Biogenesis of the Yeast Mitochondrial ATPase Complex

描述（由申请人提供）：有氧细胞的能量需求主要通过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。该酶的催化单元（F1）是一个亚基组成的多聚体蛋白。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β亚基就会在细胞器内聚集成蛋白质。先前的研究表明，Atp11p与F1 β亚基结合，Atp12p与F1 α亚基结合。Aim 1 （F1组装途径中中间体的表征）下的一部分研究旨在确定F1亚基从伴侣蛋白释放后发生的情况。Atp11p和Atp12p的结构/功能关系将通过化学修饰和诱变来探索。Aim 1下的其他研究主要关注Atp11p和Atp12p与线粒体Hsp60/Hsp10折叠机制的关系。在Aim 2（鉴定F1组装过程中的蛋白质-蛋白质相互作用）的工作中，我们将检查Atp11p， Atp12p以及F1 α和β亚基在F1组装过程中作为蛋白质-蛋白质相互作用介质的作用。我们还将描述导致组装缺陷表型的α和β亚基基因突变，并筛选线粒体中可能与Atp11p和Atp12p协同作用的其他蛋白质。在Aim 3（结构研究的Atp11p和Atp12p工程）下的工作是我们小组和两个结构生物学实验室之间的合作，通过x射线晶体学和核磁共振确定Atp11p和Atp12p的三维结构。

DESCRIPTION (provided by applicant): The energy 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 subunit composition alpha3beta3gamma-delta-epsilon. 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 alpha and beta subunits of the F1 accumulate as aggregated proteins inside the organelle. Previous studies have shown that Atp11p binds the F1 beta subunit and Atp12p binds the F1 alpha subunit. A subset of studies under Aim 1 (Characterization of intermediates in the F1 assembly pathway) are designed to determine what happens to the F1 subunits following their release from the chaperone proteins. Structure/function relationships in Atp11p and Atp12p will be probed by chemical modification and mutagenesis. Other studies under Aim 1 focus on the relationship of Atp11p and Atp12p with the Hsp60/Hsp10 folding machinery of mitochondria. In work under Aim 2 (Identification of protein protein interactions durinq F1 assembly) we will examine Atp11p, Atp12p, and the F1 alpha and Beta subunits for actions as mediators of protein-protein interactions during the process of F1 assembly. We will also characterize mutations in the genes for the alpha and beta subunits that cause an assembly defective phenotype and screen for additional proteins in mitochondria that may act in concert with Atp11p and Atp12p. Work under Aim 3 (Engineerinq of Atp11p and Atp12p for structural studies) is a collaboration between our group and two laboratories of structural biology to determine the three dimensional structure for Atp11p and for Atp12p by means of X-ray crystallography and nuclear magnetic resonance.