Structure, mechanism and regulation of the V-ATPases

描述（由申请人提供）：本研究的长期目标是确定液泡(H+)- atp酶（v - atp酶）的机制和调控。v - atp酶是atp依赖的质子泵，在正常和疾病过程中都起作用，包括膜传输、病毒感染、尿液酸化、骨吸收和肿瘤侵袭。v -ATP酶是一种多亚基复合物，由水解ATP的外周V1结构域和转运质子的完整V0结构域组成。v - atp酶在体内通过V1和V0结构域的可逆解离来调节。本提案的第一个目标是测试V0域内螺旋旋转在质子输运中的作用。我们已经获得了在亚基a和蛋白脂亚基中存在螺旋旋转（螺旋绕其长轴旋转）的证据。我们假设含有转运关键残基的螺旋旋转通过V0在质子易位中起作用。本提案的第二个目标是阐明葡萄糖调节酵母中v - atp酶组装的机制。我们开发了一种新的V-ATPase组装调节因子的遗传筛选，并使用该筛选确定Ras/cAMP/PKA通路作为关键调节因子。我们假设除了PKA之外，还有新的调节因子控制v - atp酶的组装，并且最近已经确定蛋白磷酸酶PP1是这样的调节因子之一。我们将检验这些假设，并通过追求以下具体目标来实现我们的目标。为了验证V0结构域中的螺旋旋转在质子传输中起作用的假设，我们将确定分子内，亚基a和亚基c'内相邻螺旋之间的二硫化物介导的交联对质子传输活性的影响。我们预计，如果质子输运需要螺旋旋转，那么通过亚基a或亚基c'内的相邻螺旋交联来阻止螺旋旋转将抑制活性。具体目标2 -为了确定葡萄糖调节酵母中v - atp酶组装的机制，我们将使用我们改良的基因筛选来识别和表征v - atp酶组装的其他新调节剂。这些调节因子包括必需和非必需基因，其突变可阻断V- atp酶解离或逆转pka介导的组装。这项研究具有重要意义，因为它将极大地促进我们对V-ATPase进行质子运输的机制的理解，并有助于鉴定V-ATPase组装的新调节剂。由于v - atp酶在酵母和哺乳动物细胞之间高度保守，使用调节组装来控制v - atp酶活性也是如此，并且由于葡萄糖、PKA和醛缩酶等调节因子控制酵母和高等真核生物的组装，这些研究可能会为哺乳动物系统中v - atp酶组装的控制提供重要的见解。这些见解将反过来促进调节V- atp酶活性的疗法的发展，这些疗法可能被证明对V- atp酶参与的疾病（如病毒感染、骨质疏松症和癌症）的治疗有效。

DESCRIPTION (provided by applicant): The long term goals of this research are to determine the mechanism and regulation of the vacuolar (H+)- ATPases (V-ATPases). The V-ATPases are ATP-dependent proton pumps that function in both normal and disease processes, including membrane traffic, viral infection, urinary acidification, bone resorption and tumor invasion. V-ATPases are multisubunit complexes composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that translocates protons. V-ATPases are regulated in vivo by reversible dissociation of the V1 and V0 domains. The first objective of this proposal is to test the role of helical swiveling within the V0 domain in proton transport. We have obtained evidence for helical swiveling (rotation of a helix about its long axis) in both subunit a and the proteolipid subunits. We hypothesize that swiveling of helices containing transport critical residues functions in proton translocation through V0. The second objective of this proposal is to elucidate the mechanism by which glucose regulates V-ATPase assembly in yeast. We have developed a novel genetic screen for regulators of V-ATPase assembly and used this screen to identify the Ras/cAMP/PKA pathway as a key regulator. We hypothesize that there are novel regulators in addition to PKA that control V-ATPase assembly, and have recently identified protein phosphatase PP1 as one such regulator. We will test these hypotheses and achieve our objectives by pursuing the following Specific Aims. Specific Aim 1 - To test the hypothesis that helical swiveling within the V0 domain functions in proton transport, we will determine the effect of intramolecular, disulfide-mediated cross-linking between adjacent helices within both subunit a and subunit c' on proton transport activity. We expect that if helical swiveling is required for proton transport, preventing helical swiveling by cross-linking adjacent helices within subunit a or subunit c' will inhibit activity. Specific Aim 2 - To determine the mechanism by which glucose regulates V-ATPase assembly in yeast, we will identify and characterize additional novel regulators of V-ATPase assembly using our modified genetic screen. These regulators include both essential and non-essential genes whose mutation blocks V- ATPase dissociation or reverses PKA-mediated assembly. The proposed research is significant because it will greatly advance our understanding of the mechanism by which V-ATPases carry out proton transport and facilitate the identification of novel regulators of V-ATPase assembly. Because V-ATPases are highly conserved between yeast and mammalian cells, as is the use of regulated assembly to control V-ATPase activity, and because regulators such as glucose, PKA and aldolase control assembly in both yeast and higher eukaryotes, these studies will likely provide important insight into control of V-ATPase assembly in mammalian systems. These insights will in turn facilitate the development of therapies to modulate V-ATPase activity that could prove effective in the treatment of diseases, such as viral infection, osteoporosis and cancer, in which V- ATPases participate.