SUBUNIT STRUCTURE/FUNCTION IN VACUOLAR H+ ATPASES

空泡质子转运ATPase(V-ATPase)存在于ALL 真核细胞，似乎在所有细胞中都扮演着两个组成部分的角色 在某些细胞类型中扮演更特殊的角色。V-ATPase是 能偶联三磷酸腺苷的多亚单位酶 通过膜进行质子运输的水解液。初级阶段 V-ATPase在所有人类细胞中的构成作用似乎是 某些细胞内隔间的酸化，而这 酸化对于维护电池内部的 对细胞外刺激作出反应的组织和能力。细胞器 V-ATPase介导的酸化被多种 病原体，包括某些病毒和毒素，使这些 病原体进入细胞质；其他病原体操纵V- ATPase的活性，使它们存在于细胞内的隔间。 V-ATPase在血管内皮细胞分泌颗粒调控中的特殊作用 神经细胞，在那里它们参与隔离 神经递质，并在肾脏质膜上嵌插 参与的细胞、破骨细胞、巨噬细胞和中性粒细胞 在尿酸、骨吸收和胞浆调节中 PH值分别为。酿酒酵母的V-ATPase 已经证明是一种很好的V-ATPase的实验模型 真核生物，包括人类。这项研究的长期目标是 要了解的结构、功能、组装和调节 酵母V-ATPase。这项建议的具体目标是 了解外围设备V1扇区之间的交互 负责ATP水解的V-ATPase和积分 负责质子运输的膜Vo部门。这个 V1和Vo扇区之间的相互作用是催化剂的中心 V-ATPase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(Vma5p和Vma13p)的功能 已知对V1和Vo扇区之间的相互作用很重要 将在野生型酵母细胞和菌株中进行详细研究 包含每个亚单位基因的点突变。可逆的 V1-Vo复合体解离成胞质V1扇区和膜- 结合的VO扇区已被证明在体内发生对营养的响应 在酵母和昆虫细胞中被剥夺，这可能是一种普遍的 调控机制。胞质V1扇区将从 酵母细胞以及它们的生化和酶特性 将对行业进行审查。催化活性、核苷酸之间的联系 V-ATPase的结合、胞液pH变化和组装状态 将在生化研究中探索，以及生理上的好处 营养剥夺条件下V-ATPase的解离 将通过分离在分解过程中有缺陷的酵母突变株来进行检测 这种酶。

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.