Mitochondrial Hsp78, a member of the Clp/Hsp100 family in Saccharomyces cerevisiae, cooperates with Hsp70 in protein refolding

The molecular chaperone protein Hsp78, a member of the C1p/Hsp100 family lo calized in the mitochondria of Saccharomyces cerevisiae, is required for ma intenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneit y and its role in the reactivation of chemically and heat-denatured substra te protein was analyzed in vitro. Hsp78 alone was not able to mediate react ivation of firefly luciferase. Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates w ith the Hsp78 homolog, C1pB in Escherichia coli, could not substitute for t he mitochondrial Hsp70 system. However, efficient Hsp78-dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experime nts, suggesting a specific functional interaction of both chaperone protein s. These findings establish the cooperation of Hsp78 with the Hsp70 machine ry in the refolding of heat-inactivated proteins and demonstrate a conserve d mode of action of C1pB homologs. (C) 2001 Federation of European Biochemi cal Societies. Published by Elsevier Science B.V. All rights reserved.