SUBUNIT STRUCTURE/FUNCTION IN VACUOLAR H+ ATPASES

液泡 H ATP酶中的亚基结构/功能

• 批准号: 6164790
• 负责人: PATRICIA M KANE
• 金额: $ 19.84万
• 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 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6164790
• 关键词: Saccharomyces cerevisiae; acidity /alkalinity; active sites; adenosinetriphosphatase; conformation; cytoplasm; enzyme activity; enzyme mechanism; enzyme structure; epitope mapping; hydrogen transport; hydrogen transporting ATP synthase; membrane proteins; molecular assembly /self assembly; mutant; point mutation; site directed mutagenesis; structural genes; vesicle /vacuole

Vacuolar proton-translocating ATPases (V-ATPases) are found in all eukaryotic cells and appear to play both constitutive roles in all cells and more specialized roles in certain cell types. V-ATPases are multisubunit enzymes capable of coupling ATP hydrolysis to proton transport across membranes. The primary constitutive role of V-ATPases in all human cells appears to be acidification of certain intracellular compartments, and this acidification is critical for maintenance of the cell's internal organization and ability to respond to extracellular stimuli. Organelle acidification mediated by V-ATPases is exploited by a variety of pathogens, including certain viruses and toxins, to allow these pathogens to enter the cell cytoplasm; other pathogens manipulate V- ATPase activity to allow them to exist in intracellular compartments. V-ATPases play specialized roles in regulated secretory granules of neural cells, where they are involved in sequestration of neurotransmitters, and at the plasma membrane of kidney intercalated cells, osteoclasts, macrophages and neutrophils, where they are involved in urinary acidification, bone resorption, and regulation of cytoplasmic pH, respectively. The V-ATPase of the yeast Saccharomyces cerevisiae has proven to be an excellent experimental model for V-ATPases of other eukaryotes, including humans. The long-term goals of this research are to understand the structure, function, assembly, and regulation of the yeast V-ATPase. The specific aims of this proposal are directed toward understanding the interaction between the peripheral V1 sector of the V-ATPase, which is responsible for ATP hydrolysis, and the integral membrane Vo sector, which is responsible for proton transport. The interaction between the V1 and Vo sectors is central to the catalytic activity of V-ATPases and is also a major site of enzyme regulation. Toward this goal, the functions of two subunits (Vma5p and Vma13p) that are known to be important for interaction between the V1 and Vo sectors will be studied in detail, both in wild-type yeast cells and in strains containing point mutations in each subunit gene. Reversible dissociation of V1-Vo complexes into cytosolic V1 sectors and membrane- bound Vo sectors has been shown to occur in vivo in response to nutrient deprivation in yeast and in insect cells, and is probably a general mechanism of regulation. Cytosolic V1 sectors will be isolated from yeast cells, and the biochemical and enzymatic properties of these sectors will be examined. Links between catalytic activity, nucleotide binding, changes in cytosolic pH, and assembly state of the V-ATPase will be explored in biochemical studies, and the physiological benefits of dissociation of the V-ATPase under conditions of nutrient deprivation will be examined by isolating yeast mutants defective in disassembly of the enzyme.

在所有 真核细胞，似乎在所有细胞中都起着两种构成作用 以及在某些细胞类型中更专业的角色。 V-ATPases是 能够耦合ATP的多育酶 水解向质子跨膜传输的水解。 主要 V-ATPases在所有人类细胞中的构成作用似乎是 某些细胞内室的酸化，这 酸化对于维持细胞的内部至关重要 组织和对细胞外刺激做出反应的能力。细胞器 通过V-ATPases介导的酸化被多种 病原体，包括某些病毒和毒素，以允许这些病原体 病原体进入细胞质；其他病原体操纵V- ATPase活性使它们可以存在于细胞内室中。 V-ATPases在受监管的分泌颗粒中扮演专业角色 神经细胞，它们参与隔离 神经递质，以及肾脏插入的质膜 细胞，破骨细胞，巨噬细胞和中性粒细胞，它们涉及 在尿酸化，骨吸收和细胞质调节中 pH分别。 酿酒酵母的V-ATPase 事实证明，已成为其他V-ATP酶的极好的实验模型 真核生物，包括人类。 这项研究的长期目标是 了解结构，功能，组装和调节 酵母V-ATPase。 该提议的具体目的是针对 了解该外围V1部门之间的相互作用 V-ATPase，负责ATP水解和积分 膜VO部门，负责质子运输。 这 V1与Vo扇区之间的相互作用是催化的核心 V-ATP酶的活性，也是酶调节的主要部位。 为了实现这一目标，两个亚基的功能（VMA5P和VMA13P）的功能 众所周知，对于V1和VO部门之间的相互作用很重要 将在野生型酵母细胞和菌株中详细研究 每个亚基基因中包含点突变。 可逆 V1-VO复合物分解为胞质V1扇区和膜 已显示绑定的VO扇区在体内响应养分而发生 酵母和昆虫细胞中的剥夺，可能是一般 调节机制。 胞质V1扇区将从 酵母细胞，以及这些细胞的生化和酶促特性 将检查部门。 催化活性，核苷酸之间的联系 结合，胞质pH的变化和V-ATPase的组装状态 将在生化研究和生理益处中探讨 在营养剥夺条件下，V-ATPase解离 将通过隔离酵母突变体的拆卸来检查 酶。