Subunit Structure and Function in Vacuolar H+-ATPases

液泡 H-ATP 酶的亚基结构和功能

• 批准号: 8330154
• 负责人: PATRICIA M KANE
• 金额: $ 4.79万
• 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 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8330154
• 关键词: ATP Hydrolysis; ATP Synthesis Pathway; ATP phosphohydrolase; Affect; Biological Models; Calcium; Cell membrane; Cells; Chronic; Complex; Cytosol; Defect; Diploid Cells; Diploidy; Elements; Energy Supply; Enzyme Precursors; Enzymes; Eukaryota; Eukaryotic Cell; Genetic; Genetic Screening; Glucose; Grant; Heavy Metals; Homeostasis; Ions; Kidney; Link; Malignant Neoplasms; Measurement; Measures; Membrane; Membrane Proteins; Metabolic; Metals; Methods; Mining; Neoplasm Metastasis; Organelles; Osteoclasts; Oxidative Stress; Pathway interactions; Peripheral; Phosphotransferases; Physiological; Play; Positioning Attribute; Property; Protein Kinase; Proteins; Proton Pump; Protons; Recruitment Activity; Regulation; Research Personnel; Role; Signal Transduction; Site; Sorting - Cell Movement; Structure; Testing; Time; Toxin; Vacuole; Virus; Work; Yeasts; base; deprivation; enzyme activity; extracellular; flexibility; fungus; in vivo; interest; male; mutant; novel; pH Homeostasis; programs; reproductive; research study; response; sensor; trafficking; uptake; vacuolar H+-ATPase

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.

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，并募集到质膜上的质膜，从而在破骨细胞，肾脏和雄性生殖道中具有专门功能。 V-ATPase活性与内吞和生物合成途径，酶原激活以及pH，钙和金属离子稳态中的蛋白质分类密切相关。 V-ATPase活性也被颠覆以支持某些癌症和病毒的转移，以及从内吞途径释放到细胞溶胶中的转移。 V-ATP酶非常保守，由含有ATP水解位点的植物膜蛋白组成，该膜v1附着于含有质子孔的膜络合物上。 V1和VO部门必须与质子泵送相关，但在能量限制条件下它们也可逆地解离。我们建议通过以下三个目的继续使用酵母作为模型系统，以探索V-ATPase机制和调节的结构基础，以及V-ATPase活性的细胞环境：1）我们将定义V1 C和Hubunits与V1和VO型的相互作用，并探测这些相互作用在Glucose glucose glucosivativativation过程中的相互作用变化。当前赠款期间的工作表明，V1部门有两个定子茎，我们假设C和H将这些茎桥接到膜部门的不同区域，并且从该位置则调节V1的V1可逆拆卸。 2）对葡萄糖的反应可逆拆卸表明，通过较知的代谢信号，V- ATPase活性与细胞的不同需求保持一致。我们将通过测试最近发现与Vo扇区相互作用并利用二倍体细胞对H亚基单倍倍足够的敏感性的几种蛋白质的功能作用来探索该比对的细胞基础，以此作为识别对V1-VO重新组装的基因产物的一种手段。 3）我们将确定V-ATPase既是细胞中的一般pH调节剂，又是pH传感器的程度。使用最近开发的酵母中柔性和鲁棒性液泡和胞质pH测量的方法，我们将确定V-ATPase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-ATPase活性的酵母突变体的深远生理缺陷。我们还将检查V-ATPase本身的活性，以响应测得的胞质和液泡pH变化。