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

利用非天然氨基酸分析线粒体蛋白质转运的分子机制

基本信息

批准号：
09044070
负责人：
ENDO Toshiya
金额：
$ 3.52万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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.

大多数线粒体蛋白在细胞质中作为前体蛋白合成，并借助外膜和内膜（分别称为 TOM 复合物和 TIM 复合物）的蛋白质易位机制导入线粒体。在本研究中，人工氨酰化抑制性tRNA用于将光反应性非天然氨基酸引入模型线粒体前体蛋白中，以绘制前体蛋白与输入途径中的易位机制之间的相互作用。模型前体蛋白pSu9-DHFR在两个不同的阶段被捕获，阶段A（在0℃下累积）和阶段B（在0℃下累积） 30℃），以前所未有的高分辨率分析了跨外膜的易位以及被捕获的前体蛋白和 TOM 蛋白之间的相互作用。虽然A阶段和B阶段中间体先前被指定为分别与TOM复合物的顺式位点和反式位点结合的形式，但交联的结果表明A阶段和B阶段中间体的前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。从 TOM 复合体解离后，Tom22 的膜间空间结构域促进 B 阶段中间体（而非 A 阶段中间体）跨内膜易位。这些结果表明前序列的易位和成熟结构域的解折叠不一定是耦合的。我们还将位点特异性光交联方法应用于载体蛋白转运至线粒体内膜以及蛋白质跨叶绿体包膜膜易位的过程。