Subunit Structure and Function in Vacuolar H+-ATPase

描述(申请人提供)：液泡质子转运ATPase(V-ATPase)将胞质中的三磷酸腺苷的水解作用结合到质子转运到所有真核细胞的细胞器和某些细胞类型的质膜上。细胞器酸化是V-ATPase的主要组成功能，在许多生理过程中是必不可少的，但也与许多疾病状态有关。例如，吞噬小体的酸化对于杀死入侵的细菌是必不可少的，但许多病毒和毒素利用V-ATPase产生的酸性环境来帮助它们从细胞器逃逸到细胞质中，在那里它们变得具有生物活性。质膜V-ATPase参与肾酸分泌和破骨细胞骨溶解；这些过程所必需的组织特异性V-ATPase亚基突变导致以代谢性酸中毒和骨质疏松为特征的遗传性疾病。该实验室的长期目标是通过研究酵母V-ATPase来了解V-ATPase的结构、功能、组装和调控，酵母V-ATPase已被证明是所有真核V-ATPase的优秀模型。所有的V-ATPase都由两个多亚单位结构域组成，一个是参与ATP水解的外周膜复合体，另一个是质子转运所需的完整膜复合体。在这个提案中，我们重点关注在结构和功能上连接这两个结构域的“茎”亚单位。这些亚基负责将ATP水解引起的构象变化传递到质子孔中，也是V-ATPase的调节分解的关键角色，这是一种重要的调节机制。这项建议的目的是：1)通过结合电子显微镜、亚复合体的流体动力学研究、突变和交联实验来定位酵母V-ATPase中的茎亚基，2)阐明C和H亚基的作用，特别是在伴随着外周部分从膜部分释放而发生的功能上的重要构象变化中的作用，3)检测蛋白质与“a”亚基的两种异构体的相互作用，并测试它们在调节拆卸中的重要性，以及4)使用GFP标记的V-ATPase亚基跟踪V-ATPase在体内的组装和拆卸。

DESCRIPTION (provided by applicant): Vacuolar proton-translocating ATPases (V-ATPases) couple hydrolysis of cytosolic ATP to proton transport into organelles of all eukaryotic cells and across the plasma membrane of some cell types. Organelle acidification, the major constitutive function of V-ATPases, is essntial for many physiological processes, but is also linked to a number of disease states. For example, acidification of phagosomes is essential for killing invading bacteria, but many viruses and toxins exploit the acidic environment generated by V-ATPases to facilitate their escape from organelles into the cytoplasm where they become biologically active. Plasma membrane V-ATPases are involved in renal acid secretion and osteoclast bone dissolution; mutations in tissue-specific V-ATPase subunit isoforms necessary for these processes result in genetic diseases characterized by metabolic acidosis and osteoporosis. The long-term goal of the lab is to understand the structure, function, assembly and regulation of V-ATPases by studying the yeast V-ATPase, which has proven to be an excellent model for all eukaryotic V-ATPases. All V-ATPases are composed of two multisubunit domains, a peripheral membrane complex involved in ATP hydrolysis and an integral membrane complex required for proton transport. In this proposal, we focus on the "stalk" subunits that structurally and functionally bridge these two domains. These subunits are responsible for transmission of conformational changes resulting from ATP hydrolysis to the proton pore, and are also key players in regulated disassembly of V-ATPases, an important regulatory mechanism. The aims of this proposal are: 1) to position the stalk subunits in the yeast V-ATPase, by a combination of electron microscopy, hydrodynamic studies of subcomplexes, mutagenesis, and crosslinking experiments, 2) to elucidate the roles of the C and H subunits, particularly in the functionally important conformational change accompanying release of the peripheral sector from the membrane sector, 3) to examine protein-protein interactions with two isoforms of the "a" subunit and test their importance in regulated disassembly, and 4) to follow V-ATPase assembly and disassembly in vivo using GFP-tagged V-ATPase subunits.