Subunit Structure and Function in Vacuolar H+-ATPases

描述（由申请人提供）：V-ATP酶是ATP驱动的质子泵，负责所有真核细胞中的细胞器酸化，并被募集到质膜，在破骨细胞、肾脏和男性生殖道中发挥专门功能。V-ATP酶活性与内吞和生物合成途径中的蛋白质分选、酶原活化以及pH、钙和金属离子稳态密切相关。V-ATP酶活性也被破坏以支持某些癌症的转移以及病毒和毒素从内吞途径释放到胞质溶胶中。V-ATP酶是非常高度保守的，并且由包含ATP水解位点的质膜蛋白复合物V1组成，所述质膜蛋白复合物连接到包含质子孔Vo的膜复合物。V1和Vo扇区必须关联以发生质子泵送，但它们在能量限制的条件下也可逆地解离。我们建议继续使用酵母作为模型系统来探索V-ATP酶机制和调节的结构基础，沿着V-ATP酶活性的细胞背景，通过以下三个目标：1）我们将定义V1 C和H亚基与V1和Vo部分的相互作用，并探索这些相互作用在葡萄糖剥夺期间如何变化。在目前的资助期间的工作表明，V1部门有两个定子柄，我们假设，C和H桥这些柄膜部门的不同区域，并从这个位置，调节可逆拆卸V1从Vo。2)响应于葡萄糖的可逆分解表明，V-ATP酶活性通过知之甚少的代谢信号与细胞的不同需求相一致。我们将通过测试最近发现的与Vo部分相互作用的几种蛋白质的功能作用，并利用二倍体细胞对H亚基单倍性的敏感性作为鉴定对V1-Vo重组重要的基因产物的手段，来探索这种对齐的细胞基础。3)我们将确定V-ATP酶既是细胞中的一般pH调节剂又是pH传感器的程度。使用最近开发的方法，灵活和强大的液泡和胞质pH值测量酵母，我们将确定V-ATP酶活性影响整体pH稳态的程度。这些实验可能有助于解释缺乏V-ATP酶活性的酵母突变体的深远的生理缺陷。我们还将检查V-ATPase本身的活性，以响应测量的胞质和液泡pH值的变化。

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.