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

大多数线粒体蛋白在细胞质中作为前体蛋白合成，并通过分别称为TOM复合物和TIM复合物的外膜和内膜中的蛋白质转运机制导入线粒体。在本研究中，使用人工氨基酰化抑制trna将光反应性非天然氨基酸引入模型线粒体前体蛋白中，以绘制前体蛋白与转运机制之间的相互作用。模型前体蛋白pSu9-DHFR在跨外膜易位的A阶段（0℃积累）和B阶段（30℃积累）被捕获，并以高分辨率分析了捕获的前体蛋白与TOM蛋白之间的相互作用。虽然A期和B期中间体先前被分配到与TOM复合物的顺式位点和反式位点结合的形式，但更重要的是，交联结果表明A期和B期中间体的序列已经在外膜的反侧。成熟结构域在B阶段展开并与Tom40结合，而在a阶段保持折叠。与TOM复合物解离后，Tom22的膜间空间结构域促进了B阶段中间体（而不是a阶段中间体）跨内膜的易位。这些结果表明，前序的易位和成熟结构域的展开并不一定是耦合的。我们还应用了位点特异性光交联的方法来研究载体蛋白运输到线粒体内膜的三个过程。光反应性非天然氨基酸被引入到ADP/ATP载体（AAC）的不同位置，在其进入线粒体内膜的不同阶段被阻止。随后的光交联实验表明，AAC与外膜上的Tom70和Tom40、膜间的Tim9和tim10以及内膜上的Tim22以阶段依赖的方式相互作用。少

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