The small heat shock protein Hsp31 cooperates with Hsp104 to modulate Sup35 prion aggregation

The yeast homolog of DJ-1, Hsp31, is a multifunctional protein that is involved in several cellular pathways including detoxification of the toxic metabolite methylglyoxal and as a protein deglycase. Prior studies ascribed Hsp31 as a molecular chaperone that can inhibit α-Syn aggregation in vitro and alleviate its toxicity in vivo. It was also shown that Hsp31 inhibits Sup35 aggregate formation in yeast, however, it is unknown if Hsp31 can modulate [PSI⁺] phenotype and Sup35 prionogenesis. Other small heat shock proteins, Hsp26 and Hsp42 are known to be a part of a synergistic proteostasis network that inhibits Sup35 prion formation and promotes its disaggregation. Here, we establish that Hsp31 inhibits Sup35 [PSI⁺] prion formation in collaboration with a well-known disaggregase, Hsp104. Hsp31 transiently prevents prion induction but does not suppress induction upon prolonged expression of Sup35 indicating that Hsp31 can be overcome by larger aggregates. In addition, elevated levels of Hsp31 do not cure [PSI⁺] strains indicating that Hsp31 cannot intervene in a pre-existing prion oligomerization cycle. However, Hsp31 can modulate prion status in cooperation with Hsp104 because it inhibits Sup35 aggregate formation and potentiates [PSI⁺] prion curing upon overexpression of Hsp104. The absence of Hsp31 reduces [PSI⁺] prion curing by Hsp104 without influencing its ability to rescue cellular thermotolerance. Hsp31 did not synergize with Hsp42 to modulate the [PSI⁺] phenotype suggesting that both proteins act on similar stages of the prion cycle. We also showed that Hsp31 physically interacts with Hsp104 and together they prevent Sup35 prion toxicity to greater extent than if they were expressed individually. These results elucidate a mechanism for Hsp31 on prion modulation that suggest it acts at a distinct step early in the Sup35 aggregation process that is different from Hsp104. This is the first demonstration of the modulation of [PSI⁺] status by the chaperone action of Hsp31. The delineation of Hsp31's role in the chaperone cycle has implications for understanding the role of the DJ-1 superfamily in controlling misfolded proteins in neurodegenerative disease and cancer.     

Peptidase activity of the Escherichia coli Hsp31 chaperone

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys(184), His(185), and Asp(213) catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His(85), His(122), and Glu(90) metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8-12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.     

Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H₂O₂, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins.     

Structural alteration of Escherichia coli Hsp31 by thermal unfolding increases chaperone activity

Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more β-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.     

Structural and energetic insight into the isoform-selective inhibitors of tumour marker Hsp90 against Grp94

The heat shock protein 90 (Hsp90) represents a new avenue for cancer therapy. A novel benzolactam inhibitor, compound 31, showed a great selectivity for Hsp90α and Hsp90β against Grp94. However, the structural basis for the great selectivity of compound 31 for Hsp90α/β versus Grp94 remains poorly understood. In this study, we carried out molecular docking, molecular dynamics simulations and binding free energy calculations (MM-GBSA) to address the isoform selective property. Molecular docking studies indicated the different binding modes of the Hsp90 and Grp94 with compound 31. The MM-GBSA calculations revealed that the hydrophobic interactions between compound 31 and proteins contributed the most to the binding affinity and the Grp94/compound 31 complex could result in a less energy-favourable complex compared with the Hsp90α/compound 31 and the Hsp90β/compound 31 complexes. This may render the weak binding of compound 31 to the Grp94. This study may be helpful for the future design of novel and selective Hsp90 inhibitors.     

Crystal structures of human DJ-1 and Escherichia coli Hsp31, which share an evolutionarily conserved domain

Human DJ-1 and Escherichia coli Hsp31 belong to ThiJ/PfpI family, whose members contain a conserved domain. DJ-1 is associated with autosomal recessive early onset parkinsonism and Hsp31 is a molecular chaperone. Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity of DJ-1 and the proteolytic activity of Hsp31. Moreover, the comparisons provide insights into how the functional diversity is realized in proteins that share an evolutionarily conserved domain. On the basis of the chaperone activity the possible role of DJ-1 in the pathogenesis of Parkinson's disease is discussed.     

Backbone resonance assignments of the <Emphasis Type="Italic">Escherichia coli</Emphasis> 62 kDa protein,Hsp31

Dimeric Hsp31 protein was first characterized as a holding chaperone of Escherichia coli (E. coli), and has been suggested as having protease activity due to the presence of a potential catalytic triad, Cys185, His186, and Asp214. However, it has recently been reported that Hsp31 displays a relatively strong glyoxalase III activity that can decompose reactive carbonyl species (methylglyoxal and glyoxal) in the absence of additional cofactor. Hsp31 is a representative member of the DJ-1/ThiJ/PfpI protein superfamily, and the importance of DJ-1 protein in Parkinson’s disease has been well known. The structural flexibility of the long loop region, which encompasses from the P- to the A-domain, is important for the chaperone activity of Hsp31. The backbone chemical shifts (CSs) would be useful for studying the structural changes of Hsp31 that are critical for the holding chaperone activity, and also for deciphering the switching mechanism between the glyoxalase III and the chaperone. Here, we report the backbone CSs (H^N, N, CO, C_α, and C_β) of the deuterated Hsp31 protein (62 kDa). The CS analysis showed that the predicted regions of secondary structures are in good agreement with those observed in the previous crystal structure of Hsp31.     

Integrity of N‐ and C‐termini is important for E. coli Hsp31 chaperone activity

Hsp31 is a stress‐inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high‐affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20‐residue‐long hexahistidine tag to the N‐termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C‐termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging.     

Robust Glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI Family Member Protein,Is Critical for Oxidative Stress Resistance in Saccharomyces cerevisiae

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.     

Hsp31, a member of the DJ-1 superfamily,is a multitasking stress responder with chaperone activity

Among different types of protein aggregation, amyloids are a biochemically well characterized state of protein aggregation that are associated with a large number of neurodegenerative diseases including Parkinson's disease, Alzheimer and Creutzfeldt-Jakob disease. Yeast, Saccharomyces cerevisiae is an insightful model to understand the underlying mechanism of protein aggregation. Many yeast molecular chaperones can modulate aggregation and misfolding of proteins including α-Syn and the Sup35 prion. Hsp31 is a homodimeric protein structurally similar to human DJ-1, a Parkinson's disease-linked protein, and both are members of the DJ-1/ThiJ/PfpI superfamily. An emerging view is that Hsp31 and its associated superfamily members each have divergent multitasking functions that have the common theme of responding and managing various types of cellular stress. Hsp31 has several biochemical activities including chaperone and detoxifying enzyme activities that modulate at various points of a stress pathway such as toxicity associated with protein misfolding. However, we have shown the protective role of Hsp31's chaperone activity can operate independent of detoxifying enzyme activities in preventing the early stages of protein aggregate formation and associated cellular toxicities. We provide additional data that collectively supports the multiple functional roles that can be accomplished independent of each other. We present data indicating Hsp31 purified from yeast is more active compared to expression and purification from E. coli suggesting that posttranslational modifications could be important for Hsp31 to be fully active. We also compare the similarities and differences in activities among paralogs of Hsp31 supporting a model in which this protein family has overlapping but diverging roles in responding to various sources of cellular stresses.     

Absence of the Yeast Hsp31 Chaperones of the DJ-1 Superfamily Perturbs Cytoplasmic Protein Quality Control in Late Growth Phase

The Saccharomyces cerevisiae heat shock proteins Hsp31, Hsp32, Hsp33 and Hsp34 belong to the DJ-1/ThiJ/PfpI superfamily which includes the human protein DJ-1 (PARK7) as the most prominent member. Mutations in the DJ-1 gene are directly linked to autosomal recessive, early-onset Parkinson’s disease. DJ-1 acts as an oxidative stress-induced chaperone preventing aggregation and fibrillation of α-synuclein, a critical factor in the development of the disease. In vivo assays in Saccharomyces cerevisiae using the model substrate ΔssCPY*Leu2myc (ΔssCL*myc) as an aggregation-prone misfolded cytoplasmic protein revealed an influence of the Hsp31 chaperone family on the steady state level of this substrate. In contrast to the ubiquitin ligase of the N-end rule pathway Ubr1, which is known to be prominently involved in the degradation process of misfolded cytoplasmic proteins, the absence of the Hsp31 chaperone family does not impair the degradation of newly synthesized misfolded substrate. Also degradation of substrates with strong affinity to Ubr1 like those containing the type 1 N-degron arginine is not affected by the absence of the Hsp31 chaperone family. Epistasis analysis indicates that one function of the Hsp31 chaperone family resides in a pathway overlapping with the Ubr1-dependent degradation of misfolded cytoplasmic proteins. This pathway gains relevance in late growth phase under conditions of nutrient limitation. Additionally, the Hsp31 chaperones seem to be important for maintaining the cellular Ssa Hsp70 activity which is important for Ubr1-dependent degradation.     

Methylglyoxal detoxification by a DJ-1 family protein provides dual abiotic and biotic stress tolerance in transgenic plants

Methylglyoxal (MG) is a key signaling molecule resulting from glycolysis and other metabolic pathways. During abiotic stress, MG levels accumulate to toxic levels in affected cells. However, MG is routinely detoxified through the action of DJ1/PARK7/Hsp31 proteins that are highly conserved across kingdoms and mutations in such genes are associated with neurodegenerative diseases. Here, we report for the first time that, similar to abiotic stresses, MG levels increase during biotic stresses in plants, likely contributing to enhanced susceptibility to a wide range of stresses. We show that overexpression of yeast Heat shock protein 31 (Hsp31), a DJ-1 homolog with robust MG detoxifying capabilities, confers dual biotic and abiotic stress tolerance in model plant Nicotiana tabacum. Strikingly, overexpression of Hsp31 in tobacco imparts robust stress tolerance against diverse biotic stress inducers such as viruses, bacteria and fungi, in addition to tolerance against a range of abiotic stress inducers. During stress, Hsp31 was targeted to mitochondria and induced expression of key stress-related genes. These results indicate that Hsp31 is a novel attractive tool to engineer plants against both biotic and abiotic stresses.     

Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditions

Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48 degrees C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50 degrees C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB-DnaK-DnaJ-GrpE system to clear thermally aggregated proteins at 30 degrees C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, Delta clpB or Delta ibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK-DnaJ-GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding.     

A new native EcHsp31 structure suggests a key role of structural flexibility for chaperone function

Heat shock proteins and proteases play a crucial role in cell survival under conditions of environmental stress. The heat shock protein Hsp31, produced by gene hchA at elevated temperatures in Escherichia coli, is a homodimeric protein consisting of a large A domain and a smaller P domain connected by a linker. Two catalytic triads are present per dimer, with the Cys and His contributed by the A domain and an Asp by the P domain. A new crystal Form II confirms the dimer and catalytic triad arrangement seen in the earlier crystal Form I. In addition, several loops exhibit increased flexibility compared to the previous Hsp31 dimer structure. In particular, loops D2 and D3 are intriguing because their mobility leads to the exposure of a sizable hydrophobic patch made up by surface areas of both subunits near the dimer interface. The residues creating this hydrophobic surface are completely conserved in the Hsp31 family. At the same time, access to the catalytic triad is increased. These observations lead to the hypothesis for the functioning of Hsp31 wherein loops D2 and D3 play a key role: first, at elevated temperatures, by becoming mobile and uncovering a large hydrophobic area that helps in binding to client proteins, and second, by removing the client protein from the hydrophobic patch when the temperature decreases and the loops adopt their low-temperature positions at the Hsp31 surface. The proposed mode of action of flexible loops in the functioning of Hsp31 may be a general principle employed by other chaperones.     

Hsp70 protects macrophages infected with Salmonella choleraesuis against TNF-α-induced cell death

Hsp70 plays an important role in cytoprotection against tumor necrosis factor (TNF) α-mediated cytotoxicity. To investigate the role of Hsp70 in cytoprotein during Salmonella infection, we examined endogenous Hsp70 induction and TNF-α production in a monocyte/macrophage line, J774A.1, after infection with a virulent strain of Salm.choleraesuis RF-1 carrying a 50 kb virulent plasmid or the plasmid-cured avirulent strain 31N-1. Intracellular bacteria progressively increased in J774A.1 cells phagocytosing avirulent 31N-1 bacteria, whereas such progressive growth was not evident in J774A.1 cells phagocytosing avirulent 31N-1 bacteria. On the contrary, J774A.1 cells infected with virulent RF-1 bacteria expressed less Hsp70 than those infected with avirulent 31N-1 bacteria. The level of TNF-α production by J774A.1 infected with virulent RF-1 was much the same as that by J774A.1 infected with avirulent 31N-1. J774A.1 infected with virulent RF-1 died spontaneously; death was inhibited by the addition of anti-TNF-α mAb. Although the frequency of dead J774A.1 with hypodiploid DNA content increased only marginally after infection with avirulent 31N-1, treatment with Hsp70 anti-sense oligonucleotide resulted in a dramatic increase of dead cells in the infected macrophages. Taken together, these results suggest that Hsp70 induced macrophages plays an important role in host defense against Salmonella infection by protecting the macrophages against TNF α-induced cell death. Furthermore, cell death due to impaired endogenous Hsp synthesis in the phagocytes implies a novel pathogenic mechanism for virulence of Salm. choleraesuis RF-1.     

Hsp31, the Escherichia coli yedU gene product,is a molecular chaperone whose activity is inhibited by ATP at high temperatures

The Escherichia coli chromosome contains several uncharacterized heat-inducible loci that may encode novel molecular chaperones or proteases. Here we show that the 31-kDa product of the yedU gene is an efficient homodimeric molecular chaperone that is conserved in a number of pathogenic eubacteria and fungi. Heat shock protein (Hsp) 31 relies on temperature-driven conformational changes to expose structured hydrophobic domains that are likely responsible for substrate binding. Complementing the function of refolding, remodeling, and holding chaperones, Hsp 31 preferentially interacts with early unfolding intermediates and rapidly releases them in an active form after transfer to low temperatures. Although Hsp 31 does not appear to exhibit intrinsic ATPase activity, binding of ATP at high temperatures restricts the size or availability of the substrate binding site, thereby modulating chaperone activity. The possible role of ATP in coordinating the function of the cellular complement of molecular chaperones is discussed.     

Bisecting GlcNAc mediates the binding of annexin V to Hsp47

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.     

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.