Fujiki M, Verner K
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
J Biol Chem. 1991 Apr 15;266(11):6841-7.
We made use of a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae to investigate the coupling of protein synthesis and import. Mitochondrial precursor proteins were synthesized in a yeast lysate either in the presence or absence of isolated yeast mitochondria. We were, therefore, able to analyze protein import into mitochondria either in a strictly posttranslational reaction (when isolated mitochondria were added only after protein synthesis has been arrested by the addition of cycloheximide) or in a reaction in which synthesis and import were permitted to occur simultaneously. We found that the import of a precursor protein consisting of the amino-terminal mitochondrial targeting sequence of cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase is very inefficient in a strictly posttranslational reaction, whereas efficient import is observed if precursor synthesis and import are coupled. The same result was obtained when we analyzed the import of bulk endogenous yeast mitochondrial proteins in this system. Finally, we found that the insertion of the yeast outer membrane protein porin is also several times more efficient when synthesis and insertion are coupled.
我们利用源自酿酒酵母的同源无细胞线粒体蛋白导入系统来研究蛋白质合成与导入的偶联。线粒体前体蛋白在酵母裂解物中合成，合成过程中存在或不存在分离的酵母线粒体。因此，我们能够在严格的翻译后反应（即仅在加入环己酰亚胺使蛋白质合成停止后才添加分离的线粒体时）或允许合成与导入同时发生的反应中分析蛋白质向线粒体的导入。我们发现，由细胞色素氧化酶亚基IV的氨基末端线粒体靶向序列与小鼠二氢叶酸还原酶融合而成的前体蛋白在严格的翻译后反应中导入效率非常低，而在前体蛋白合成与导入偶联时则观察到高效导入。当我们在该系统中分析大量内源性酵母线粒体蛋白的导入时，也得到了相同的结果。最后，我们发现当合成与插入偶联时，酵母外膜蛋白孔蛋白的插入效率也会提高几倍。
