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Most mitochondrial preproteins are maintained in a loosely folded import-competent conformation by cytosolic chaperones, and are imported into mitochondria by translocator complexes containing a pre-protein receptor, termed translocase of the outer membrane of mitochondria (Tom) 20. Using two-hybrid screening, we identified arylhydrocarbon receptor-interacting protein (AIP), an FK506-binding protein homologue, interacting with Tom20. The extreme COOH-terminal acidic segment of Tom20 was required for interaction with tetratricopeptide repeats of AIP. An in vitro import assay indicated that AIP prevents preornithine transcarbamylase from the loss of import competency. In cultured cells, overexpression of AIP enhanced preornithine transcarbamylase import, and depletion of AIP by RNA interference impaired the import. An in vitro binding assay revealed that AIP specifically binds to mitochondrial preproteins. Formation of a ternary complex of Tom20, AIP, and preprotein was observed. Hsc70 was also found to bind to AIP. An aggregation suppression assay indicated that AIP has a chaperone-like activity to prevent substrate proteins from aggregation. These results suggest that AIP functions as a cytosolic factor that mediates preprotein import into mitochondria.

An investigation of the roles of heat shock cognate 70 protein (hsc70) and the two cytosolic DnaJ homologues dj1 and dj2 of mammals indicates that both hsc70 and dj2 are required for efficient mitochondrial import of preornithine transcarbamylse (pOTC) during its synthesis. It was established that dj2, but not dj1, facilitates productive folding in cooperation with hsc70. It is possible that contranslational interaction of hsc70 and dj2 exists.