STRUCTURE, MECHANISM AND REGULATION OF THE V-ATPASES

申请是为了支持对该结构的继续研究， V-型膜上质子转运ATPase的机制及其调控 一家人。V型ATPase负责酸化 在真核细胞中的细胞内隔室和服务于重要的 在多种细胞过程中的作用，包括受体介导的 内吞作用，细胞内膜运输，大分子加工， 以及降解和耦合运输。质膜中的V-ATPase 在肾脏酸化、骨质疏松症中也起作用 吸收和肿瘤转移。了解V-ATPase是如何 受监管对于理解这些过程很重要。 本实验室先前已经证明，来自笼状蛋白的V-ATPase- 包被的囊泡被组织成外围V1区域，负责 ATP水解与质子的积分Vo结构域 易位。已使用化学修饰来探测该结构 在核苷酸结合位点中，这个小组提出了二硫键 在体内，键的形成可能在调节V-ATPase活性中起作用。 重组研究已经被用来测试个体的功能 亚基，包括V-ATPase和AP-2接头之间共有的蛋白质 复合体。最近，他们开始了对这种基因的突变研究 酵母V-ATPase用于鉴定对V-ATPase活性重要的残基。 拟议的研究将追求四个具体目标。要确定 非催化核苷酸结合位点的结构和功能 将采用B亚基半胱氨酸扫描诱变。角色 V-ATPase B亚基异构体的活性和细胞内靶向性 也将接受测试。为了进一步确定催化剂A的结构 亚基，参与核苷酸结合的残基将被识别 通过定点突变。守恒A的近似性及其作用 半胱氨酸残基在调节空泡酸化中的作用 也有待进一步探讨。对100 kDa亚基的研究将集中在 突变的第二位点抑制子的鉴定 情感作用，对100 kDa地貌的澄清 亚基，并鉴定与刀豆素有关的突变 抵抗。最后，辅助亚单位的排列和功能， 包括AP50和VMA6基因产物，将进行研究。这些 研究应该提供对结构和监管的进一步洞察 这个重要的H+-ATPase家族的成员。

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.