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Molecular anatomy of protein translocation machineries in yeast mitochondria

酵母线粒体中蛋白质易位机制的分子解剖学

基本信息

批准号：
08458180
负责人：
ENDO Toshiya
金额：
$ 4.8万
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：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458180/
关键词：
yeast mitochondria tarnslocation machinery the TOM complex the TIM complex precursor protein タンパク質輸送光架橋

项目摘要

Most of 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, we analyzed subunit arrangements, structures, and precursor interactions of the mitochondrial translocation machineries.The TOM complex consists of receptor proteins, Tom7O, Tom37, Tom2O and Tom22, a channel protein, Tom4O, and small Tom proteins, Tom5, Tom6 and Tom7, By using differential solubilization methods, we found that Tom7OiTom37 and Tom2O/Tom22/Tom4O form distinct subcomplex structures in the TOM complex. We prepared cytosolic domains of Tom7O and Tom22 in large amounts and analyzed Tom7O-Tom2O interactions by glycerol gradient centrifugation. The results showed that a positively charged segment just downstream of the N-terminal transmembrane segment of Tom22 is important for interactions of Tom2O with possibly a negatively charged domain of Tom7O.Next, a tertiary structure of the cytosolic domain of Tom2O was determined by NMR spectroscopy. A binding site for presequences is also mapped on the NMR structure. In the last, we introduced a photoreactive unnatural amino acids into various positions along mitochondrial precursor protein by suppressor *RNA method, By using these labeled precursor proteins, interactions of the translocating polypeptide chains with components of the mitochondrial translocation machineries were analyzed by photocrosslinking at a high resolution that had not been obtained so far.

大多数线粒体蛋白质在胞质中合成为前体蛋白，并借助于分别位于外膜和内膜的蛋白质转运机制（称为TOM复合物和TIM复合物）转运到线粒体中。Tom 37、Tom 20和Tom 22，通道蛋白Tom 40，以及小的Tom蛋白Tom 5、Tom 6和Tom 7。通过微分增溶方法，我们发现Tom 7、Tom 37和Tom 20/Tom 22/Tom 40在TOM复合物中形成不同的亚复合物结构。我们大量制备了Tom 7 O和Tom 22的胞质结构域，并通过甘油梯度离心分析了Tom 7 O-Tom 2 O相互作用。结果表明，Tom 22 N端跨膜区下游的一个带正电荷的片段对Tom 2 O与Tom 7 O的负电荷结构域的相互作用具有重要作用。前序列的结合位点也映射在NMR结构上。最后，我们利用抑制子RNA法在线粒体前体蛋白的不同沿着引入了一个光反应性的非天然氨基酸，并利用这些标记的前体蛋白，通过光交联技术，以迄今为止尚未获得的高分辨率分析了易位多肽链与线粒体易位机制组分的相互作用。