Subunit Structure and Function in Vacuolar H+-ATPases

描述(申请人提供)：V-ATPase是由ATP驱动的质子泵，负责所有真核细胞中的细胞器酸化，并被招募到质膜上，用于破骨细胞、肾脏和男性生殖道的特殊功能。V-ATPase活性与蛋白质在胞内和生物合成途径中的分选、酶原激活以及pH、钙和金属离子的动态平衡密切相关。V-ATPase活性也被颠覆，以支持某些癌症的转移，并支持病毒和毒素从内吞途径释放到胞浆中。V-ATPase非常保守，由含有ATP水解点的外周膜蛋白复合体V1结合到含有质子孔的膜复合体Vo组成。V1和Vo扇区必须联合才能发生质子泵浦，但在能量限制的条件下，它们也会可逆地解离。我们建议继续使用酵母作为一个模型系统，通过以下三个目的来探索V-ATPase机制和调控的结构基础以及V-ATPase活性的细胞背景：1)我们将定义V1、C和H亚基与V1和Vo部分的相互作用，并探讨这些相互作用在缺糖过程中的变化。当前授权期的工作表明，V1扇区有两个定子柄，我们假设C和H将这些柄连接到膜扇区的不同区域，并从这个位置调控V1从Vo可逆地拆解。2)对葡萄糖反应的可逆分解表明，V-ATPase的活性与细胞的不同需求通过鲜为人知的代谢信号相一致。我们将通过测试最近发现的几种与Vo部分相互作用的蛋白质的功能作用，并利用二倍体细胞对H亚基单倍体充足的敏感性作为一种手段来识别对V1-Vo重组至关重要的基因产物，来探索这种比对的细胞基础。3)我们将确定V-ATPase在多大程度上既是细胞中的一般pH调节器，又是pH传感器。使用最近开发的灵活而强大的酵母液泡和胞液pH测量方法，我们将确定V-ATPase活性影响总体pH动态平衡的程度。这些实验可能有助于解释缺乏V-ATPase活性的酵母突变株的深远的生理缺陷。我们还将检测V-ATPase本身的活性对测得的胞浆和空泡pH变化的响应。

DESCRIPTION (provided by applicant): V-ATPases are ATP-driven proton pumps that are responsible for organelle acidification in all eukaryotic cells and are recruited to the plasma membrane for specialized functions in osteoclasts, kidney, and the male reproductive tract. V-ATPase activity is intimately linked to protein sorting in the endocytic and biosynthetic pathways, zymogen activation, and pH, calcium, and metal ion homeostasis. V-ATPase activity is also subverted to support metastasis of certain cancers and virus and toxin release from the endocytic pathway into the cytosol. V-ATPases are very highly conserved, and are comprised of a complex of peri- pheral membrane proteins containing the sites of ATP hydrolysis, V1, attached to a membrane complex containing the proton pore, Vo. V1 and Vo sectors must associate for proton pumping to occur, but they also reversibly dissociate under conditions of energy limitation. We propose to continue to use yeast as a model system to explore the structural basis of V-ATPase mechanism and regulation, along with the cellular context of V-ATPase activity, through the following three aims: 1) We will define the interactions of the V1 C and H subunits with the V1 and Vo sectors and probe how these interactions change during glucose deprivation. Work in the current grant period indicated that the V1 sector has two stator stalks, and we hypothesize that the C and H bridge these stalks to different regions of the membrane sector, and from this position, regulate reversible disassembly of V1 from Vo. 2) Reversible disassembly in response to glucose suggests that V- ATPase activity is aligned to the varying needs of the cell through poorly understood metabolic signals. We will explore the cellular basis of this alignment by testing the functional roles of several proteins recently found to interact with the Vo sector and exploiting the sensitivity of diploid cells to H subunit haploin- sufficiency as a means to identify gene products important for V1-Vo reassembly. 3) We will determine the extent to which the V-ATPase is both a general pH regulator in the cell and a pH sensor. Using recently developed methods for flexible and robust vacuolar and cytosolic pH measurement in yeast, we will deter- mine the extent to which V-ATPase activity affects overall pH homeostasis. These experiments may help explain the far-reaching physiological defects of yeast mutants lacking V-ATPase activity. We will also examine the activity of the V-ATPase itself in response to measured cytosolic and vacuolar pH changes.