Subunit Structure and Function of Vacuolar H+-ATPases

液泡H-ATP酶的亚基结构和功能

• 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
• 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

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酶是普遍存在且高度保守的质子泵，负责几乎所有真核细胞中的细胞器酸化以及少数细胞类型中的质子输出。通过其在细胞器酸化中的作用，V-ATP 酶影响大分子降解、蛋白质分选、pH 稳态以及离子和营养物质的隔离。最近的数据表明，V-ATP 酶在多种病理生理状况中发挥着核心作用。例如，它们有助于防御某些类型的神经退行性疾病，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点，但它们的复杂性也使它们变得困难。 V-ATP 酶是多亚基酶，由外周复合物（V1 部分）和完整膜复合物（Vo 部分）组成；这两个部分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-ATP 酶模型系统无与伦比的灵活性来解决哺乳动物 V-ATP 酶中至关重要但实验上难以解决的几个问题。在目标 1 中，我们将测试以下假设：内体/溶酶体信号脂质 PI(3,5)P2 直接与 V-ATP 酶的 Vo 部分相互作用，并通过稳定 V1-Vo 相互作用来调节酶活性。哺乳动物中这种脂质的消耗会导致神经变性。我们的实验可能表明细胞器酸化的丧失是否是一个潜在的原因。在目标 2 中，我们将使用隔室特异性比率荧光探针来测试两种不同的酵母亚基亚型对体内 pH 调节的贡献。高等真核细胞通常具有多种 V-ATP 酶亚基异构体，其对细胞器 pH 控制和调节的各自贡献尚不清楚；实验上更容易处理的酵母系统的结果可以作为其他细胞中同工型依赖性 pH 控制的范例。最后，我们发现 V-ATP 酶活性的急性和慢性丧失都会引发主要质膜质子输出蛋白的下调，这表明主要细胞器和质膜 pH 控制机制之间存在意想不到的协调水平。在目标 3 中，我们将检验以下假设：细胞器酸化的丧失（可能在内体中感知到）会诱导质膜上质子输出机制的泛素依赖性内化，作为一种补偿机制。平衡细胞器酸化、细胞质 p​​H 控制和质子输出需求的机制可能存在于所有细胞中，但很大程度上尚未被探索。这些实验将开始解决细胞器酸化如何被感知和保存的问题。