STRUCTURE, MECHANISM AND REGULATION OF THE V-ATPASES

申请是为了支持继续研究结构， 机制和调节质子转运ATP酶在V型 家人 V型ATP酶负责酸化 真核细胞中的细胞内区室， 在多种细胞过程中发挥作用，包括受体介导的 内吞作用，细胞内膜运输，大分子加工， 以及降解和耦合传输。 质膜中的V-ATP酶 特化细胞也在肾脏酸化，骨 吸收和肿瘤转移。 了解V-ATP酶是如何 对理解这些过程很重要。 该实验室先前已经表明，来自网格蛋白的V-ATP酶- 包被的囊泡被组织成一个外周V1结构域， ATP水解和一个完整的Vo结构域负责质子 易位 化学修饰已被用来探测结构 在核苷酸结合位点中，该小组认为二硫键 成键可能在体内V-ATP酶活性的调节中起作用。 重组研究已被用于测试个体的功能 亚基，包括V-ATP酶和AP-2接头之间共享的蛋白质 配合物 最近，他们已经开始了突变研究， 酵母V-ATP酶，以鉴定在V-ATP酶活性中重要的残基。 拟议的研究将追求四个具体目标。 以确定 非催化核苷酸结合位点的结构和功能 将使用B亚基半胱氨酸扫描诱变。 的作用 B亚基亚型在V-ATP酶活性和细胞内靶向中的作用 也将受到考验。 为了进一步确定催化剂A的结构， 亚基，将鉴定参与核苷酸结合的残基 通过定点突变。 保守A的邻近性和作用 亚基半胱氨酸残基在液泡酸化的调节将 也要进一步探索。 100 kDa亚基的研究将集中在 鉴定突变的第二位点抑制子， 影响函数，在 说明100 kDa的拓扑结构 亚基，并鉴定赋予康卡霉素的突变 阻力 最后介绍了附属亚单位的配置和功能， 包括AP50和VMA6基因产物。 这些 研究应提供进一步的了解结构和监管 这个重要的H +-ATP酶家族。

The application is for support to continue studies of the structure, mechanism, and regulation of proton-translocating ATPases in the V-type family. The V-type ATPases are responsible for acidification of intracellular compartments in eukaryotic cells and serve an important function in a variety of cellular processes, including receptor-mediated endocytosis, intracellular membrane traffic, macromolecular processing, and degradation and coupled transport. V-ATPases in the plasma membrane of specialized cells also play a role in renal acidification, bone resorption, and tumor metastasis. Understanding how V-ATPases are regulated is important to understanding these processes. This laboratory has previously shown that the V-ATPase from clathrin- coated vesicles is organized into a peripheral V1 domain responsible for ATP hydrolysis and an integral Vo domain responsible for proton translocation. Chemical modification has been used to probe the structure of the nucleotide binding sites, this group has suggested that disulfide bond formation may play a role in regulation of V-ATPase activity in vivo. Reassembly studies have been used to test the function of individual subunits, including a protein shared between the V-ATPase and AP-2 adaptor complexes. More recently, they have begun mutagenesis studies of the yeast V-ATPase to identify residues important in V-ATPase activity. Four specific aims will be pursued in the proposed studies. To determine the structure and function of the noncatalytic nucleotide binding sites on the B subunit, cysteine-scanning mutagenesis will be employed. The role of B subunit isoforms in activity and intracellular targeting of V-ATPases will also be tested. To further define the structure of the catalytic A subunit, residues participating in nucleotide binding will be identified by site-directed mutagenesis. The proximity and role of conserved A subunit cysteine residues in regulation of vacuolar acidification will also be further probed. Studies of the 100 kDa subunit will focus on identification of second-site suppressors of mutations demonstrated to affect function, on elucidation of the topography of the 100 kDa subunit, and on identification of mutations which confer concanamycin resistance. Finally, the arrangement and function of accessory subunits, including AP50 and the VMA6 gene product, will be investigated. These studies should provide further insight into the structure and regulation of this important family of H+-ATPase.