Subunit Structure and Function in Vacuolar H+-ATPase

描述（由申请人提供）：液泡质子易位ATP酶（v -ATP酶）结合胞质ATP的水解，将质子运输到所有真核细胞的细胞器中，并穿过某些细胞类型的质膜。细胞器酸化是v - atp酶的主要组成功能，对许多生理过程至关重要，但也与许多疾病状态有关。例如，吞噬体的酸化对于杀死入侵细菌至关重要，但许多病毒和毒素利用v - atp酶产生的酸性环境，促进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 - atp酶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 - atp酶亚基亚型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。实验室的长期目标是通过研究酵母v - atp酶来了解v - atp酶的结构、功能、组装和调控，酵母v - atp酶已被证明是所有真核生物v - atp酶的优秀模型。所有v -ATP酶都由两个多亚基结构域组成，一个是参与ATP水解的外周膜复合物，另一个是质子运输所需的整体膜复合物。在本提案中，我们将重点关注在结构和功能上连接这两个域的“stalk”亚基。这些亚基负责将ATP水解引起的构象变化传递到质子孔，并且也是v -ATP酶的调节分解的关键参与者，这是一种重要的调节机制。本建议的目的是：1)通过结合电子显微镜、亚复合物的流体动力学研究、诱变和交联实验来定位酵母V-ATPase中的杆亚基；2)阐明C和H亚基的作用，特别是在伴随外周区从膜区释放而发生的重要功能构象变化中；3)检查与a亚基的两种同工异构体的蛋白质-蛋白质相互作用，并测试它们在调节拆卸中的重要性。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.