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

使用非天然氨基酸分析蛋白质导入线粒体的机制。

• 批准号: 10480156
• 负责人: ENDO Toshiya
• 金额: $ 8.7万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10480156/
• 关键词: translocation across the membranes; mitochondria; yeast; suppressor tRNA method; photocrosslinking; unnatural amino acid

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

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。尽管A阶段和B阶段中间体先前被指定为与TOM复合体的顺式和反式结合的形式，但请尊重…更生动的是，交联的结果表明，A阶段和B阶段的中间体的前序都已经在外膜的反面。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。我们还应用了定点光交联法来研究载体蛋白运输到线粒体内膜的过程。光反应性非天然氨基酸被引入到ADP/ATP载体(AAC)的不同位置，并沿其进入线粒体内膜的途径在不同阶段被阻止。随后的光交联实验表明，AAC在外膜与Tom70和Tom40相互作用，在膜间隙与Tim9和Tim10相互作用，在内膜与Tim22以阶段依赖的方式相互作用。较少

项目成果

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Shuichi Nishikawa、Toshiya Endo（撰稿人）：“分子伴侣控制细胞功能（由 Kazuhiro Nagata、Masataka Mori、Kensuke Yoshida 编辑）”运输：内质网 66-74 (2001)。

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Shuichi Nishikawa、Toshiya Endo（合著者）：“新版微生物实验方法（Koichi Owada、Tsuneyoshi Kuroiwa、Junta Sugiyama、Hideo Takahashi、Hajime Tokuda、Makoto Watanabe 等）”制备酵母细胞内结构的方法（正在印刷中） ，（1999）

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