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 Hsp70ΔEEVD 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.

Novel Entropically Driven Conformation-specific Interactions with Tomm34 Protein Modulate Hsp70 Protein Folding and ATPase Activities

“…The Hsp40 used in our assays is encoded by gene DNAJB1. The cooperation of this Hsp40 variant with Hsp70 in refolding assays was reported to be dependent on the presence of the EEVD motif (32,75,76). The association between Hsp40s and the C-terminal structures of Hsp70 seems to represent a more general phenom- FIG.…”

Functionality of Class A and Class B J‐protein co‐chaperones with Hsp70

“…1B). In addition, alteration of either E50 or R73 to alanine overcame the dependence of Sis1 on EEVD(Hsp70) [11]. Sequence comparison revealed that the charge of these residues is conserved in Sis1 orthologs across species (Fig.…”

“…The templates for Hsp70A1A (Hsp72), DnaJB1, DnaJA2 and Apg2 constructs were obtained from Addgene (Cambridge, MA) cloned into pMAL-His-TEV [17] and expressed in Rosetta 2 (DE3) pLys E. coli cells. DnaJB1, DnaJA2 and Apg2 were purified in a manner similar to that described for Ydj1/Sis1 [11], using Ni chromatography and subsequent removal of MBP-His tag with TEV protease. Hsp72 was purified in a similar manner with an additional ATP agarose chromatography step after TEV cleavage.…”

“…Luciferase refolding assay for DnaJA2/Ydj1 with Ssa1 ( Fig. 2B) was carried out as described [11]. Briefly, luciferase was denatured for 1 hr 20 min at 30°C in buffer A [25 mM HEPES (pH 7.4), 50 mM KCl and 5 mM MgCl 2 ] containing DTT (5 mM) and guanidinium hydrochloride (6 M), and then, to give a final concentration of 30 nM, diluted in refolding buffer [buffer A containing 2 mM ATP].…”

How Do J-Proteins Get Hsp70 to Do So Many Different Things?

“…The β-barrel domain specifically interacts with this tetrapeptide. Cytosolic systems of both yeast and humans require this interaction for robust in vitro protein refolding activity [39, 40]. However, this activity of an Hsp70 lacking its EEVD is restored if an intramolecular salt bridge between the J-domain and the adjacent glycine-rich region of its J-protein partner is disrupted, suggesting that intramolecular interactions regulate activity.…”