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.     

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.     

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.     

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.     

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.     

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.     

Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates.     

Multiple domains of the co-chaperone Hop are important for Hsp70 binding

The Hop/Sti1 co-chaperone binds to both Hsp70 and Hsp90. Biochemical and co-crystallographic studies have suggested that the EEVD-containing C terminus of Hsp70 or Hsp90 binds specifically to one of the Hop tetratricopeptide repeat domains, TPR1 or TPR2a, respectively. Mutational analyses of Hsp70 and Hop were undertaken to better characterize interactions between the C terminus of Hsp70 and Hop domains. Surprisingly, truncation of EEVD plus as many as 34 additional amino acids from the Hsp70 C terminus did not reduce the ability of Hsp70 mutants to co-immunoprecipitate with Hop, although further truncation eliminated Hop binding. Hop point mutations targeting a carboxylate clamp position in TPR1 disrupted Hsp70 binding, as was expected; however, similar point mutations in TPR2a or TPR2b also inhibited Hsp70 binding in some settings. Using a yeast-based in vivo assay for Hop function, wild type Hop and TPR2b mutants could fully complement deletion of Sti1p; TPR1 and TPR2a point mutants could partially restore activity. Conformations of Hop and Hop mutants were probed by limited proteolysis. The TPR1 mutant digested in a similar manner to wild type; however, TPR2a and TPR2b mutants each displayed greater resistance to chymotryptic digestion. All point mutants retained an ability to dimerize, and none appeared to be grossly misfolded. These results raise questions about current models for Hop/Hsp70 interaction.     

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.     

Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

In the eukaryotic cytosol, Hsp70 and Hsp90 cooperate with various co-chaperone proteins in the folding of a growing set of substrates, including the glucocorticoid receptor (GR). Here, we analyse the function of the co-chaperone Tpr2, which contains two chaperone-binding TPR domains and a DnaJ homologous J domain. In vivo, an increase or decrease in Tpr2 expression reduces GR activation, suggesting that Tpr2 is required at a narrowly defined expression level. As shown in vitro, Tpr2 recognizes both Hsp70 and Hsp90 through its TPR domains, and its J domain stimulates ATP hydrolysis and polypeptide binding by Hsp70. Furthermore, unlike other co-chaperones, Tpr2 induces ATP-independent dissociation of Hsp90 but not of Hsp70 from chaperone-substrate complexes. Excess Tpr2 inhibits the Hsp90-dependent folding of GR in cell lysates. We propose a novel mechanism in which Tpr2 mediates the retrograde transfer of substrates from Hsp90 onto Hsp70. At normal levels substoichiometric to Hsp90 and Hsp70, this activity optimizes the function of the multichaperone machinery.     

Identification of Novel Host Factors via Conserved Domain Search: Cns1 Cochaperone Is a Novel Restriction Factor of Tombusvirus Replication in Yeast

A large number of host-encoded proteins affect the replication of plus-stranded RNA viruses by acting as susceptibility factors. Many other cellular proteins are known to function as restriction factors of viral infections. Previous studies with tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the inhibitory function of TPR (tetratricopeptide repeat) domain-containing cyclophilins, which are members of the large family of host prolyl isomerases, in TBSV replication. In this paper, we tested additional TPR-containing yeast proteins in a cell-free TBSV replication assay and identified the Cns1p cochaperone for heat shock protein 70 (Hsp70) and Hsp90 chaperones as a strong inhibitor of TBSV replication. Cns1p interacted with the viral replication proteins and inhibited the assembly of the viral replicase complex and viral RNA synthesis in vitro. Overexpression of Cns1p inhibited TBSV replication in yeast. The use of a temperature-sensitive (TS) mutant of Cns1p in yeast revealed that at a semipermissive temperature, TS Cns1p could not inhibit TBSV replication. Interestingly, Cns1p and the TPR-containing Cpr7p cyclophilin have similar inhibitory functions during TBSV replication, although some of the details of their viral restriction mechanisms are different. Our observations indicate that TPR-containing cellular proteins could act as virus restriction factors.     

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.     

Two tetratricopeptide repeat proteins facilitate human aryl hydrocarbon receptor signalling in yeast

A human aryl hydrocarbon (Ah) receptor signalling pathway was constructed in yeast and used to identify regulatory proteins that may be related to those present in mammalian cells. The sequence similarity of human hepatitis B protein X-associated protein 2 (XAP2) protein to yeast Cpr7 and Cns1 proteins suggested that these proteins might be involved in Ah receptor signalling in this model system. Ah receptor signalling from a lacZ reporter gene was reduced by approximately 60% in cells that lacked Cpr7. In vitro interaction experiments indicated that a Cpr7-GST fusion protein and Ah receptor formed a complex. Expression of Cpr7, Cns1 and the isolated tetratricopeptide repeat (TPR) region of Cpr7 from plasmids restored Ah receptor signalling function in the Cpr7-deficient strain. Thus, Cpr7 and Cns1 proteins facilitate the signalling of human Ah receptor expressed in yeast, perhaps in the same manner as the TPR-containing XAP2 protein and related chaperone proteins in mammalian cells.     

Hsp90-Associated Immunophilin Homolog Cpr7 Is Required for the Mitotic Stability of [URE3] Prion in Saccharomyces cerevisiae

The role of Hsp70 chaperones in yeast prion propagation is well established. Highly conserved Hsp90 chaperones participate in a number of cellular processes, such as client protein maturation, protein degradation, cellular signalling and apoptosis, but little is known about their role in propagation of infectious prion like aggregates. Here, we examine the influence of Hsp90 in the maintenance of yeast prion [URE3] which is a prion form of native protein Ure2, and reveal a previously unknown role of Hsp90 as an important regulator of [URE3] stability. We show that the C-terminal MEEVD pentapeptide motif, but not the client maturation activity of Hsp90, is essential for [URE3] prion stability. In testing deletions of various Hsp90 co-chaperones known to bind this motif, we find the immunophilin homolog Cpr7 is essential for [URE3] propagation. We show that Cpr7 interacts with Ure2 and enhances its fibrillation. The requirement of Cpr7 is specific for [URE3] as its deletion does not antagonize both strong and weak variant of another yeast prion [PSI ⁺], suggesting a distinct role of the Hsp90 co-chaperone with different yeast prions. Our data show that, similar to the Hsp70 family, the Hsp90 chaperones also influence yeast prion maintenance, and that immunophilins could regulate protein multimerization independently of their activity as peptidyl-prolyl isomerases.     

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.     

Functional similarity between the chloroplast translocon component, Tic40, and the human co-chaperone, Hsp70-interacting protein (Hip)

Tic40 is a component of the protein import apparatus of the inner envelope of chloroplasts, but its role in the import mechanism has not been clearly defined. The C terminus of Tic40 shares weak similarity with the C-terminal Sti1 domains of the mammalian Hsp70-interacting protein (Hip) and Hsp70/Hsp90-organizing protein (Hop) co-chaperones. Additionally, Tic40 may possess a tetratricopeptide repeat (TPR) protein-protein interaction domain, another characteristic feature of Hip/Hop co-chaperones. To investigate the functional importance of different parts of the Tic40 protein and to determine whether the homology between Tic40 and co-chaperones is functionally significant, different Tic40 deletion and Tic40:Hip fusion constructs were generated and assessed for complementation activity in the Arabidopsis Tic40 knock-out mutant, tic40. Interestingly, all Tic40 deletion constructs failed to complement tic40, indicating that each part removed is essential for Tic40 function; these included a construct lacking the Sti1-like domain (DeltaSti1), a second lacking a central region, including the putative TPR domain (DeltaTPR), and a third lacking the predicted transmembrane anchor region. Moreover, the DeltaSti1 and DeltaTPR constructs caused strong dominant-negative, albino phenotypes in tic40 transformants, indicating that the truncated Tic40 proteins interfere with the residual chloroplast protein import that occurs in tic40 plants. Remarkably, the Tic40:Hip fusion constructs showed that the Sti1 domain of human Hip is functionally equivalent to the Sti1-like region of Tic40, strongly suggesting a co-chaperone role for the Tic40 protein. Supporting this notion, yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated the in vivo interaction of Tic40 with Tic110, a protein believed to recruit stromal chaperones to protein import sites.     

Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine

The adaptor protein Hop mediates the association of the molecular chaperones Hsp70 and Hsp90. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70 while the TPR2A domain binds the C-terminal pentapeptide of Hsp90. Both sequences end with the motif EEVD. The crystal structures of the TPR-peptide complexes show the peptides in an extended conformation, spanning a groove in the TPR domains. Peptide binding is mediated by electrostatic interactions with the EEVD motif, with the C-terminal aspartate acting as a two-carboxylate anchor, and by hydrophobic interactions with residues upstream of EEVD. The hydrophobic contacts with the peptide are critical for specificity. These results explain how TPR domains participate in the ordered assembly of Hsp70-Hsp90 multichaperone complexes.     

Ligand discrimination by TPR domains. Relevance and selectivity of EEVD-recognition in Hsp70 x Hop x Hsp90 complexes

Protein-protein interaction modules containing so-called tetratricopeptide repeats (TPRs) mediate the assembly of Hsp70/Hsp90 multi-chaperone complexes. The TPR1 and TPR2A domains of the Hsp70/Hsp90 adapter protein p60/Hop specifically bind to short peptides corresponding to the C-terminal tails of Hsp70 and Hsp90, respectively, both of which contain the highly conserved sequence motif EEVD-COOH. Here, we quantitatively assessed the contribution of TPR-mediated peptide recognition to Hsp70.Hop.Hsp90 complex formation. The interaction of TPR2A with the C-terminal pentapeptide of Hsp90 (MEEVD) is identified as the core contact for Hop binding to Hsp90. (In peptide sequences, italics are used to highlight residues specific for Hsp70 or Hsp90.) In contrast, formation of the Hsp70.Hop complex depends not only on recognition of the C-terminal Hsp70 heptapeptide (PTIEEVD) by TPR1 but also on additional contacts between Hsp70 and Hop. The sequence motifs for TPR1 and TPR2A binding were defined by alanine scanning of the C-terminal octapeptides of Hsp70 and Hsp90 and by screening of combinatorial peptide libraries. Asp0 and Val-1 of the EEVD motif are identified as general anchor residues, but the highly conserved glutamates of the EEVD sequence, which are critical in Hsp90 binding by TPR2A, do not contribute appreciably to the interaction of Hsp70 with TPR1. Rather, TPR1 prefers hydrophobic amino acids in these positions. Moreover, the TPR domains display a pronounced tendency to interact preferentially with hydrophobic aliphatic and aromatic side chains in positions -4 and -6 of their respective peptide ligands. Ile-4 in Hsp70 and Met-4 in Hsp90 are most important in determining the specific binding of TPR1 and TPR2A, respectively.     

Plasticity of the Hsp90 chaperone machine in divergent eukaryotic organisms

Hsp90 is critical for the regulation and activation of numerous client proteins critical for diverse functions such as cell growth, differentiation, and reproduction. Cytosolic Hsp90 function is dependent on a battery of co-chaperone proteins that regulate the ATPase activity of Hsp90 function or direct Hsp90 to interact with specific client proteins. Little is known about how Hsp90 complexes vary between different organisms and how this affects the scope of clients that are activated by Hsp90. This study determined whether ten distinct Hsp90 co-chaperones were encoded by genes in 19 disparate eukaryotic organisms. Surprisingly, none of the co-chaperones were present in all organisms. The co-chaperone Hop/Sti1 was most widely dispersed (18 out of 19 species), while orthologs of Cdc37, which is critical for the stability and activation of diverse protein kinases in yeast and mammals, were identified in only nine out of 19 species examined. The organism with the smallest proteome, Encephalitozoon cuniculi, contained only three of these co-chaperones, suggesting a correlation between client diversity and the complexity of the Hsp90 co-chaperone machine. Our results suggest co-chaperones are critical for cytosolic Hsp90 function in vivo, but that the composition of Hsp90 complexes varies depending on the specialized protein folding requirements of divergent species.