Molecular anatomy of protein translocation machineries in yeast mitochondria

大多数线粒体蛋白都是在细胞质中作为前体蛋白合成的，并通过细胞膜外的蛋白质转运机制TOM复合物和细胞膜内的蛋白质转运机制TIM复合物进入线粒体。在本研究中，我们分析了线粒体转运机制的亚基排列、结构和前体相互作用。TOM复合物由受体蛋白tom70、Tom37、Tom2O和Tom22、通道蛋白tom40和小分子TOM蛋白Tom5、Tom6和Tom7组成。通过差异增溶方法，我们发现Tom7OiTom37和Tom2O/Tom22/Tom4O在TOM复合物中形成不同的亚复合物结构。我们大量制备了tom70和Tom22的胞质结构域，并通过甘油梯度离心分析了tom70 - tom2o的相互作用。结果表明，Tom22的n端跨膜段下游的一个带正电的片段对于Tom2O与可能带负电的tom70结构域的相互作用很重要。其次，通过核磁共振光谱测定了Tom2O的细胞质结构域的三级结构。前导序列的结合位点也被映射到NMR结构上。最后，我们通过抑制RNA方法将光反应性非天然氨基酸引入线粒体前体蛋白的不同位置，利用这些标记的前体蛋白，通过光交联分析了易位多肽链与线粒体易位机制组分的相互作用，这是迄今为止尚未获得的高分辨率。

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.