Subunit Structure and Function of Vacuolar H+-ATPases

描述(申请人提供)：V-ATPase是普遍存在的高度保守的质子泵，负责几乎所有真核细胞的细胞器酸化和少数细胞类型的质子输出。通过它们在细胞器酸化中的作用，V-ATPase影响大分子降解、蛋白质分类、pH动态平衡以及离子和营养物质的隔离。最近的数据显示，V-ATPase在多种病理生理条件下发挥着核心作用。例如，它们有助于防止某些类型的神经退化，但可能会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是由一个外周复合体(V1区段)和一个完整的膜复合体(Vo区段)组成的多亚单位酶，这两个区段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模型系统无与伦比的灵活性来解决哺乳动物V-ATPase中几个至关重要但在实验上难以解决的问题。在目标1中，我们将验证这样的假设，即内切体/溶酶体信号转导脂质PI(3，5)P2直接与V-ATPase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。哺乳动物体内这种脂质离子的耗尽会导致神经退化；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单位亚基异构体在体内对pH调节的贡献。高等真核细胞通常有几种V-ATPase亚基异构体，它们对细胞器pH控制和调节的单独作用尚不清楚；更易于实验控制的酵母系统的结果可能为其他细胞中依赖于异构体的pH控制提供范例。最后，我们发现，急性和慢性V-ATPase活性的丧失都会触发主要质膜质子输出器的下调，这表明主要细胞器和质膜pH控制机制之间存在着意想不到的协调。在目标3中，我们将检验一种假设，即细胞器酸化的丧失可能是在内体感受到的，诱导质膜上质子输出机制的泛素依赖的内化作为一种补偿机制。细胞器酸化、胞浆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。这些实验将开始研究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.