SUBUNIT STRUCTURE/FUNCTION IN VACUOLAR H+ ATPASES

在所有的细胞中都发现了质子转运ATP酶（V-ATP酶）。 真核细胞，并似乎在所有细胞中发挥组成性作用 在某些细胞类型中扮演更特殊的角色。V-ATP酶是 能偶联ATP的多亚基酶 水解到质子跨膜转运。 主 V-ATP酶在所有人类细胞中的组成性作用似乎是 某些细胞内区室的酸化， 酸化对于维持细胞内部的 对细胞外刺激作出反应的组织和能力。细胞器 由V-ATP酶介导的酸化被各种 病原体，包括某些病毒和毒素， 病原体进入细胞质;其他病原体操纵V- ATP酶的活性，使它们存在于细胞内区室。 V-ATP酶在调节分泌颗粒中发挥特殊作用， 神经细胞，在那里它们参与隔离 神经递质，并在肾脏的质膜上插入 细胞、破骨细胞、巨噬细胞和嗜中性粒细胞， 在尿酸化、骨吸收和细胞质调节中的作用 pH值，分别。 酿酒酵母的V-ATPase 已被证明是一个很好的实验模型的V-ATP酶的其他 真核生物，包括人类。 这项研究的长期目标是 了解的结构，功能，组装，和调节的 酵母V-ATPase 本提案的具体目标是 了解周围V1扇区之间的相互作用 负责ATP水解的V-ATP酶，以及 膜Vo部门，负责质子运输。 的 V1和Vo扇区之间的相互作用是催化剂的核心。 V-ATP酶的活性，也是酶调节的主要位点。 为了实现这一目标，研究了两个亚基（Vma 5 p和Vma 13 p）的功能， 已知对于V1和Vo扇区之间的相互作用是重要的 将详细研究，无论是在野生型酵母细胞和菌株 每个亚基基因中含有点突变。 可逆 V1-Vo复合物解离成胞质V1部分和膜- 结合Vo扇区已被证明在体内响应于营养素而发生 在酵母和昆虫细胞中， 调控机制。 细胞溶质V1部分将从 酵母细胞，以及这些细胞的生物化学和酶性质， 将对各部门进行检查。 催化活性、核苷酸 结合、胞质pH变化和V-ATP酶组装状态 将在生物化学研究中进行探索， 在营养剥夺的条件下， 将通过分离在分解中有缺陷的酵母突变体来检查， 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.