title: Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone Sis1.
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 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.
authors: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
authors: Hyun Young
authors: HY
authors: Yu
authors: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
authors: Thomas
authors: T
authors: Ziegelhoffer
authors: Midwest Center for Structural Genomics, Department of Biosciences, Argonne National Laboratory, Building 202, 9700 South Cass Avenue, Argonne, IL 60439, USA.
authors: Jerzy
authors: J
authors: Osipiuk
authors: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
authors: Szymon J
authors: SJ
authors: Ciesielski
authors: Laboratory of Biopolymer Structure, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk 80822, Poland.
authors: Maciej
authors: M
authors: Baranowski
authors: Midwest Center for Structural Genomics, Department of Biosciences, Argonne National Laboratory, Building 202, 9700 South Cass Avenue, Argonne, IL 60439, USA.
authors: Min
authors: M
authors: Zhou
authors: Midwest Center for Structural Genomics, Department of Biosciences, Argonne National Laboratory, Building 202, 9700 South Cass Avenue, Argonne, IL 60439, USA; Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
authors: Andrzej
authors: A
authors: Joachimiak
authors: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA. Electronic address: ecraig@wisc.edu.
authors: Elizabeth A
authors: EA
authors: Craig
mesh_terms: D000595:Amino Acid Sequence; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D021381:Protein Transport; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; C070320:SIS1 protein, S cerevisiae; C090869:SSA3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C070319:YDJ1 protein, S cerevisiae
keywords: EEVD motif; Hsp40; J domain; molecular chaperone; protein folding