The molecular chaperone function of the secretory vesicle cysteine string proteins

The "J" domains of eukaryotic DnaJ-like proteins specify interaction with various Hsp70s. The conserved tripeptide, HPD, present in all J domains has been shown to be important for the interaction between yeast and bacterial DnaJ/Hsp70 protein pairs. We have characterized mutations in the HPD motif of the synaptic vesicle protein cysteine-string protein (Csp). Mutation of the histidine (H43Q) or aspartic acid (D45A) residues of this motif reduced the ability of Csp to stimulate the ATPase activity of mammalian Hsc70. The H43Q and D45A mutant proteins were not able to stimulate the ATPase activity of Hsc70 to any significant extent. The mutant proteins were characterized by competition assays, tryptic digestion analysis, and direct binding analysis from which it was seen that these proteins were defective in binding to Hsc70. Thus, the HPD motif of Csp is required for binding to Hsc70. We also analyzed the interaction between Csp and a model substrate protein, denatured firefly luciferase. Both Csp1 and the C-terminally truncated isoform Csp2 were able to prevent aggregation of heat-denatured luciferase, and they also cooperated with Hsc70 to prevent aggregation. In addition, complexes of Csp1 or Csp2 with Hsc70 and luciferase were isolated, confirming that these proteins interact and that Csps can bind directly to denatured proteins. Csp1 and Csp2 isoforms must differ in some aspect other than interaction with Hsc70 and substrate protein. These results show that both Csp1 and Csp2 can bind a partially unfolded protein and act as chaperones. This suggests that Csps may have a general chaperone function in regulated exocytosis.