Prion propagation by Hsp40 molecular chaperones

Molecular chaperones regulate essential steps in the propagation of yeast prions. Yeast prions possess domains enriched in glutamines and asparagines that act as templates to drive the assembly of native proteins into beta-sheet-rich, amyloid-like fibrils. Several recent studies highlight a significant and complex function for Hsp40 co-chaperones in propagation of prion elements in yeast. Hsp40 co-chaperones bind non-native polypeptides and transfer these clients to Hsp70s for refolding or degradation. How Hsp40 co-chaperones bind amyloid-like prion conformers that are enriched in hydrophilic residues such as glutamines and asparagines is a significant question in the field. Interestingly, selective recognition of amyloid-like conformers by distinct Hsp40s appears to confer opposing actions on prion assembly. For example, the Type I Hsp40 Ydj1 and Type II Hsp40 Sis1 bind different regions within the prion protein Rnq1 and function respectively to inhibit or promote [RNQ⁺] prion assembly. Thus, substrate selectivity enables distinct Hsp40s to act at unique steps in prion propagation.     

Prion propagation by Hsp40 molecular chaperones

Molecular chaperones regulate essential steps in the propagation of yeast prions. Yeast prions possess domains enriched in glutamines and asparagines that act as templates to drive the assembly of native proteins into beta-sheet-rich, amyloid-like fibrils. Several recent studies highlight a significant and complex function for Hsp40 co-chaperones in propagation of prion elements in yeast. Hsp40 co-chaperones bind non-native polypeptides and transfer these clients to Hsp70s for refolding or degradation. How Hsp40 co-chaperones bind amyloid-like prion conformers that are enriched in hydrophilic residues such as glutamines and asparagines is a significant question in the field. Interestingly, selective recognition of amyloid-like conformers by distinct Hsp40s appears to confer opposing actions on prion assembly. For example, the Type I Hsp40 Ydj1 and Type II Hsp40 Sis1 bind different regions within the prion protein Rnq1 and function respectively to inhibit or promote [RNQ⁺] prion assembly. Thus, substrate selectivity enables distinct Hsp40s to act at unique steps in prion propagation.Key words: Hsp40, Ydj1, Sis1, amyloid, prion, Rnq1, J-protein, Hsp70     

A structure-based mutational analysis of cyclophilin 40 identifies key residues in the core tetratricopeptide repeat domain that mediate binding to Hsp90

Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90beta construct and qualitatively assessed for binding using a beta-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90beta using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding.     

The crystal structure of the peptide-binding fragment from the yeast Hsp40 protein Sis1

BACKGROUND: Molecular chaperone Hsp40 can bind non-native polypeptide and facilitate Hsp70 in protein refolding. How Hsp40 and other chaperones distinguish between the folded and unfolded states of proteins to bind nonnative polypeptides is a fundamental issue. RESULTS: To investigate this mechanism, we determined the crystal structure of the peptide-binding fragment of Sis1, an essential member of the Hsp40 family from Saccharomyces cerevisiae. The 2.7 A structure reveals that Sis1 forms a homodimer in the crystal by a crystallographic twofold axis. Sis1 monomers are elongated and consist of two domains with similar folds. Sis1 dimerizes through a short C-terminal stretch. The Sis1 dimer has a U-shaped architecture and a large cleft is formed between the two elongated monomers. Domain I in each monomer contains a hydrophobic depression that might be involved in binding the sidechains of hydrophobic amino acids. CONCLUSIONS: Sis1 (1-337), which lacks the dimerization motif, exhibited severe defects in chaperone activity, but could regulate Hsp70 ATPase activity. Thus, dimer formation is critical for Sis1 chaperone function. We propose that the Sis1 cleft functions as a docking site for the Hsp70 peptide-binding domain and that Sis1-Hsp70 interaction serves to facilitate the efficient transfer of peptides from Sis1 to Hsp70.     

Conserved central domains control the quaternary structure of type I and type II Hsp40 molecular chaperones

Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed.     

Exchangeable chaperone modules contribute to specification of type I and type II Hsp40 cellular function

Hsp40 family members regulate Hsp70s ability to bind nonnative polypeptides and thereby play an essential role in cell physiology. Type I and type II Hsp40s, such as yeast Ydj1 and Sis1, form chaperone pairs with cytosolic Hsp70 Ssa1 that fold proteins with different efficiencies and carry out specific cellular functions. The mechanism by which Ydj1 and Sis1 specify Hsp70 functions is not clear. Ydj1 and Sis1 share a high degree of sequence identity in their amino and carboxyl terminal ends, but each contains a structurally unique and centrally located protein module that is implicated in chaperone function. To test whether the chaperone modules of Ydj1 and Sis1 function in the specification of Hsp70 action, we constructed a set of chimeric Hsp40s in which the chaperone domains of Ydj1 and Sis1 were swapped to form YSY and SYS. Purified SYS and YSY exhibited protein-folding activity and substrate specificity that mimicked that of Ydj1 and Sis1, respectively. In in vivo studies, YSY exhibited a gain of function and, unlike Ydj1, could complement the lethal phenotype of sis1 Delta and facilitate maintenance of the prion [RNQ+]. Ydj1 and Sis1 contain exchangeable chaperone modules that assist in specification of Hsp70 function.     

Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex

Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40-Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a beta-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge-charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.     

The glycine-phenylalanine-rich region determines the specificity of the yeast Hsp40 Sis1.

Hsp40s are ubiquitous, conserved proteins which function with molecular chaperones of the Hsp70 class. Sis1 is an essential Hsp40 of the cytosol of Saccharomyces cerevisiae, thought to be required for initiation of translation. We carried out a genetic analysis to determine the regions of Sis1 required to perform its key function(s). A C-terminal truncation of Sis1, removing 231 amino acids but retaining the N-terminal 121 amino acids encompassing the J domain and the glycine-phenylalanine-rich (G-F) region, was able to rescue the inviability of a Deltasis1 strain. The yeast cytosol contains other Hsp40s, including Ydj1. To determine which regions carried the critical determinants of Sis1 function, we constructed chimeric genes containing portions of SIS1 and YDJ1. A chimera containing the J domain of Sis1 and the G-F region of Ydj1 could not rescue the lethality of the Deltasis1 strain. However, a chimera with the J domain of Ydj1 and the G/F region of Sis1 could rescue the strain's lethality, indicating that the G-F region is a unique region required for the essential function of Sis1. However, a J domain is also required, as mutants expected to cause a disruption of the interaction of the J domain with Hsp70 are inviable. We conclude that the G-F region, previously thought only to be a linker or spacer region between the J domain and C-terminal regions of Hsp40s, is a critical determinant of Sis1 function.     

The Type II Hsp40 Sis1 Cooperates with Hsp70 and the E3 Ligase Ubr1 to Promote Degradation of Terminally Misfolded Cytosolic Protein

Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.     

Specificity of class II Hsp40 Sis1 in maintenance of yeast prion [RNQ+

Sis1 and Ydj1, functionally distinct heat shock protein (Hsp)40 molecular chaperones of the yeast cytosol, are homologs of Hdj1 and Hdj2 of mammalian cells, respectively. Sis1 is necessary for propagation of the Saccharomyces cerevisiae prion [RNQ(+)]; Ydj1 is not. The ability to function in [RNQ(+)] maintenance has been conserved, because Hdj1 can function to maintain Rnq1 in an aggregated form in place of Sis1, but Hdj2 cannot. An extended glycine-rich region of Sis1, composed of a region rich in phenylalanine residues (G/F) and another rich in methionine residues (G/M), is critical for prion maintenance. Single amino acid alterations in a short stretch of amino acids of the G/F region of Sis1 that are absent in the otherwise highly conserved G/F region of Ydj1 cause defects in prion maintenance. However, there is some functional redundancy within the glycine-rich regions of Sis1, because a deletion of the adjacent glycine/methionine (G/M) region was somewhat defective in propagation of [RNQ(+)] as well. These results are consistent with a model in which the glycine-rich regions of Hsp40s contain specific determinants of function manifested through interaction with Hsp70s.     

The crystal structure of the putative peptide-binding fragment from the human Hsp40 protein Hdj1

Background  

The mechanism by which Hsp40 and other molecular chaperones recognize and interact with non-native polypeptides is a fundamental question. How Hsp40 co-operates with Hsp70 to facilitate protein folding is not well understood. To investigate the mechanisms, we determined the crystal structure of the putative peptide-binding fragment of Hdj1, a human member of the type II Hsp40 family.     

Convergent evolution of clamp-like binding sites in diverse chaperones

Molecular chaperones have evolved diverse tertiary and quaternary structures to stabilize non-native polypeptides and facilitate their transition to the native state. Indeed, different families of chaperones lack sequence similarity, and few are represented ubiquitously in all three domains of life. Despite their discrete evolutionary paths, recent crystal structures reveal that many chaperones use seemingly convergent strategies to bind non-native proteins. This crystallographic evidence shows, or strongly suggests, that chaperones including prefoldin, Skp, trigger factor, Hsp40 and Hsp90 have clamp-like structural features used to grip substrate proteins. We explore the notion that clamp-like structures are evolutionarily favored by both ATP-dependent and ATP-independent molecular chaperones. Presumably, clamps present a multivalent binding surface ideal for protecting unstable protein conformers until they reach the native state or are transferred to another component of the folding machinery.     

Functions of yeast Hsp40 chaperone Sis1p dispensable for prion propagation but important for prion curing and protection from prion toxicity

Replication of amyloid-based yeast prions [PSI(+)], [URE3], and [PIN(+)] depends on the protein disaggregation machinery that includes Hsp104, Hsp70, and Hsp40 molecular chaperones. Yet, overexpressing Hsp104 cures cells of [PSI(+)] prions. An Hsp70 mutant (Ssa1-21p) antagonizes propagation of [PSI(+)] in a manner resembling elevated Hsp104. The major cytosolic Hsp40 Sis1p is the only Hsp40 required for replication of these prions, but its role in [PSI(+)] curing is unknown. Here we find that all nonessential functional regions of Sis1p are dispensable for [PSI(+)] propagation, suggesting that other Hsp40's might provide Hsp40 functions required for [PSI(+)] replication. Conversely, several Sis1p functions were important for promoting antiprion effects of both Ssa1-21p and Hsp104, which implies a link between the antiprion effects of these chaperones and suggests that Sis1p is a specific Hsp40 important for [PSI(+)] curing. These contrasting findings suggest that the functions of Hsp104 that are important for propagation and elimination of [PSI(+)] are either distinct or specified by different Hsp40's. This work also uncovered a growth inhibition caused by [PSI(+)] when certain functions of Sis1p were absent, suggesting that Sis1p protects cells from cytotoxicity caused by [PSI(+)] prions.     

An essential role for the substrate-binding region of Hsp40s in Saccharomyces cerevisiae

In addition to regulating the ATPase cycle of Hsp70, a second critical role of Hsp40s has been proposed based on in vitro studies: binding to denatured protein substrates, followed by their presentation to Hsp70 for folding. However, the biological importance of this model is challenged by the fact that deletion of the substrate-binding domain of either of the two major Hsp40s of the yeast cytosol, Ydj1 and Sis1, leads to no severe defects, as long as regions necessary for Hsp70 interaction are retained. As an in vivo test of this model, requirements for viability were examined in a strain having deletions of both Hsp40 genes. Despite limited sequence similarity, the substrate-binding domain of either Sis1 or Ydj1 allowed cell growth, indicating they share overlapping essential functions. Furthermore, the substrate-binding domain must function in cis with a functional Hsp70-interacting domain. We conclude that the ability of cytosolic Hsp40s to bind unfolded protein substrates is an essential function in vivo.     

Identification of Regions Involved in Substrate Binding and Dimer Stabilization within the Central Domains of Yeast Hsp40 Sis1

Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I) deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1) Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2) The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3) The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4) Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein.     

The crystal structure of the yeast Hsp40 Ydj1 complexed with its peptide substrate

The mechanisms by which Hsp40 functions as a molecular chaperone to recognize and bind non-native polypeptides is not understood. We have identified a peptide substrate for Ydj1, a member of the type I Hsp40 from yeast. The structure of the Ydj1 peptide binding fragment and its peptide substrate complex was determined to 2.7 A resolution. The complex structure reveals that Ydj1 peptide binding fragment forms an L-shaped molecule constituted by three domains. The domain I exhibits a similar protein folds as domain III while the domain II contains two Zinc finger motifs. The peptide substrate binds Ydj1 by forming an extra beta strand with domain I of Ydj1. The Leucine residue in the middle of the peptide substrate GWLYEIS inserts its side chain into a hydrophobic pocket formed on the molecular surface of Ydj1 domain I. The Zinc finger motifs located in the Ydj1 domain II are not in the vicinity of peptide substrate binding site.     

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.     

Hsp40s Specify Functions of Hsp104 and Hsp90 Protein Chaperone Machines

Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI⁺] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN⁺] prions was less sensitive than [URE3], but more sensitive than [PSI⁺]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.     

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.     

A domain in the N-terminal part of Hsp26 is essential for chaperone function and oligomerization

Small heat-shock proteins (Hsps) are ubiquitous molecular chaperones which prevent the unspecific aggregation of non-native proteins. For Hsp26, a cytosolic sHsp from of Saccharomyces cerevisiae, it has been shown that, at elevated temperatures, the 24 subunit complex dissociates into dimers. This dissociation is required for the efficient interaction with non-native proteins. Deletion analysis of the protein showed that the N-terminal half of Hsp26 (amino acid residues 1-95) is required for the assembly of the oligomer. Limited proteolysis in combination with mass spectrometry suggested that this region can be divided in two parts, an N-terminal segment including amino acid residues 1-30 and a second part ranging from residues 31-95. To analyze the structure and function of the N-terminal part of Hsp26 we created a deletion mutant lacking amino acid residues 1-30. We show that the oligomeric state and the structure, as determined by size exclusion chromatography and electron microscopy, corresponds to that of the Hsp26 wild-type protein. Furthermore, this truncated version of Hsp26 is active as a chaperone. However, in contrast to full length Hsp26, the truncated version dissociates at lower temperatures and complexes with non-native proteins are less stable than those found with wild-type Hsp26. Our results suggest that the N-terminal segment of Hsp26 is involved in both, oligomerization and chaperone function and that the second part of the N-terminal region (amino acid residues 31-95) is essential for both functions.