Loss of expression of the methylation-controlled J gene, MCJ (DNAJC15), is observed in cases of several tumors and plays a crucial role in the chemoresistance of ovarian cancer cells. Aside from the pathophysiological effects, almost nothing is known about the cellular function of MCJ. Here, we provide the first evidence that MCJ acts in the biogenesis of mitochondria. Our results demonstrate that MCJ is located in mitochondria. It is anchored in the mitochondrial inner membrane with the C-terminal J domain facing the matrix space. We show that MCJ forms a stable subcomplex with a component of the mitochondrial import motor, MAGMAS, a protein overexpressed in cells treated with granulocyte-macrophage colony-stimulating factor and in prostate carcinomas. In addition, MCJ and MAGMAS interact with the core components of the TIM23 pre-protein translocase. We demonstrate that the recombinant soluble MCJ domain stimulates the ATPase activity of the human mtHsp70 chaperone, mortalin, the central component of the import motor of the TIM23 translocase. This stimulation is counteracted by MAGMAS. Moreover, pre-protein import into mitochondria is impaired in the absence of MCJ. Interestingly, MCJ is able to take over the function of Tim14, the essential J co-chaperone of the mitochondrial protein import motor in yeast. In summary, our results show that MCJ functions as J co-chaperone of the human TIM23 pre-protein translocase, suggesting a link between mitochondrial pre-protein import and tumorigenesis.

The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in ⌬hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.Hsp70 chaperones mediate important processes in prokaryotes and eukaryotes such as folding, translocation, and prevention of aggregation of proteins (1-3). They are highly conserved in respect to both, amino acid sequence and structure. An N-terminal ATP binding domain (ATPase domain) and a C-terminal peptide binding domain (PBD) 3 are connected by a short hydrophobic interdomain linker. The PBD binds unfolded polypeptides. It acts in cooperation with the ATPase domain, which binds and hydrolyzes ATP. The nucleotide state of the ATPase domain determines the properties of the PBD. In the ATP state the PBD has a low affinity for substrates and binds and releases substrates rapidly. Hydrolysis leads to the ADP bound state of the ATPase domain in which the PBD adopts a closed conformation and binds substrates with high affinity. On the other hand, substrate binding to the PBD stimulates the hydrolysis rate of the ATPase domain. Thus, structural changes in one domain induce conformational alterations in the other domain (4 -10). This interdomain communication is due to changing interdomain contacts. Such contacts have been reported for all nucleotide states (11-15); however, it is not clear whether contacts of Hsp70 proteins observed in the absence of ATP are physiologically relevant (11,13,15). In the ATP bound state the interdomain linker binds to a hydrophobic cleft present in the ATPase domain and stimulates the ATPase activity (15). Together with other studies this suggests an important role of the linker in the mechanism of interdomain communication (5,(15)(16)(17)(18). Mutations within the interdomain linker inhibit the allosteric control of the ATPase domain by the PBD (5, 16, 18). DnaK constructs consisting of the ATPase domain and the in...

Hep1 acts as a specialized chaperone to mediate the de novo folding of yeast mitochondrial Hsp70.