Molecular mechanism of recognition of mitochondrial protein precursor by mitochondria

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Most of mitochondrial proteins are synthesized on free polysomes in cytosol as precursors with a presequence at their N-terminal portions, imported into mitochondria and located in their own locations in various mitochondrial compartments (outer membrane, intermembrane space, inner membrane, and matrix). In this import process, many cellular proteins such as a cytosolic factor and receptor for the mitochondrial protein precursors are required as import machinery.From a rabbit reticulocyte lysate, we purified homogeneously 28 kDa protein which were required for import of the precursor proteins with an affinity column using synthetic peptide containing the presequence of ornithine aminotransferase precursor as a ligand. This 28 kDa protein (28 kDa targeting factor) was a component constructing a factor carrying the precursor to mitochondria. Anti-28 kDa targeting factor IgG specifically inhibited the import of the precursor as well as its binding to mitochondria.The components of import … More machinery were extensively purified from the mitochondrial membrane fraction by affinity column chromatography using the same synthetic peptide as that used for purification of 28 kDa targeting factor. The purified fraction contained two major proteins with molecular masses of 29 and 52 kDa. Although these two proteins had an affinity to the presequence of omithine aminotransferase, the 29 kDa protein could more tightly bind the presequence than the 52 kDa protein. Furthermore, 42 kDa protein was also purified as complex with 29 and/or 52 kDa protein. It was also shown that anti-29, 42, and 52 kDa protein Fab fragments strongly inhibited the import of precursor of ornithine aminotransferase into mitochondria. The localization of 29 and 52 kDa proteins in the mitochondriawas studied, then it was found that most of these proteins were located in the distinct area, so called "the contact site" in the outer mitochondrial membrane. And it was comfirmed by further analysis that these three proteins function as translocation machinery for the mitochondrial protein precursors. Less