MAMMALIAN CYTOSOLIC DNAJ HOMOLOGS AFFECT THE HSP70 CHAPERONE-SUBSTRATE REACTION CYCLE, BUT DO NOT INTERACT DIRECTLY WITH NASCENT OR NEWLY SYNTHESIZED PROTEINS

Members of the hsp70 family of molecular chaperones interact with and stabilize nascent polypeptides during synthesis and/or translocation i nto organelles. The bacterial hsp70 homologue DnaK requires the DnaJ c ofactor for its reaction cycle with polypeptide substrates. DnaJ stimu lates the ATPase activity of the DnaK chaperone and thereby is thought to regulate the affinity of DnaK for its protein target. Herein we ha ve analyzed some of the biochemical properties of two mammalian cytoso lic DnaJ homologues, the hdj-1 and hdj-2 proteins. We were particularl y interested in examining the proposal that DnaJ homologues are the fi rst molecular chaperones to interact directly with nascent polypeptide s. Nascent/newly synthesized proteins, nascent polypeptides released f rom the ribosome by puromycin, or polypeptides misfolded as a result o f incorporation of an amino acid analogue were not found in complexes with either of the two HeLa cell DnaJ homologues. We still were unable to demonstrate any interactions between hdj-1p and nascent/newly synt hesized proteins even after chemical cross-linking. We did find that h dj-1p, like bacterial DnaJ, stimulated the ATPase activity of hsp70. S table complex formation between hsp70 and an unfolded polypeptide subs trate in vitro was found to be reduced in the presence of hdj-1p and A TP. Thus, while hdj-1p likely does function as a cofactor for the hsp7 0 chaperone, having effects on hsp70's ATPase activity and conformatio n/oligomeric structure and the stability of hsp70-substrate complexes, it was not observed to interact directly with nascent/newly synthesiz ed proteins. Rather, hdj-1p likely serves a regulatory role, governing the reaction cycle of hsp70 with polypeptide substrates.