Biogenesis of the Yeast Mitochondrial ATPase Complex

描述（由申请人提供）：有氧细胞的生物能量需求主要通过F1F0 ATP合成酶的作用来满足，该酶在氧化磷酸化过程中催化ATP合成。酶的催化单元（F1）是亚基组成的多聚体蛋白¿3¿3？在我们的实验室中，使用酵母酿酒酵母作为线粒体功能研究的模型系统，已经提供了证据，证明F1寡聚物在线粒体中的组装需要两种分子伴侣蛋白，称为Atp11p和Atp12p。没有这些蛋白质，F1的α和α亚基就会在细胞器内聚集成蛋白质。在下一个资助期内，我们将继续努力提供有关Atp11p和Atp12p伴侣作用的详细信息。Aim 1下的研究重点是这些蛋白与天然配体和模型配体的相互作用。Atp11p的伴侣结构域的结构/功能关系将通过使用已被证明在该区域结合的荧光化合物，以及通过保守序列元件的定点诱变来探测；后者的后果将在体内和体外进行评估。在其他工作中，Atp11p， Atp12p及其底物蛋白（F1和¿亚基）将被检查作为蛋白质-蛋白质相互作用的介质的作用。研究还将继续进行，以确定线粒体中是否有其他蛋白质与Atp11p和Atp12p协同作用。Aim 2的目标是用x射线晶体学的方法求解Atp11p和Atp12p的三维结构。F1伴侣被认为通过互补表面与其蛋白质底物相互作用。在这种观点下，结构分析有望在使我们能够解决Atp11p和Atp12p的机制方面发挥重要作用。Aim 3关注的是在不存在Atp11p或Atp12p的情况下发生的F1亚基聚集过程。现在我们知道，Atp11p和Atp12p是人类细胞的伴侣蛋白，当这些蛋白缺乏或有缺陷时会发生什么，详细的信息是感兴趣的。

DESCRIPTION (provided by applicant): The bioenergetic needs of aerobic cells are met principally through the action of the F1F0 ATP synthase, which catalyzes ATP synthesis during oxidative phosphorylation. The catalytic unit of the enzyme (F1) is a multimeric protein of the subunit composition ¿3¿3?¿¿. Work in our laboratory, which employs the yeast Saccharomyces cerevisiae as a model system for studies of mitochondrial function, has provided evidence that assembly of the F1 oligomer in mitochondria requires two molecular chaperone proteins called Atp11p and Atp12p. Without these proteins, the ¿ and ¿ subunits of the F1 accumulate as aggregated proteins inside the organelle. In the next funding period we will continue our efforts to provide detailed information about the chaperone actions of Atp11p and Atp12p. Studies under Aim 1 focus on the interactions of these proteins with natural and model ligands. Structure/function relationships in the chaperone domain of Atp11p will be probed through the use of a fluorescent compound that has been shown to bind in this region, and through site-directed mutagenesis of conserved sequence elements; the ramifications of the latter will be assessed both in vivo and in vitro. In other work, Atp11p, Atp12p, and their substrate proteins (the F1 ¿ and ¿ subunits) will be examined for actions as mediators of protein-protein interactions. Studies will also be pursued to determine if there are additional proteins in mitochondria that act in concert with Atp11p and Atp12p. The goal of Aim 2 is to solve the three-dimensional structure of Atp11p and of Atp12p by means of X-ray crystallography. The F1 chaperones are proposed to interact with their protein substrates via complementary surfaces. Under this point of view, the structural analysis is expected to play a major role in enabling us to solve the mechanism of Atp11p and Atp12p. Aim 3 focuses on the process of F1 subunit aggregation that occurs under conditions in which Atp11p or Atp12p is not present. As it is now known that Atp11p and Atp12p are chaperone proteins of human cells, detailed information about what occurs when these proteins are deficient or defective is of interest.