Structure of Hsp90-p23-GR reveals the Hsp90 client-remodelling mechanism.

Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins1-3. The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity4-9. GR ligand binding was previously shown to nr inhibited by Hsp70 and restored by Hsp90, aided by the co-chaperone p2310. However, a molecular understanding of the chaperone-mediated remodelling that occurs between the inactive Hsp70-Hsp90 'client-loading complex' and an activated Hsp90-p23 'client-maturation complex' is lacking for any client, including GR. Here we present a cryo-electron microscopy (cryo-EM) structure of the human GR-maturation complex (GR-Hsp90-p23), revealing that the GR ligand-binding domain is restored to a folded, ligand-bound conformation, while being simultaneously threaded through the Hsp90 lumen. In addition, p23 directly stabilizes native GR using a C-terminal helix, resulting in enhanced ligand binding. This structure of a client bound to Hsp90 in a native conformation contrasts sharply with the unfolded kinase-Hsp90 structure11. Thus, aided by direct co-chaperone-client interactions, Hsp90 can directly dictate client-specific folding outcomes. Together with the GR-loading complex structure12, we present the molecular mechanism of chaperone-mediated GR remodelling, establishing the first, to our knowledge, complete chaperone cycle for any Hsp90 client.

Structure of Hsp90-Hsp70-Hop-GR reveals the Hsp90 client-loading mechanism.

Maintaining a healthy proteome is fundamental for the survival of all organisms1. Integral to this are Hsp90 and Hsp70, molecular chaperones that together facilitate the folding, remodelling and maturation of the many 'client proteins' of Hsp902. The glucocorticoid receptor (GR) is a model client protein that is strictly dependent on Hsp90 and Hsp70 for activity3-7. Chaperoning GR involves a cycle of inactivation by Hsp70; formation of an inactive GR-Hsp90-Hsp70-Hop 'loading' complex; conversion to an active GR-Hsp90-p23 'maturation' complex; and subsequent GR release8. However, to our knowledge, a molecular understanding of this intricate chaperone cycle is lacking for any client protein. Here we report the cryo-electron microscopy structure of the GR-loading complex, in which Hsp70 loads GR onto Hsp90, uncovering the molecular basis of direct coordination by Hsp90 and Hsp70. The structure reveals two Hsp70 proteins, one of which delivers GR and the other scaffolds the Hop cochaperone. Hop interacts with all components of the complex, including GR, and poises Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized through an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR-maturation complex structure9, we present a complete molecular mechanism of chaperone-dependent client remodelling, and establish general principles of client recognition, inhibition, transfer and activation.

Hsp90 mutants with distinct defects provide novel insights into cochaperone regulation of the folding cycle.

Molecular chaperones play a key role in maintaining proteostasis and cellular health. The abundant, essential, cytosolic Hsp90 (Heat shock protein, 90 kDa) facilitates the folding and activation of hundreds of newly synthesized or misfolded client proteins in an ATP-dependent folding pathway. In a simplified model, Hsp70 first helps load client onto Hsp90, ATP binding results in conformational changes in Hsp90 that result in the closed complex, and then less defined events result in nucleotide hydrolysis, client release and return to the open state. Cochaperones bind and assist Hsp90 during this process. We previously identified a series of yeast Hsp90 mutants that appear to disrupt either the 'loading', 'closing' or 'reopening' events, and showed that the mutants had differing effects on activity of some clients. Here we used those mutants to dissect Hsp90 and cochaperone interactions. Overexpression or deletion of HCH1 had dramatically opposing effects on the growth of cells expressing different mutants, with a phenotypic shift coinciding with formation of the closed conformation. Hch1 appears to destabilize Hsp90-nucleotide interaction, hindering formation of the closed conformation, whereas Cpr6 counters the effects of Hch1 by stabilizing the closed conformation. Hch1 and the homologous Aha1 share some functions, but the role of Hch1 in inhibiting progression through the early stages of the folding cycle is unique. Sensitivity to the Hsp90 inhibitor NVP-AUY922 also correlates with the conformational cycle, with mutants defective in the loading phase being most sensitive and those defective in the reopening phase being most resistant to the drug. Overall, our results indicate that the timing of transition into and out of the closed conformation is tightly regulated by cochaperones. Further analysis will help elucidate additional steps required for progression through the Hsp90 folding cycle and may lead to new strategies for modulating Hsp90 function.

EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde.

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.

Functional and physical interaction between yeast Hsp90 and Hsp70.

Heat shock protein 90 (Hsp90) is a highly conserved ATP-dependent molecular chaperone that is essential in eukaryotes. It is required for the activation and stabilization of more than 200 client proteins, including many kinases and steroid hormone receptors involved in cell-signaling pathways. Hsp90 chaperone activity requires collaboration with a subset of the many Hsp90 cochaperones, including the Hsp70 chaperone. In higher eukaryotes, the collaboration between Hsp90 and Hsp70 is indirect and involves Hop, a cochaperone that interacts with both Hsp90 and Hsp70. Here we show that yeast Hsp90 (Hsp82) and yeast Hsp70 (Ssa1), directly interact in vitro in the absence of the yeast Hop homolog (Sti1), and identify a region in the middle domain of yeast Hsp90 that is required for the interaction. In vivo results using Hsp90 substitution mutants showed that several residues in this region were important or essential for growth at high temperature. Moreover, mutants in this region were defective in interaction with Hsp70 in cell lysates. In vitro, the purified Hsp82 mutant proteins were defective in direct physical interaction with Ssa1 and in protein remodeling in collaboration with Ssa1 and cochaperones. This region of Hsp90 is also important for interactions with several Hsp90 cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface.

Rad25 protein is targeted for degradation by the Ubc4-Ufd4 pathway.

Proteasome-mediated proteolysis provides dynamic spatial and temporal modulation of protein concentration in response to various intrinsic and extrinsic challenges. To gain a better understanding of the role of the proteasome in DNA repair, we systematically monitored the stability of 26 proteins involved in nucleotide excision repair (NER) under normal growth conditions. Among six NER factors found to be regulated by the proteasome, we further delineated the specific pathway involved in the degradation of Rad25, a subunit of TFIIH. We demonstrate that Rad25 turnover requires the ubiquitin-conjugating enzyme Ubc4 and the ubiquitin ligase Ufd4. Interestingly, the deletion of UFD4 specifically suppresses the rad25 mutant defective in transcription. Our results reveal a novel function of the Ufd4 pathway and another tie between the proteasome and NER regulators.

The Hsp90 cochaperones Cpr6, Cpr7, and Cns1 interact with the intact ribosome.

The abundant molecular chaperone Hsp90 is essential for the folding and stabilization of hundreds of distinct client proteins. Hsp90 is assisted by multiple cochaperones that modulate Hsp90's ATPase activity and/or promote client interaction, but the in vivo functions of many of these cochaperones are largely unknown. We found that Cpr6, Cpr7, and Cns1 interact with the intact ribosome and that Saccharomyces cerevisiae lacking CPR7 or containing mutations in CNS1 exhibited sensitivity to the translation inhibitor hygromycin. Cpr6 contains a peptidyl-prolyl isomerase (PPIase) domain and a tetratricopeptide repeat (TPR) domain flanked by charged regions. Truncation or alteration of basic residues near the carboxy terminus of Cpr6 disrupted ribosome interaction. Cns1 contains an amino-terminal TPR domain and a poorly characterized carboxy-terminal domain. The isolated carboxy-terminal domain was able to interact with the ribosome. Although loss of CPR6 does not cause noticeable growth defects, overexpression of CPR6 results in enhanced growth defects in cells expressing the temperature-sensitive cns1-G90D mutation (the G-to-D change at position 90 encoded by cns1). Cpr6 mutants that exhibit reduced ribosome interaction failed to cause growth defects, indicating that ribosome interaction is required for in vivo functions of Cpr6. Together, these results represent a novel link between the Hsp90 molecular-chaperone machine and protein synthesis.

The cochaperone SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) demonstrates regulatory specificity for the androgen, glucocorticoid, and progesterone receptors.

Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity. However, the specificity of SGTA for additional members of the steroid hormone receptor superfamily and the mechanism by which SGTA regulates receptor activity remain unclear. Here we report that SGTA associates with and specifically regulates the androgen, glucocorticoid, and progesterone receptors and has no effect on the mineralocorticoid and estrogen receptors in both yeast and mammalian cell-based reporter assays. In both systems, SGTA knockdown/deletion enhances receptor activity, whereas SGTA overexpression suppresses receptor activity. We demonstrate that SGTA binds directly to Hsp70 and Hsp90 in vitro with similar affinities yet predominately precipitates with Hsp70 from cell lysates, suggesting a role for SGTA in early, Hsp70-mediated folding. Furthermore, SGTA expression completely abrogates the regulation of receptor function by FKBP52 (52-kDa FK506-binding protein), which acts at a later stage of the chaperone cycle. Taken together, our data suggest a role for SGTA at distinct steps in the chaperone-dependent modulation of androgen, glucocorticoid, and progesterone receptor activity.

Interaction of heat shock protein 90 and the co-chaperone Cpr6 with Ura2, a bifunctional enzyme required for pyrimidine biosynthesis.

The molecular chaperone heat shock protein 90 (Hsp90) is an essential protein required for the activity and stability of multiple proteins termed clients. Hsp90 cooperates with a set of co-chaperone proteins that modulate Hsp90 activity and/or target clients to Hsp90 for folding. Many of the Hsp90 co-chaperones, including Cpr6 and Cpr7, contain tetratricopeptide repeat (TPR) domains that bind a common acceptor site at the carboxyl terminus of Hsp90. We found that Cpr6 and Hsp90 interacted with Ura2, a protein critical for pyrimidine biosynthesis. Mutation or inhibition of Hsp90 resulted in decreased accumulation of Ura2, indicating it is an Hsp90 client. Cpr6 interacted with Ura2 in the absence of stable Cpr6-Hsp90 interaction, suggesting a direct interaction. However, loss of Cpr6 did not alter the Ura2-Hsp90 interaction or Ura2 accumulation. The TPR domain of Cpr6 was required for Ura2 interaction, but other TPR containing co-chaperones, including Cpr7, failed to interact with Ura2 or rescue CPR6-dependent growth defects. Further analysis suggests that the carboxyl-terminal 100 amino acids of Cpr6 and Cpr7 are critical for specifying their unique functions, providing new information about this important class of Hsp90 co-chaperones.

Mutation of essential Hsp90 co-chaperones SGT1 or CNS1 renders yeast hypersensitive to overexpression of other co-chaperones.

The essential molecular chaperone Hsp90 functions with over ten co-chaperones in Saccharomyces cerevisiae, but the in vivo roles of many of these co-chaperones are poorly understood. Two of these co-chaperones, Cdc37 and Sgt1, target specific types of clients to Hsp90 for folding. Other co-chaperones have general roles in supporting Hsp90 function, but the degree of overlapping or competing functions is unclear. None of the chaperones, when overexpressed, were able to rescue the lethality of an SGT1 disruption strain. However, overexpression of SBA1, PPT1, AHA1 or HCH1 caused varying levels of growth defects in an sgt1-K360E strain. Negative effects of CPR6 overexpression were similarly observed in cells expressing the temperature-sensitive mutation cns1-G90D. In all cases, alterations within co-chaperones designed to disrupt Hsp90 interaction relieved the negative growth defects. Sgt1-K360E and Cns1-G90D were previously shown to exhibit reduced Hsp90 interaction. Our results indicate that overexpression of other co-chaperones further disrupts the essential functions of Cns1 and Sgt1. However, the specificity of the negative effects indicates that only a subset of co-chaperones competes with Sgt1 or Cns1 for binding to Hsp90. This provides new evidence that co-chaperones selectively compete for binding to subpopulations of cellular Hsp90 and suggest that changes in the relative levels of co-chaperones may have dramatic effects on Hsp90 function.

Insights into Hsp90 mechanism and in vivo functions learned from studies in the yeast, Saccharomyces cerevisiae.

The molecular chaperone Hsp90 (Heat shock protein, 90 kDa) is an abundant and essential cytosolic protein required for the stability and/or folding of hundreds of client proteins. Hsp90, along with helper cochaperone proteins, assists client protein folding in an ATP-dependent pathway. The laboratory of Susan Lindquist, in collaboration with other researchers, was the first to establish the yeast Saccharomyces cerevisiae as a model organism to study the functional interaction between Hsp90 and clients. Important insights from studies in her lab were that Hsp90 is essential, and that Hsp90 functions and cochaperone interactions are highly conserved between yeast and mammalian cells. Here, we describe key mechanistic insights into the Hsp90 folding cycle that were obtained using the yeast system. We highlight the early contributions of the laboratory of Susan Lindquist and extend our analysis into the broader use of the yeast system to analyze the understanding of the conformational cycle of Hsp90 and the impact of altered Hsp90 function on the proteome.

Quantitative proteomic analysis reveals unique Hsp90 cycle-dependent client interactions.

Hsp90 is an abundant and essential molecular chaperone that mediates the folding and activation of client proteins in a nucleotide-dependent cycle. Hsp90 inhibition directly or indirectly impacts the function of 10-15% of all proteins due to degradation of client proteins or indirect downstream effects. Due to its role in chaperoning oncogenic proteins, Hsp90 is an important drug target. However, compounds that occupy the ATP-binding pocket and broadly inhibit function have not achieved widespread use due to negative effects. More selective inhibitors are needed; however, it is unclear how to achieve selective inhibition. We conducted a quantitative proteomic analysis of soluble proteins in yeast strains expressing wild-type Hsp90 or mutants that disrupt different steps in the client folding pathway. Out of 2,482 proteins in our sample set (approximately 38% of yeast proteins), we observed statistically significant changes in abundance of 350 (14%) of those proteins (log2 fold change ≥ 1.5). Of these, 257/350 (∼73%) with the strongest differences in abundance were previously connected to Hsp90 function. Principal component analysis of the entire dataset revealed that the effects of the mutants could be separated into 3 primary clusters. As evidence that Hsp90 mutants affect different pools of clients, simultaneous co-expression of 2 mutants in different clusters restored wild-type growth. Our data suggest that the ability of Hsp90 to sample a wide range of conformations allows the chaperone to mediate folding of a broad array of clients and that disruption of conformational flexibility results in client defects dependent on those states.

Evolution and function of diverse Hsp90 homologs and cochaperone proteins.

Members of the Hsp90 molecular chaperone family are found in the cytosol, ER, mitochondria and chloroplasts of eukaryotic cells, as well as in bacteria. These diverse family members cooperate with other proteins, such as the molecular chaperone Hsp70, to mediate protein folding, activation and assembly into multiprotein complexes. All examined Hsp90 homologs exhibit similar ATPase rates and undergo similar conformational changes. One of the key differences is that cytosolic Hsp90 interacts with a large number of cochaperones that regulate the ATPase activity of Hsp90 or have other functions, such as targeting clients to Hsp90. Diverse Hsp90 homologs appear to chaperone different types of client proteins. This difference may reflect either the pool of clients requiring Hsp90 function or the requirement for cochaperones to target clients to Hsp90. This review discusses known functions, similarities and differences between Hsp90 family members and how cochaperones are known to affect these functions. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the Glucocorticoid Receptor.

Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins, including the glucocorticoid receptor (GR)1-3. Previously, we revealed that GR ligand binding activity is inhibited by Hsp70 and restored by Hsp90, aided by co-chaperones4. We then presented cryo-EM structures mechanistically detailing how Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding5,6. In vivo, GR-chaperone complexes are found associated with numerous Hsp90 co-chaperones, but the most enigmatic have been the immunophilins FKBP51 and FKBP52, which further regulate the activity of GR and other steroid receptors7-9. A molecular understanding of how FKBP51 and FKBP52 integrate with the GR chaperone cycle to differentially regulate GR activation in vivo is lacking due to difficulties reconstituting these interactions. Here, we present a 3.01 Å cryo-EM structure of the GR:Hsp90:FKBP52 complex, revealing , for the first time, that FKBP52 directly binds to the folded, ligand-bound GR using three novel interfaces, each of which we demonstrate are critical for FKBP52-dependent potentiation of GR activity in vivo. In addition, we present a 3.23 Å cryo-EM structure of the GR:Hsp90:FKBP51 complex, which, surprisingly, largely mimics the GR:Hsp90:FKBP52 structure. In both structures, FKBP51 and FKBP52 directly engage the folded GR and unexpectedly facilitate release of p23 through an allosteric mechanism. We also reveal that FKBP52, but not FKBP51, potentiates GR ligand binding in vitro, in a manner dependent on FKBP52-specific interactions. Altogether, we reveal how FKBP51 and FKBP52 integrate into the GR chaperone cycle to advance GR to the next stage of maturation and how FKBP51 and FKBP52 compete for GR:Hsp90 binding, leading to functional antagonism.

Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the glucocorticoid receptor.

Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins, including the glucocorticoid receptor (GR). Previously, we revealed that Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding, aided by co-chaperones. In vivo, the co-chaperones FKBP51 and FKBP52 antagonistically regulate GR activity, but a molecular understanding is lacking. Here we present a 3.01 Å cryogenic electron microscopy structure of the human GR:Hsp90:FKBP52 complex, revealing how FKBP52 integrates into the GR chaperone cycle and directly binds to the active client, potentiating GR activity in vitro and in vivo. We also present a 3.23 Å cryogenic electron microscopy structure of the human GR:Hsp90:FKBP51 complex, revealing how FKBP51 competes with FKBP52 for GR:Hsp90 binding and demonstrating how FKBP51 can act as a potent antagonist to FKBP52. Altogether, we demonstrate how FKBP51 and FKBP52 integrate into the GR chaperone cycle to advance GR to the next stage of maturation.

Identification of an Hsp90 mutation that selectively disrupts cAMP/PKA signaling in Saccharomyces cerevisiae.

The molecular chaperone Hsp90 cooperates with multiple cochaperone proteins as it promotes the folding and activation of diverse client proteins. Some cochaperones regulate the ATPase activity of Hsp90, while others appear to promote Hsp90 interaction with specific types of client proteins. Through its interaction with the adenylate cyclase Cyr1, the Sgt1 cochaperone modulates the activity of the cAMP pathway in Saccharomyces cerevisiae. A specific mutation in yeast Hsp90, hsc82-W296A, or a mutation in Sgt1, sgt1-K360E, resulted in altered transcription patterns genetically linked to the cAMP pathway. Hsp90 interacted with Cyr1 in vivo and the hsc82-W296A mutation resulted in reduced accumulation of Cyr1. Hsp90-Sgt1 interaction was altered by either the hsc82-W296A or sgt1-K360E mutation, suggesting defective Hsp90-Sgt1 cooperation leads to reduced Cyr1 activity. Microarray analysis of hsc82-W296A cells indicated that over 80 % of all transcriptional changes in this strain may be attributed to altered cAMP signaling. This suggests that a majority of the cellular defects observed in hsc82-W296A cells are due to altered interaction with one specific essential cochaperone, Sgt1 and one essential client, Cyr1. Together our results indicate that specific interaction of Hsp90 and Sgt1 with Cyr1 plays a key role in regulating gene expression, including genes involved in polarized morphogenesis.

Glaucoma-associated WDR36 variants encode functional defects in a yeast model system.

Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. POAG is associated with a characteristic progression of changes to ocular morphology and degeneration at the optic nerve head with the loss of visual fields. Physical mapping efforts identified genomic loci in which to search for causative POAG gene mutations. WDR36, at locus GLC1G, was initially identified as a gene with a low frequency of non-synonymous sequence variations that were exclusive to adult-onset POAG patients. It has since been shown that rare WDR36 sequence variants are also present in the normal population at similarly low frequencies. The lack of a consistent genotype:phenotype correlation prompted us to investigate the functional consequences of WDR36 sequence variations. WDR36 is involved in rRNA processing, a critical step in ribosome biogenesis, and is very similar to yeast Utp21p which is a member of the small subunit (SSU) processome complex responsible for maturation of 18S rRNA. We, therefore, developed a yeast model system to test the functional and phenotypic consequences of POAG-associated sequence variants introduced into UTP21. Alone, the POAG variants did not produce any significant defects in cell viability or rRNA processing. However, when combined with disruption of STI1 (which synthetically interacts with UTP21), 5 of the 11 tested variants had increased or decreased cell viability which corresponded to reduced or elevated levels of pre-rRNA, respectively. These results demonstrate that, in the correct genetic background, WDR36 sequence variants can lead to an altered cellular phenotype, supporting the theory that WDR36 participates in polygenic forms of glaucoma.

Mutations in Hsp90 Cochaperones Result in a Wide Variety of Human Disorders.

The Hsp90 molecular chaperone, along with a set of approximately 50 cochaperones, mediates the folding and activation of hundreds of cellular proteins in an ATP-dependent cycle. Cochaperones differ in how they interact with Hsp90 and their ability to modulate ATPase activity of Hsp90. Cochaperones often compete for the same binding site on Hsp90, and changes in levels of cochaperone expression that occur during neurodegeneration, cancer, or aging may result in altered Hsp90-cochaperone complexes and client activity. This review summarizes information about loss-of-function mutations of individual cochaperones and discusses the overall association of cochaperone alterations with a broad range of diseases. Cochaperone mutations result in ciliary or muscle defects, neurological development or degeneration disorders, and other disorders. In many cases, diseases were linked to defects in established cochaperone-client interactions. A better understanding of the functional consequences of defective cochaperones will provide new insights into their functions and may lead to specialized approaches to modulate Hsp90 functions and treat some of these human disorders.

Human Hsp90 cochaperones: perspectives on tissue-specific expression and identification of cochaperones with similar in vivo functions.

The Hsp90 molecular chaperone is required for the function of hundreds of different cellular proteins. Hsp90 and a cohort of interacting proteins called cochaperones interact with clients in an ATP-dependent cycle. Cochaperone functions include targeting clients to Hsp90, regulating Hsp90 ATPase activity, and/or promoting Hsp90 conformational changes as it progresses through the cycle. Over the last 20 years, the list of cochaperones identified in human cells has grown from the initial six identified in complex with steroid hormone receptors and protein kinases to about fifty different cochaperones found in Hsp90-client complexes. These cochaperones may be placed into three groups based on shared Hsp90 interaction domains. Available evidence indicates that cochaperones vary in client specificity, abundance, and tissue distribution. Many of the cochaperones have critical roles in regulation of cancer and neurodegeneration. A more limited set of cochaperones have cellular functions that may be limited to tissues such as muscle and testis. It is likely that a small set of cochaperones are part of the core Hsp90 machinery required for the folding of a wide range of clients. The presence of more selective cochaperones may allow greater control of Hsp90 activities across different tissues or during development.

Disrupting progression of the yeast Hsp90 folding pathway at different transition points results in client-specific maturation defects.

The protein molecular chaperone Hsp90 (Heat shock protein, 90 kilodalton) plays multiple roles in the biogenesis and regulation of client proteins impacting myriad aspects of cellular physiology. Amino acid alterations located throughout Saccharomyces cerevisiae Hsp90 have been shown to result in reduced client activity and temperature-sensitive growth defects. Although some Hsp90 mutants have been shown to affect activity of particular clients more than others, the mechanistic basis of client-specific effects is unknown. We found that Hsp90 mutants that disrupt the early step of Hsp70 and Sti1 interaction, or show reduced ability to adopt the ATP-bound closed conformation characterized by Sba1 and Cpr6 interaction, similarly disrupt activity of three diverse clients, Utp21, Ssl2, and v-src. In contrast, mutants that appear to alter other steps in the folding pathway had more limited effects on client activity. Protein expression profiling provided additional evidence that mutants that alter similar steps in the folding cycle cause similar in vivo consequences. Our characterization of these mutants provides new insight into how Hsp90 and cochaperones identify and interact with diverse clients, information essential for designing pharmaceutical approaches to selectively inhibit Hsp90 function.