Protein Folding Activity of Hsp70 Is Modified Differentially by the Hsp40 Co-chaperones Sis1 and Ydj1

University of Alabama at Birmingham, Birmingham, Alabama, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 11/1998; 273(43):27824-30. DOI: 10.1074/jbc.273.43.27824
Source: PubMed


Specification of Hsp70 action in cellular protein metabolism may occur through the formation of specialized Hsp70:Hsp40 pairs. To test this model, we compared the ability of purified Sis1 and Ydj1 to regulate the ATPase and protein-folding activity of Hsp70 Ssa1 and Ssb1/2 proteins. Ydj1 and Sis1 could both functionally interact with Ssa1, but not the Ssb1/2 proteins, to refold luciferase. Interestingly, Ydj1:Ssa1 could promote up to four times more luciferase folding than Sis1:Ssa1. This functional difference was explored and could not be accounted for by differences in the ability of Sis1 and Ydj1 to regulate Ssa1 ATPase activity. Instead, differences in the chaperone function of Ydj1 and Sis1 were observed. Ydj1 was dramatically more effective than Sis1 at suppressing the thermally induced aggregation of luciferase. Paradoxically, Sis1 and Ydj1 could bind similar quantities of chemically denatured luciferase. The polypeptide binding domain of Sis1 was found to lie between residues 171-352 and correspond to its conserved carboxyl terminus. The conserved carboxyl terminus of Ydj1 is also known to participate in the binding of nonnative polypeptides. Thus, Ydj1 appears more efficient at assisting Ssa1 in folding luciferase because its contains a zinc finger-like region that is absent from Sis1. Ydj1 and Sis1 are structurally and functionally distinct Hsp40 proteins that can specify Ssa1 action by generating Hsp70:Hsp40 pairs that exhibit different chaperone activities.

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    • "between Sis1-driven and Ydj1/Xdj1-driven refolding by Hsp70 and Hsp70 ΔEEVD , we carried out refolding assays in which luciferase was partially denatured by guanidinium hydrochloride. This is a milder treatment than the urea denaturation method, and, as expected [21], refolding occurred when only Hsp70 and J-protein were included in the reaction (Fig. 1b). As with the assay containing Hsp104 and Sse1, Sis1 did not partner with Hsp70 ΔEEVD . "
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    ABSTRACT: 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 interaction(s) between the J-domain and glycine-rich region controls co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. Yet, 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. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Feb 2015 · Journal of Molecular Biology
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    • "Several studies have been made to elucidate the reason for the excess number of the co-chaperons in a cell. The researchers suggested that Hsp70 pairs with Hsp40 to form specific functions and currently details of the mechanism are under investigation [17]. "
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    ABSTRACT: Heat shock proteins (Hsps) are highly conserved proteins and have cytoprotective role for maintaining cellular protein conformation. Hsps not only keep proteins in their native state but also involve in several essential biochemical process. This review summarizes structural properties of Hsps (Hsp70, Hsp40, Hsp90, Hsp100, Hsp60, sHsps, and Nucleotide Exchange Factors) and explains their roles in aging, apoptosis, cancer, neurodegeneration, cardio-vascular diseases, obesity and diabetes mellitus, and housekeeping.
    Full-text · Chapter · Jul 2014
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    • "The most abundant Hsp40s are members of the Type I and Type II sub-families who partner with Hsp70 to promote protein folding [12], [16], protein degradation [17], [18], [19], [20], translation [21], translocation across membranes [22] and assembly of amyloid-like fibers [23]. In eukaryotes such as yeast, the Type I and Type Hsp40s Ydj1 and Sis1 utilize their unique structural features, substrate specificity, post-translational modification, and localization to direct Hsp70 to function in different aspects of protein metabolism [10], [24], [25], [26], [27], [28]. Yet, it is still unclear how specialized Hsp70:Hsp40 pairs function in PQC networks to triage non-native clients for folding, degradation, or sequestration into misfolded protein handling centers. "
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    ABSTRACT: 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.
    Full-text · Article · Jan 2013 · PLoS ONE
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