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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在跨外膜易位的两个不同阶段，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阶段和B阶段的中间体先前分别被分配到与TOM复合物的顺式位点和反式位点结合的形式上，但交联的结果表明，A阶段和B阶段的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阶段保持折叠状态。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。我们还将位点特异性光交联的方法应用于载体蛋白运输到线粒体内膜和蛋白质在叶绿体包膜上的易位过程。