Subunit Structure and Function of Vacuolar H+-ATPases

描述（由申请人提供）：v - atp酶是普遍存在的高度保守的质子泵，在几乎所有真核细胞中负责细胞器酸化，并在少数细胞类型中负责质子输出。通过它们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。最近的数据显示，v - atp酶在多种病理生理条件中发挥核心作用。例如，它们有助于预防某些类型的神经变性，但可能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难以实现。v - atp酶是一种多亚基酶，由外周复合物（V1区）和完整膜复合物（Vo区）组成；这两个部分之间的相互作用是酶调控的主要目标。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Aim 1中，我们将验证内核体/溶酶体信号脂质PI(3,5)P2直接与v - atp酶的Vo区相互作用并通过稳定V1- Vo相互作用来调节酶活性的假设。哺乳动物体内这种脂质的消耗会导致神经退化；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Aim 2中，我们将使用室特异性比例荧光探针来测试两种不同酵母亚基异构体对体内pH调节的贡献。高等真核细胞通常有几种v - atp酶亚基异构体，其对细胞器pH控制和调节的个体贡献尚不清楚；实验上更易处理的酵母系统的结果可以作为其他细胞中依赖于同工异构体的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触发主要质膜质子输出的下调，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Aim 3中，我们将验证一个假设，即细胞器酸化的丧失（可能在核内体感知）诱导了质膜上依赖泛素的质子输出机制的内化作为一种补偿机制。细胞器酸化、胞质pH控制和质子输出的平衡机制可能存在于所有细胞中，但在很大程度上尚未被探索。这些实验将开始解决细胞器酸化是如何被感知和保存的。

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.