A Conserved HPD Sequence of the J-domain Is Necessary for YDJ1 Stimulation of Hsp70 ATPase Activity at a Site Distinct from Substrate Binding

Abstract

The 46-kDa protein YDJ1 is one of several known yeast homologues of the Escherichia coli DnaJ protein. Like all J homologues, it shares homology with the highly conserved NH2-terminal "J-domain" of DnaJ. A component of the DnaK (Hsp70) chaperone machinery that mediates protein folding, DnaJ is necessary for survival at elevated temperatures. It stimulates ATP hydrolysis by DnaK and effects the release of DnaK-bound polypeptides. Previous genetic and biochemical studies indicate that the J-domain is necessary for these functions. Using peptides corresponding to J-domain sequence, we show that a peptide containing the highly conserved His-Pro-Asp sequence at positions 34-36 in the J-domain competes off YDJ1 stimulation of Hsp70 ATPase activity. Inhibitory concentrations of peptide do not prevent binding of folding substrates, therefore YDJ1 must interact with Hsp70 at a site distinct from that for substrate binding. This interaction is critical for Hsp70 activity, since a mutant YDJ1 protein harboring a H34Q change (ydj1Q34) stimulates neither Hsp70 ATPase nor substrate release. The importance of the proper function of this region of the protein is supported by the poor growth and temperature-sensitive phenotype of yeast expressing ydj1Q34.

Interaction of Auxilin with the Molecular Chaperone, Hsc70

“…This is consistent with our previous observation that auxilin still binds to clathrin baskets after its J-domain is removed, suggesting that auxilin has separate clathrin-binding and Hsc70-binding domains (12). However, the auxilin J-peptides containing the HDPK region do not show the same nucleotide dependence as auxilin in their interaction with Hsc70; in contrast to a similar peptide from the J-domain of YDJ1 (28), they appear to interact with Hsc70 like any other peptide substrate. Therefore, more work is required to determine exactly which portions of the J-domain are crucial to its proper interaction with Hsc70.…”

“…Therefore, we synthesized the two similar length peptides (auxilin J-peptides) shown in Fig. 6, analogous to the peptides prepared from YDJ1 by Tsai and Douglas (28). We found that both J-peptides caused about a 3-fold activation of the Hsc70 ATPase activity, but in contrast to auxilin, both peptides bound very weakly in ATP.…”

“…Tsai and Douglas (28) recently suggested that a 20-amino acid peptide, corresponding to positions 21-40 of the J-domain of YDJ1 and containing the highly conserved HPD sequence at positions 34 -36, bound to the YDJ1 binding site of Hsc70 rather than to the peptide binding site. Therefore, we synthesized the two similar length peptides (auxilin J-peptides) shown in Fig.…”

TID1, a mammalian homologue of the drosophila tumor suppressor lethal(2) tumorous imaginal discs, regulates activation-induced cell death in Th2 cells

“…Modulation of activation-induced cell death J Syken et al dnaJ and Hsp70 family proteins (Wall et al, 1994;Tsai and Douglas, 1996). The H121Q TidS protein will efficiently interact with TidS-specific substrates but because of its lack of Hsp70 interaction cannot function as a chaperone and acts as a dominant-negative mutant (Syken et al, 1999).…”

Identification of a hTid‐1 mutation which sensitizes gliomas to apoptosis

“…A modest inhibition of growth is observed in U373 following infection with control virus, indicating a cytotoxic effect of the virus on the cells. DnaJ protein function is dependent upon a highly conserved histidine-proline-aspartate (HPD) sequence located within the J-domain [29]. Mutation of one or all of the amino acids completely abolishes J-domain-mediated stimulation of Hsp70 ATPase activity.…”