BIOGENESIS OF THE YEAST MITOCHONDRIAL ATPASE COMPLEX

酵母线粒体F1 F0 ATP合成酶的研究进展 酿酒酵母鉴定了两种蛋白质（Atp 11 p和Atp 12 p），其表观 功能是陪伴F1部分的形成。酵母是 这些“装配因子”中的任何一个有缺陷的， 和β亚基聚集而不是形成酶寡聚体。 尽管有证据表明Atp 11 p和Atp 12 p在类似的点起作用， 在F1组装途径中，这些“组装”的个体活动 只有Atp 11 p出现的事实表明， 直接与F1的α和β亚基相互作用。的一个目的 我们的工作是确定Atp 11 p和Atp 12 p的作用是否 主要是为了保护α和β亚基免于聚集，或者如果 组装因素的作用更为活跃， 形成α异源寡聚体。此外，感兴趣的是， 确定Atp 11 p和Atp 12 p是否对文件夹发挥其活动，或 F1亚基的未折叠形式。这些问题将 使用纯化的Atp 11 p、Atp 12 p和F1进行体外试验研究 α和β亚基。光散射测量将用于 在不存在/存在添加的效应物的情况下F1蛋白聚集后， 荧光共振能量转移（FRET）实验 为了监测α和β亚基之间的异源寡聚体形成 （从折叠状态或从展开状态开始） 不同的荧光团。在其他工作中，使用可裂解的化学物质， 与质谱肽图谱偶联的交联试剂，和 还将使用遗传测定（酵母双杂交、噬菌体展示）来作图 在Atp 11 p和α和β亚基上的相互作用区域。 探讨Atp 11 p和Atp 12 p时间序列的相关问题 在F1 alpha Beta之后，将进行脉冲追踪实验 在酵母中体内亚基组装是Atp 11 p条件， atp 12 p活动。蛋白质-蛋白质序列相关信息 在F1装配期间的相互作用也将来自工作， 与F1 α和β亚基形成的复合物， 光活化基团将及时跟踪，当蛋白质 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.