating a critical in vivo role for the TPR domains of Sti1 has been reported. We used site-directed mutagenesis of the TPR domains combined with a genetic screen to isolate mutations that disrupt Sti1 function. A single amino acid alteration in TPR2A disrupted Hsp90 interaction in vivo but did not significantly affect function. However, deletion of a conserved residue in TPR2A or mutations in the carboxy-terminal DP2 domain completely disrupted Sti1 function. Surprisingly, mutations in TPR1, previously shown to interact with Hsp70, were not sufficient to disrupt in vivo functions unless combined with mutations in TPR2B, suggesting that TPR1 and TPR2B have redundant or overlapping in vivo functions. We further examined the genetic and physical interaction of Sti1 with a mutant form of Hsp90, providing insight into the importance of the TPR2A domain of Sti1 in regulating Hsp90 function.