PROTEIN IMPORT INTO YEAST MITOCHONDRIA

描述(申请者的描述)：本研究的长期目标 建议理解线粒体内膜和线粒体的蛋白质是如何 膜间隙调节两个事件：蛋白质移位到细胞内 线粒体基质，并将蛋白质分选到内膜。线粒体 是必不可少的细胞器，其功能需要进口数百个 在细胞核中编码、在胞浆中合成的不同蛋白质 并被输入细胞器。蛋白质输入是一个多步骤的途径， 包括前体蛋白与表面受体的结合、易位 跨一个或两个线粒体膜，以及折叠和组装 线粒体内的进口蛋白质。为了了解这些过程是如何 ，我们已经分离并分析了酵母中的突变株--酵母 在线粒体蛋白输入方面存在缺陷的酿酒酵母。 许多研究表明，酵母中的线粒体将蛋白质输入到他们的体内 线粒体的过程实际上与哺乳动物线粒体的方式相同 进口蛋白质。因此，我们对酵母的研究将产生有价值的信息。 关于线粒体在所有生物体中的生物发生。支持这一观点的人， 一种名为莫尔-特拉布雅格综合征的疾病，是一种神经退行性疾病 以耳聋、肌张力障碍、智力低下和失明为特征，有 最近被证明是由于线粒体蛋白进口的缺陷造成的。 人类的突变是在编码一种名为DDP的蛋白质的基因中发生的，该蛋白质与 酵母菌TIM8蛋白。我们预计，我们未来的研究将继续 深入了解线粒体的正常功能及其 与人类疾病的关系。 我们最近发现线粒体内膜包含两个 转位复合体：需要转位的TIM23复合体 穿过内膜进入基质和TIM22复合体的前体， 这是将多聚体蛋白插入到内部所必需的 薄膜。Tim54p、Tim22p和Tim18p组成TIM22的膜亚单位 很复杂。 在这项提案中，我们提出了几个问题，旨在了解这一机制 通过它蛋白质被插入到内膜。(1)还有哪些蛋白质 组成TIM22复合体？(2)Tim18p在导入中起直接作用吗？(3) Tim54p在插入过程中起到对接作用吗？(4)什么是 蛋白质在其输入和插入过程中穿过的精确路径 内膜？(5)内膜蛋白插入能重组吗？ 使用提纯的成分？

DESCRIPTION (applicant's description): The long range goal of this research proposal is to understand how proteins of the mitochondrial inner membrane and intermembrane space mediate two events: translocation of proteins into the mitochondrial matrix, and protein sorting to the inner membrane. Mitochondria are essential organelles, whose function requires the import of hundreds of different proteins that are encoded in the nucleus, synthesized in the cytosol and imported into the organelle. Protein import is a multistep pathway which includes the binding of precursor proteins to surface receptors, translocation across one or both mitochondrial membranes, and folding and assembly of the imported protein inside the mitochondrion. To understand how these processes occur, we have isolated and are analyzing mutants in the yeast, Saccharomyces cerevisiae, that are defective in mitochondrial protein import. Many studies indicate that mitochondria in yeast import proteins into their mitochondria by a process virtually identical to the way mammalian mitochondria import proteins. Hence our studies with yeast will yield valuable information about mitochondria biogenesis in all organisms. Supporting this view, a human disease called Mohr-Tranebjaerg syndrome, a neurodegenerative disease characterized by deafness, dystonia , mental retardation and blindness, has recently been shown to result from a defect in mitochondrial protein import. The human mutation is in a gene encoding a protein called DDP, homologous to the yeast Tim8 protein. We anticipate that our future studies will continue to provide insights into the normal functions of mitochondria and their relationships to human disease. We have recently shown that the mitochondrial inner membrane contains two translocation complexes: the TIM23 complex, which is required to translocate precursors across the inner membrane into the matrix and the TIM22 complex, which is required for the insertion of polytopic proteins into the inner membrane. Tim54p, Tim22p and Tim18p comprise the membrane subunits of the TIM22 complex. In this proposal, we ask several questions aimed at understanding the mechanism by which proteins are inserted into the inner membrane. (1) What other proteins comprise the TIM22 complex? (2) Does Tim18p play a direct role in import? (3) Does Tim54p play a docking role in the insertion process? (4) What is the precise pathway that proteins traverse during their import and insertion into the inner membrane? (5) Can inner membrane protein insertion be reconstituted using purified components?