Molecular chaperones perform vital functions in mitochondrial protein import and folding. In yeast mitochondria, two members of the Clp/Hsp100 chaperone family, Hsp78 and Mcx1, have been identified as homologs of the bacterial proteins ClpB and ClpX, respectively. In this report we employed a novel quantitative assay system to assess the role of Hsp78 and Mcx1 in protein degradation within the matrix. Mitochondria were preloaded with large amounts of two purified recombinant reporter proteins

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... hibiting different folding stabilities. Proteolysis of the imported substrate proteins depended on the mitochondrial level of ATP and was mediated by the matrix protease Pim1/LON. Degradation rates were found to be independent of the folding stability of the reporter proteins. Mitochondria from hsp78⌬ cells exhibited a significant defect in the degradation efficiency of both substrates even at low temperature whereas mcx1⌬ mitochondria showed wild-type activity. The proteolysis defect in hsp78⌬ mitochondria was independent from the aggregation behavior of the substrate proteins. We conclude that Hsp78 is a genuine component of the mitochondrial proteolysis system required for the efficient degradation of substrate proteins in the matrix.