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，并被募集至质膜以在破骨细胞、肾脏和男性生殖道中发挥特殊功能。 V-ATP 酶活性与内吞和生物合成途径中的蛋白质分选、酶原激活以及 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细胞质中。 V-ATP 酶非常高度保守，由含有 ATP 水解位点 V1 的外周膜蛋白复合物组成，该复合物附着在含有质子孔 Vo 的膜复合物上。 V1 和 Vo 扇区必须结合才能发生质子泵浦，但它们在能量限制的条件下也会可逆地解离。我们建议继续使用酵母作为模型系统，通过以下三个目标来探索 V-ATPase 机制和调节的结构基础，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葡萄糖剥夺期间如何变化。当前资助期间的工作表明，V1 部分有两个定子茎，我们假设 C 和 H 将这些茎桥接到膜部分的不同区域，并从这个位置调节 V1 从 Vo 的可逆分解。 2）响应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 区域相互作用的几种蛋白质的功能作用来探索这种比对的细胞基础，并利用二倍体细胞对 H 亚基单倍体充足性的敏感性作为识别对 V1-Vo 重组重要的基因产物的手段。 3) 我们将确定 V-ATP 酶在多大程度上既是细胞中的一般 pH 调节剂，又是 pH 传感器。使用最近开发的灵活且可靠的酵母液泡和细胞质 p​​H 测量方法，我们将确定 V-ATP 酶活性对整体 pH 稳态的影响程度。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-ATP 酶本身的活性，以响应测量的细胞质和液泡 pH 值变化。