Role of the Mitochondrial DnaJ Homolog Mdj1p as a Chaperone for Mitochondrially Synthesized and Imported Proteins

Abstract

Mdj1p, a DnaJ homolog in the mitochondria of Saccharomyces cerevisiae, is involved in the folding of proteins in the mitochondrial matrix. In this capacity, Mdj1p cooperates with mitochondrial Hsp70 (mt-Hsp70). Here, we analyzed the role of Mdj1p as a chaperone for newly synthesized proteins encoded by mitochondrial DNA and for nucleus-encoded proteins as they enter the mitochondrial matrix. A series of conditional mutants of mdj1 was constructed. Mutations in the various functional domains led to a partial loss of Mdj1p function. The mutant Mdj1 proteins were defective in protecting the tester protein firefly luciferase against heat-induced aggregation in isolated mitochondria. The mitochondrially encoded var1 protein showed enhanced aggregation after synthesis in mdj1 mutant mitochondria. Mdj1p and mt-Hsp70 were found in a complex with nascent polypeptide chains on mitochondrial ribosomes. Mdj1p was not found to interact with translocation intermediates of imported proteins spanning the two membranes and exposing short segments into the matrix, in accordance with the lack of requirement of Mdj1p in the mt-Hsp70-mediated protein import into mitochondria. On the other hand, precursor proteins in transit which had further entered the matrix were found in a complex with Mdj1p. Our results suggest that Mdj1p together with mt-Hsp70 plays an important role as a chaperone for mitochondrially synthesized polypeptide chains emerging from the ribosome and for translocating proteins at a late import step.

Mss51 and Ssc1 Facilitate Translational Regulation of Cytochrome c Oxidase Biogenesis

“…A small portion of Mdj1 was also consistently pulled down. Mdj1 is a mitochondrial DnaJ type chaperone that cooperates with Ssc1 in its folding functions (53). Consistent with our results, Mdj1 and Ssc1 were previously reported to form a complex with nascent mitochondrial polypeptide chains, probably to exert a chaperone function during ongoing translation and thus control the productive folding of the newly synthesized mitochondrial proteins (53).…”

“…Mdj1 is a mitochondrial DnaJ type chaperone that cooperates with Ssc1 in its folding functions (53). Consistent with our results, Mdj1 and Ssc1 were previously reported to form a complex with nascent mitochondrial polypeptide chains, probably to exert a chaperone function during ongoing translation and thus control the productive folding of the newly synthesized mitochondrial proteins (53). Shy1, which is part of large complexes and interacts with Cox1-containing subassemblies downstream from the roles of Mss51 in COX biogenesis (2,34), was detected exclusively in the supernatant but not in the pulldown material (Fig.…”

Mitochondrial Heat Shock Protein (Hsp) 70 and Hsp10 Cooperate in the Formation of Hsp60 Complexes

“…The J domain-containing protein Pam18/ Tim14, together with its partner protein Pam16/Tim16 and the nucleotide exchange factor Mge1, regulates the activity of mtHsp70 (17)(18)(19)(20)(21)(22)(23)(24). In addition, the chaperone associates with the J protein Mdj1 and Mge1 to promote the folding of nucleusencoded and mitochondrially encoded proteins in the matrix (25)(26)(27)(28)(29)(30)(31). Recent data identified further interactors of the chaperone.…”

Impaired Interdomain Communication in Mitochondrial Hsp70 Results in the Loss of Inward-directed Translocation Force

“…J-proteins not only activate the ATPase activity of Hsp70-type chaperones but contribute to the transduction of conformational signals from the NBD to the SBD, thereby enhancing substrate affinity (32). MtHsp70 has two prominent J-domain partner proteins: Mdj1, the genuine DnaJ homolog, is involved in protein folding reactions in the matrix (61,62), whereas the membrane-integrated Pam18 regulates mtHsp70 translocation activity at the import channel (12)(13)(14). In contrast to Mdj1, the Pam18-mtHsp70 interaction was sensitive to the presence of ATP (13,48), indicating a different interaction behavior of mtHsp70 with the individual J-domain partner proteins.…”