Identification of the Protein Import Components of the Rat Mitochondrial Inner Membrane, rTIM17, rTIM23, and rTIM44

We cloned rat liver mitochondrial 18.1, 21.9, and 51.0 kDa proteins with a significant structural homology to the components of the translocase of the yeast mitochondrial inner membrane, Tim17, Tim23, and Tim44. The 18.1 and 21.9 kDa proteins were synthesized as mature forms having four potential transmembrane segments and localized to the mitochondrial inner membrane. The 51.0 kDa protein is a precursor having a presequence of approximately 6 kDa which is cleaved during import into the mitochondria. The mature 45 kDa protein is located in the matrix, both in a soluble form and in a membrane-bound, alkali-extractable form. Immunofluorescence microscopy confirmed the location of all three proteins in the mitochondria. Antibodies against the 21.9 kDa protein, but not those against the 18.1 and 51.0 kDa proteins, inhibited the precursor import into the mitoplasts in vitro. Immunoprecipitation indicated that all three proteins interacted with the protein in transit to the matrix. Immunoprecipitation also revealed that the 18.1 kDa protein formed a complex with the 21.9 kDa protein and the 45 kDa protein with mHsp70; the latter complex was dissociated in an ATP- or ADP-dependent manner and the reaction was impeded by AMP-PNP or inorganic phosphate. These assays thus demonstrated the 18.1, 21.9, and 45 kDa proteins to be the translocator components of the rat mitochondrial inner membrane and, therefore, the functional homologues of Tim17, Tim23, and Tim44, respectively.