Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.
ABSTRACT: By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.
Project description: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.
Project description: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.
Project description: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.
Project description: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.
Project description:Heat shock proteins of 70 kDa (Hsp70s) and their J domain-containing Hsp40 cofactors are highly conserved chaperone pairs that facilitate a large number of cellular processes. The observation that each Hsp70 partners with many J domain-containing proteins (JDPs) has led to the hypothesis that Hsp70 function is dictated by cognate JDPs. If this is true, one might expect highly divergent Hsp70-JDP pairs to be unable to function in vivo. However, we discovered that, when a yeast cytosolic JDP, Ydj1, was targeted to the mammalian endoplasmic reticulum (ER), it interacted with the ER-lumenal Hsp70, BiP, and bound to BiP substrates. Conversely, when a mammalian ER-lumenal JDP, ERdj3, was directed to the yeast cytosol, it rescued the temperature-sensitive growth phenotype of yeast-containing mutant alleles in two cytosolic JDPs, HLJ1 and YDJ1, and activated the ATP hydrolysis rate of Ssa1, the yeast cytosolic Hsp70 that partners with Hlj1 and Ydj1. Surprisingly, ERdj3 mutants that were compromised for substrate binding were unable to rescue the hlj1ydj1 growth defect even though they stimulated the ATPase activity of Ssa1. Yet, J domain mutants of ERdj3 that were defective for interaction with Ssa1 restored the growth of hlj1ydj1 yeast. Taken together, these data suggest that the substrate binding properties of certain JDPs, not simply the formation of unique Hsp70-JDP pairs, are critical to specify in vivo function.
Project description:Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70?EEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.
Project description:Heat-shock protein 40 (Hsp40) enables Hsp70 to play critical roles in a number of cellular processes, such as protein folding, assembly, degradation and translocation in vivo. Hsp40 recognizes and binds non-native polypeptides and delivers them to Hsp70. Then Hsp40 stimulates the ATPase activity of Hsp70 to fold the polypeptides. By using yeast Hsp40 Sis1 and yeast Hsp70 Ssa1 as our model proteins, we found that the Sis1 peptide-binding fragment interacts directly with the full-length Ssa1 in vitro. Further studies showed that the C-terminal lid domain of Ssa1 could interact with Sis1 peptide-binding domain physically in vitro. The Sis1 peptide-binding fragment forms a stable complex with the Ssa1 C-terminal lid domain in solution. The interactions between these two proteins appear to be charge-charge interactions because high-ionic-strength buffer can dissociate the complex. Further mapping studies showed that the Sis1 peptide-binding fragment binds the extreme C-terminal 15 amino acid residues of Ssa1. A flexible glycine-rich region is followed by these 15 residues in the Ssa1 primary sequence. Atomic force microscopy of the Sis1-Ssa1 complex showed that only one end of the Ssa1 lid domain binds the Sis1 peptide-binding-fragment dimer at the upper level of the huge groove within the Sis1 dimer. Based on the data, we propose an "anchoring and docking" model to illustrate the mechanisms by which Hsp40 interacts with Hsp70 and delivers the non-native polypeptide to Hsp70.
Project description:Ydj1 and Sis1 are structurally and functionally distinct Hsp40 proteins of the yeast cytosol. Sis1 is an essential gene whereas the ydj1 gene is essential for growth at elevated temperatures and cannot complement sis1 gene deletion. Truncated polypeptides capable of complementing the sis1 gene deletion comprise the J-domain of either Sis1 or Ydj1 connected to the G/F region of Sis1 (but not Ydj1). Sis1 mutants in which the G/F was deleted but G/M maintained were capable of complementing the sis1 gene deletion.To investigate the relevance of central domains on the structure and function of Ydj1 and Sis1 we prepared Sis1 constructs deleting specific domains. The mutants had decreased affinity for heated luciferase but were equally capable of stimulating ATPase activity of Hsp70. Detailed low resolution structures were obtained and the overall flexibility of Hsp40 and its mutants were assessed using SAXS methods. Deletion of either the G/M or the G/M plus CTDI domains had little impact on the quaternary structure of Sis1 analyzed by the SAXS technique. However, deletion of the ZFLR-CTDI changed the relative position of the J-domains in Ydj1 in such a way that they ended up resembling that of Sis1. The results revealed that the G/F and G/M regions are not the only flexible domains. All model structures exhibit a common clamp-like conformation.Our results suggest that the central domains, previously appointed as important features for substrate binding, are also relevant keeping the J-domains in their specific relative positions. The clamp-like architecture observed seems also to be favorable to the interactions of Hsp40 with Hsp70.
Project description:The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments.
Project description: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.