Subunit Structure and Function of Vacuolar H+-ATPases

液泡H-ATP酶的亚基结构和功能

基本信息

批准号：
8415511
负责人：
PATRICIA M KANE
金额：
$ 30.78万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-03-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8415511
关键词：
ATP Hydrolysis Acute Address Alkalinization Automobile Driving Biochemical Biological Models Cell membrane Cells Chronic Complex Consensus Cytoplasmic Granules Data Dependence Development Disease Down-Regulation Drug Targeting Endosomes Enzymes Equilibrium Eukaryotic Cell Fluorescent Probes Functional disorder Future Genetic Glucose Golgi Apparatus Human Individual Ions Link Lipids Location Lysosomes Malignant Neoplasms Mammalian Cell Mammals Measurement Membrane Molecular Neoplasm Metastasis Nerve Degeneration Neurodegenerative Disorders Nutrient Organelles Organism Osteoporosis Oxidative Stress Peripheral Phosphatidylinositols Phospholipids Physiology Play Process Protein Isoforms Proteins Proton Pump Protons Regulation Relative (related person)Resistance Role Signal Transduction Site Sorting - Cell Movement Starvation Stress Structural Models Structure System Testing Time Tissues Ubiquitin Ubiquitination Vacuole Work Yeasts base cell type environmental change enzyme activity enzyme model flexibility in vivo insight macromolecule pH Homeostasis protein degradation ratiometric research study response restin therapeutic target tool tumor vacuolar H+-ATPase yeast genetics

项目摘要

DESCRIPTION (provided by applicant): V-ATPases are ubiquitous and highly conserved proton pumps responsible for organelle acidification in virtually all eukaryotic cells and for proton export in a few cell types. Through their roles in organelle acidification, V-ATPases impact macromolecular degradation, protein sorting, pH homeostasis, and sequestration of ions and nutrients. Recent data has revealed that V-ATPases play a central role in multiple pathophysiological conditions. For example, they help defend against some types of neurodegeneration, but can promote tumor metastasis and osteoporosis. They are thus attractive drug targets, but their complexity also makes them difficult. V-ATPases are multisubunit enzymes comprised of a peripheral complex, the V1 sector, attached to an integral membrane complex, the Vo sector; interaction between these two sectors is a major target of enzyme regulation. We propose to exploit the unparalleled flexibility of the yeast V-ATPase model system to address several issues that are critically important but experimentally intractable in mammalian V-ATPases. In Aim 1, we will test the hypothesis that the endosome/lysosome signaling lipid PI(3,5)P2 interacts directly with the Vo sector of the V-ATPase and regulates enzyme activity by stabilizing V1- Vo interactions. Depletion of this lipid iin mammals results in neurodegeneration; our experiments may indicate whether loss of organelle acidification is a potential cause. In Aim 2, we will use compartment-specific ratiometric fluorescent probes to test the contributions of two different yeast subunit isoforms to pH regulation in vivo. Higher eukaryotic cells often have several V-ATPase subunit isoforms whose individual contributions to organelle pH control and regulation are unclear; results from the more experimentally tractable yeast system may serve as a paradigm for isoform-dependent pH control in other cells. Finally, we have found that both acute and chronic loss of V-ATPase activity triggers downregulation of the major plasma membrane proton exporter, suggesting an unexpected level of coordination between the major organelle and plasma membrane pH control mechanisms. In Aim 3, we will test the hypothesis that loss of organelle acidification, possibly sensed at the endosome, induces ubiquitin-dependent internalization of proton export machinery at the plasma membrane as a compensatory mechanism. Mechanisms for balancing demands of organelle acidification, cytosolic pH control, and proton export are likely present in all cells but are largely unexplored. These experiments will begin to address how organelle acidification is sensed and preserved.

描述（由申请人提供）：V-ATP酶是无处不在且高度保守的质子泵，负责在几乎所有真核细胞中的细胞器酸化以及几种细胞类型的质子导出。通过它们在细胞器酸化中的作用，V-ATP酶会影响大分子降解，蛋白质排序，pH稳态以及离子和营养素的隔离。最近的数据表明，V-ATP酶在多种病理生理条件下起着核心作用。例如，它们有助于防御某些类型的神经变性，但可以促进肿瘤转移和骨质疏松症。因此，它们是有吸引力的药物靶标，但它们的复杂性也使它们变得困难。 V-ATPase是由外围络合物组成的多生物酶，该酶是连接到整体膜复合物的VO扇区的V1扇区；这两个部门之间的相互作用是酶调节的主要目标。我们建议利用酵母V-ATPase模型系统的无与伦比的灵活性，以解决几个至关重要但在哺乳动物V-ATPase中非常棘手的问题。在AIM 1中，我们将测试以下假设：内体/溶酶体信号传导脂质PI（3,5）P2与V-ATPase的Vo扇区直接相互作用，并通过稳定V1-vo相互作用来调节酶活性。这种脂质IIN哺乳动物的耗竭会导致神经退行性。我们的实验可能表明细胞器酸化的丧失是否是潜在的原因。在AIM 2中，我们将使用隔室特异性比例荧光探针来测试两种不同的酵母亚基同工型对体内pH调节的贡献。较高的真核细胞通常具有几种V-ATPase亚基同工型，其对细胞器pH控制和调节的个人贡献尚不清楚。来自实验性疗法的酵母系统的结果可能是其他细胞中同工型依赖性pH控制的范式。最后，我们发现V-ATPase活性的急性和慢性丧失触发了主要质膜质子出口商的下调，这表明主要细胞器和质膜pH控制机制之间的协调水平出乎意料。在AIM 3中，我们将检验以下假设：可能在内体处感受的细胞器酸化损失会诱导质子膜上质子导出机械的泛素依赖性内在化作为补偿机制。在所有细胞中都可能存在细胞器酸化，胞质pH控制和质子导出的平衡需求的机制，但在很大程度上没有探索。这些实验将开始解决细胞器酸化的感觉和保存方式。