Analyses of molecular mechanisms of mitochondrial protein transport by using unnatural amino acids

大多数线粒体蛋白是在胞浆中作为前体蛋白合成的，并通过外膜和内膜的蛋白质转运机制分别称为TOM复合体和TIM复合体输入到线粒体中。在本研究中，利用人工氨基酰化抑制因子tRNAs将非天然氨基酸引入到模型线粒体前体蛋白中，以描绘前体蛋白与输入途径上的易位机制之间的相互作用。模型前体蛋白pSu9-dhfr在外膜转运过程中被阻止在两个不同的阶段，A阶段(在0℃积累)和B阶段(在30℃积累)，并以以前没有达到的高分辨率分析了被捕获的前体蛋白与TOM蛋白之间的相互作用。虽然A阶段和B阶段的中间体先前分别被指定为与TOM复合体的顺式和反式结合的形式，但交联的结果表明，A阶段和B阶段的中间体的前序列都已经在外膜的反式侧。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。从TOM复合体解离后，Tom22的膜间隙结构域促进了B阶段中间体的跨内膜移位，而不是A阶段中间体的跨膜移位。这些结果表明，前序列的移位和成熟结构域的展开并不一定是耦合的。我们还将定点光交联法应用于载体蛋白向线粒体内膜运输和蛋白质跨叶绿体被膜转运的过程。

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 membranes 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, respectively, 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 membrane. The mature domain is unfolded and bound to Tom40 at stage B while 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 the process of carrier proteins transport to the mitochondrial inner membrane and of protein translocation across the chloroplast envelope membranes.