Structure and regulation of the vacuolar ATPase

项目摘要 质子泵液泡ATPase(V-ATPase；V1Vo-ATPase)是发现的一种重要的酶复合体 在所有真核生物的内膜系统和一些动物细胞的质膜中。 V-ATPase在ATP水解引起的亚细胞室或细胞外酸化中的作用 空间，这是一个对基本细胞过程至关重要的过程，包括内吞作用、蛋白质运输、骨重建、 尿液酸化、精子成熟和神经递质释放。V-ATPase在动物细胞中的丢失 胚胎致死，虽然部分功能丧失(或多动)与许多人类疾病有关， 如肾小管性酸中毒、骨质疏松症、糖尿病、男性不育、神经变性、癌症和艾滋病。 在分子水平上与这些疾病作斗争需要详细了解其结构、催化作用 真核细胞V-ATPase复合体的作用机制及其调控。 在细胞中，V-ATPase的活性受一种称为可逆分解的独特机制的调节 络合物解离为V1-ATPase和Vo质子通道扇区的条件 功能上的沉默。尽管它在V-ATPase功能中起着重要作用，但其活性的分子机制 人们对可逆拆卸的调控知之甚少，这一知识差距很大程度上是由于缺乏 高分辨率结构信息和定义的体外模型系统在受控下研究过程 条件。 该项目的直接目标是获得高分辨率的结构和机械信息。 目的是为了更好地了解V-ATPase的调控，揭示V-ATPase的非典型功能。 可能在神经元通讯中发挥作用的ATPase Vo膜区。我们将解决这些问题 具体目标如下： (1)V-ATPase组装和拆卸的分子决定因素 (2)V-ATPase Vo膜扇区的非规范功能。 我们研究了酵母V-ATPase的结构和调控，这是一个功能强大的模型系统 哺乳动物酶由于高度保守的酶的结构和机制跨越 物种，遗传操作的简易性，以及获得高纯度酶的能力 亚基组成。该项目的长期目标是制定旨在促进 或抑制人类V-ATPase的可逆解离过程，从而允许调制 为了治疗目的，该酶在组织和亚基异构体中的活性依赖于。

Project Summary The proton-pumping vacuolar ATPase (V-ATPase; V1Vo-ATPase) is an essential enzyme complex found in the endomembrane system of all eukaryotic organisms and in the plasma membrane of some animal cells. The V-ATPase functions in ATP hydrolysis-driven acidification of subcellular compartments or the extracellular space, a process vital for basic cellular processes including endocytosis, protein trafficking, bone remodeling, urine acidification, sperm maturation, and neurotransmitter release. Loss of V-ATPase in animal cells is embryonic lethal, while partial loss of function (or hyperactivity) has been linked to numerous human diseases, such as renal tubular acidosis, osteoporosis, diabetes, male infertility, neurodegeneration, cancer, and AIDS. Fighting these diseases on a molecular level will require a detailed understanding of the structure, catalytic mechanism, and regulation of the eukaryotic V-ATPase complex. In cells, V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, a condition under which the complex dissociates into V1-ATPase and Vo proton channel sectors that are both functionally silenced. Despite its important role in V-ATPase function, the molecular mechanism of activity regulation by reversible disassembly is poorly understood, a gap in knowledge that is largely due to the lack of high-resolution structural information and a defined in vitro model system to study the process under controlled conditions. The immediate goal of this project is to obtain high-resolution structural and mechanistic information aimed at a better understanding of V-ATPase regulation and to uncover non-canonical functions of the V- ATPase Vo membrane sector that may play a role in neuronal communication. We will address these questions with the following specific aims: (1) Molecular determinants of V-ATPase assembly and disassembly (2) Non-canonical functions of the V-ATPase Vo membrane sector. We study the structure and regulation of the yeast V-ATPase, a powerful model system for the mammalian enzyme due to the high level of conservation of the enzyme's structure and mechanism across species, the ease of genetic manipulation, and the ability to obtain highly purified enzyme with a defined subunit composition. The long-term objective of the project is to develop strategies aimed at either promoting or inhibiting the process of reversible disassembly of the human V-ATPase that will allow modulation of the activity of the enzyme in a tissue and subunit isoform dependent manner for therapeutic purposes.