BIOGENESIS OF THE YEAST MITOCHONDRIAL ATPASE COMPLEX

酵母菌线粒体F1F0-ATP合成酶的研究 Cerevisiae鉴定出两种蛋白质(Atp11p和Atp12p)，其明显的 其功能是陪伴F1部分的形成。酵母菌 这两个“装配系数”中的任何一个都有缺陷，累积了F1阿尔法 和聚合体中的β亚基，而不是形成酶低聚物。 尽管有证据表明Atp11p和Atp12p的作用相似 在F1组装途径中，这些“组装”的个体活动 从只有Atp11p出现这一事实可以看出，这些因素是不同的 直接与F1α和β亚基相互作用。目标之一是 我们的工作是确定Atp11p和Atp12p的动作是否 主要是为了保护α和β亚基不聚集，或者如果 集合因素具有更活跃的作用，实际上促进了 α-β杂低聚物的形成。此外，我们还感兴趣的是 确定Atp11p和Atp12p是否对文件夹执行其活动，或者 以及F1亚基的展开形式。这些问题将是 用纯化的Atp11p、Atp12p和F1进行体外检测 α亚基和β亚基。光散射测量将用于 在没有/存在添加效应器的情况下遵循F1蛋白聚集， 并将进行荧光共振能量转移(FRET)实验 监测α亚基和β亚基之间杂寡体的形成 (从折叠状态或从展开状态开始) 使用不同的荧光团。在其他工作中，使用可分解的化学物质 与质谱肽图谱相结合的交联剂，以及 遗传分析(酵母双杂交、噬菌体展示)也将被用来定位 Atp11p上以及α和β亚基上的相互作用区域。 关于Atp11p和Atp12p时间序列的探讨 活动，脉冲追逐实验将在F1 Alphabeta之后进行 酵母中对Atp11p和Atp11p有条件的亚基组装 Atp12p活动。与蛋白质-蛋白质序列相关的信息 F1组装期间的互动也将来自于 与F1α和β亚基形成的复合体被标记为 当蛋白质被激活时，可光激活基团将被及时跟踪 在体外导入线粒体。

Previous work with the mitochondrial F1F0 ATP synthase from Saccharomyces cerevisiae identified two proteins (Atp11p and Atp12p), whose apparent function is to chaperone the formation of the F1 moiety. Yeast that are defective for either of these "assembly factors" accumulate the F1 alpha and beta subunits in aggregates instead of forming the enzyme oligomer. Although evidence suggests that Atp11p and Atp12p act at a similar point in the F1 assembly pathway, the individual activities of these "assembly factors" are different as indicated by the fact that only Atp11p appears to interact directly with the F1 alpha and beta subunits. One objective of our work is to determine if the actions of Atp11p and Atp12p are principally to protect the alpha and beta subunits from aggregating, or if the assembly factors have a more activate role and actually promote the formation of alphabeta hetero-oligomers. Also, it is of interest to determine if Atp11p and Atp12p exert their activities with the folder or with the unfolded form of the F1 subunits. These issues will be investigated using in vitro assays with purified Atp11p, Atp12p, and F1 alpha and beta subunits. Light scattering measurements will be used to follow F1 protein aggregation in the absence/presence of added effectors, and fluorescence resonance energy transfer (FRET) experiments will be done to monitor hetero-oligomer formation between alpha and beta subunits (starting from the folded or from the unfolded state) that are labeled with differed fluorophores. In other work, the use of cleavable chemical cross-linking reagents coupled with mass spectral peptide mapping, and also genetic assays (yeast two-hybrid, phage display), will be used to map the regions of interactions on Atp11p and on the alpha and beta subunits. To probe questions related to the temporal sequence of Atp11p and Atp12p actives, pulse-chase experiments will be done to follow F1 alphaBeta subunit assembly in vivo in yeast that are conditional for Atp11p and Atp12p activities. Information relevant to the sequence of protein-protein interactions during F1 assembly will come also from work in which complexes formed with F1 alpha and beta subunits that are labeled with photactivatable groups will be followed in time, when the proteins are imported into mitochondria in vitro.