Analyses of mechanisms of protein import into mitochondria by using unnatural amino acids.

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Most mitochondrial proteins are synthesized as precursor proteins in the cytosol and imported into mitochondria with the aid of protein translocation machineries in the outer and the inner membranes called the TOM complex and the TIM complex, respectively. In the present study, artificially aminoacylated suppressor tRNAs were used to introduce photoreactive unnatural amino acids into model mitochondrial precursor proteins to map interactions between precursor proteins and translocation machineries along the import pathway.A model precursor protein, pSu9-DHFR, was arrested at two distinct stages, stage A (accumulated at 0℃) and stage B (accumulated at 30℃), in the translocation across the outer membrane and interactions between the arrested precursor protein and TOM proteins were analyzed at high resolution not achieved previously. Although the stage-A and the stage-B intermediates were previously assigned to the forms bound to the cis site and the trans site of the TOM complex, respect … More ively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer lembrane. The mature domain is unfolded and bound to Tom40 at stage B while it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage-B intermediate, but not of the stage-A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. These results indicate that translocation of the presequence and unfolding of the mature domain are not necessarily coupled.We have also applied the approach of site-specific photocrosslinking to trie processes of carrier proteins transport to the mitochondrial inner membrane. Photoreactive unnatural amino acids were introduced at various positions of ADP/ATP carrier (AAC), which was arrested at various stages along its import pathway to the mitochondrial inner membrane. Subsequent photcrosslinking rexperiments evealed that AAC interacts with Tom70 and Tom40 in the outer membrane, Tim9 and Tim 10 in the intermembrane space, and Tim22 in the inner membrane in a stage-dependent manner. Less