The Conserved Carboxyl Terminus and Zinc Finger-like Domain of the Co-chaperone Ydj1 Assist Hsp70 in Protein Folding

Abstract

Ydj1 is a member of the Hsp40 (DnaJ-related) chaperone family that facilitates cellular protein folding by regulating Hsp70 ATPase activity and binding unfolded polypeptides. Ydj1 contains four conserved subdomains that appear to represent functional units. To define the action of these regions, protease-resistant Ydj1 fragments and Ydj1 mutants were analyzed for activities exhibited by the unmodified protein. The Ydj1 mutant proteins analyzed were unable to support growth of yeast at elevated temperatures and were found to have alterations in the J-domain (Ydj1 H34Q), zinc finger-like region (Ydj1 C159T), and conserved carboxyl terminus (Ydj1 G315D). Fragment Ydj1 (1-90) contains the J-domain and a small portion of the G/F-rich region and could regulate Hsp70 ATPase activity but could not suppress the aggregation of the model protein rhodanese. Ydj1 H34Q could not regulate the ATPase activity of Hsp70 but could bind unfolded polypeptides. The J-domain functions independently and was sufficient to regulate Hsp70 ATPase activity. Fragment Ydj1 (179 -384) could suppress rhodanese aggregation but was unable to regulate Hsp70. Ydj1 (179 -384) contains the conserved carboxyl terminus of DnaJ but is missing the J-domain, G/F-rich region, and a major portion of the zinc finger-like region. Ydj1 G315D exhibited severe defects in its ability to suppress rhodanese aggregation and form complexes with unfolded luciferase. The conserved carboxyl terminus of Ydj1 appeared to participate in the binding of unfolded polypeptides. Ydj1 C159T could form stable complexes with unfolded proteins and suppress protein aggregation but was inefficient at refolding denatured luciferase. The zinc finger-like region of Ydj1 appeared to function in conjunction with the conserved carboxyl terminus to fold proteins. However, Ydj1 does not require an intact zinc finger-like region to bind unfolded polypeptides. These data suggest that the combined functions of the J-domain, zinc finger-like region, and the conserved carboxyl terminus are required for Ydj1 to cooperate with Hsp70 and facilitate protein folding in the cell.Hsp40 proteins make up an essential family of molecular chaperones that function to specify the cellular processes facilitated by Hsp70 (1-5). Prokaryotic and eukaryotic cells contain multiple Hsp40 proteins that act in specific pairs with Hsp70 at different cellular locations via conserved mechanisms (6 -9). Escherichia coli DnaJ, the progenitor of the Hsp40 family, functions to promote protein folding, degradation of misfolded proteins, and bacteriophage DNA replication (3, 10, 11). The mammalian Hsp40 protein Auxillin directs Hsp70 to uncoat clathrin-coated vesicles (12, 13). The cysteine string protein of Drosophilia is palmitolyated and localized to neurosecretory vesicles, where it is proposed to direct Hsp70 to catalyze putative protein assembly/disassembly events required for neurotransmitter release (14, 15). The yeast protein Tim44 is a component of the import machinery that directs the mitochondrial cognate of Hsp70 ...

Differential expression of three Paralichthys olivaceus Hsp40 genes in responses to virus infection and heat shock☆

“…In yeast, Ydj1 (Type I Hsp40 homologue) is dramatically more effective than Sis1 (Type II Hsp40 homologue) in suppressing the thermally induced aggregation of luciferase and appears more efficient in assisting Ssa1 to fold luciferase [48]. However, mutation of the CR domain of Ydj1 reduces its ability to assist Hsp70 in refolding luciferase about fourfold [20].…”

“…One categorization proposes the division of Hsp40 family into three subgroups [13]. Type I Hsp40 proteins possess all four domains similar to that of DnaJ, including the J domain with a conserved HPD tripeptide, which is responsible for regulation of the ATP hydrolytic cycle of Hsp70 [18e20], the glycine/phenylalanine-rich (G/F) domain, which appears to function as a spacer between the J-domain and other regions of Hsp40 proteins [21,22], a cysteine-rich Zn 2þ binding domain (CR) (containing four repeats of the motif CXXCXGXG where X is any amino acid), which plays a role in polypeptide binding by Hsp40 family members [23,24], and a poorly conserved C-terminal domain (CTD), which can interact with nonnative substrates [20]. Type II Hsp40 proteins lack the CR domain, and Type III proteins possess only the J domain [13].…”

The Roles of the Two Zinc Binding Sites in DnaJ

“…However, conflicting results have been reported concerning the role of the entire zinc binding region in the autonomous DnaK-independent chaperone activity (8,9,19). Furthermore, the role of the individual zinc centers in the DnaK-dependent and -independent chaperone function of DnaJ has not been investigated.…”

“…This suggested that zinc center II is important and may even be essential for the DnaK-dependent chaperone activity of class I DnaJ homologues. There was also evidence suggesting that zinc center I might be involved in this activity, because mutants in the yeast homologue Ydj1p that were lacking one of the highly conserved zinc center I cysteines, also revealed a significantly reduced ability to cooperate with yeast Hsp70 in the refolding of luciferase (8).…”

“…Together with the J-domain, the G/F domain is essential for maximal stimulation of the ATPase activity of DnaK (7). The central domain of DnaJ is the zinc-binding cysteine-rich CR-domain (residues 144 -200), which is followed by a poorly conserved C-terminal CTDdomain (8).…”