Subunit Structure and Function in Vacuolar H+-ATPase

液泡 H-ATP 酶的亚基结构和功能

• 批准号: 6752136
• 负责人: PATRICIA M KANE
• 金额: $ 25.84万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-03-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6752136
• 关键词: Saccharomyces cerevisiae; acidity /alkalinity; active sites; adenosinetriphosphatase; conformation; crosslink; cytoplasm; electron microscopy; enzyme activity; enzyme mechanism; enzyme structure; fluorescence resonance energy transfer; green fluorescent proteins; hydrogen transport; isozymes; mass spectrometry; membrane transport proteins; molecular assembly /self assembly; protein protein interaction; protein structure function; site directed mutagenesis; vesicle /vacuole; yeasts

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.

描述（由申请人提供）：极性质子转运ATP酶（V-ATP酶）将胞质ATP的水解与质子转运偶联到所有真核细胞的细胞器中和某些细胞类型的质膜上。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是必不可少的，但是许多病毒和毒素利用V-ATP酶产生的酸性环境来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酶参与肾脏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酶亚基异构体的突变导致以代谢性酸中毒和骨质疏松症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-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V-ATP酶的优秀模型。所有的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，另一个是质子转运所需的完整膜复合物。在这个建议中，我们专注于“茎”亚基的结构和功能桥梁这两个领域。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酶的调节性分解（一种重要的调节机制）中的关键参与者。这项建议的目的是：1）通过电子显微镜、亚复合物的流体动力学研究、诱变和交联实验的组合，在酵母V-ATP酶中定位茎亚基，2）阐明C和H亚基的作用，特别是在伴随外周部分从膜部分释放的功能上重要的构象变化中，3）检测与“a”亚基的两种同种型的蛋白质-蛋白质相互作用，并测试它们在调节的分解中的重要性，以及4）使用GFP标记的V-ATP酶亚基在体内跟踪V-ATP酶组装和分解。