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）的机制和调节。V-ATP酶是ATP依赖性质子泵，在正常和疾病过程中发挥作用，包括膜运输、病毒感染、尿酸化、骨吸收和肿瘤侵袭。V-ATP酶是多亚基复合物，由水解ATP的外周V1结构域和易位质子的完整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。这个建议的第一个目标是测试质子运输中的V0域内的螺旋旋转的作用。我们已经获得了在亚基a和蛋白脂亚基中螺旋旋转（螺旋绕其长轴旋转）的证据。我们假设，旋转的螺旋含有运输关键残基功能质子易位通过V0。该建议的第二个目的是阐明葡萄糖调节酵母中V-ATP酶组装的机制。我们已经开发了一种新的V-ATP酶组装调节基因筛选，并使用此筛选确定Ras/cAMP/PKA途径作为关键调节因子。我们推测，有新的监管机构除了PKA控制V-ATP酶的组装，最近已确定蛋白磷酸酶PP 1作为一个这样的调节。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。具体目标1 -为了检验V0结构域内的螺旋旋转在质子转运中起作用的假设，我们将确定亚基a和亚基c'内相邻螺旋之间的分子内二硫键介导的交联对质子转运活性的影响。我们预期，如果螺旋旋转是质子运输所必需的，那么通过交联亚基a或亚基c'内的相邻螺旋来防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遗传筛选来鉴定和表征V-ATP酶组装的其他新型调节剂。这些调节因子包括必需和非必需基因，其突变阻断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-ATP酶进行质子转运的机制的理解，并有助于鉴定V-ATP酶组装的新型调节剂。由于V-ATP酶在酵母和哺乳动物细胞之间是高度保守的，正如使用调节组装来控制V-ATP酶活性一样，并且因为调节剂如葡萄糖、PKA和醛缩酶控制酵母和高等真核生物中的组装，这些研究将可能为哺乳动物系统中V-ATP酶组装的控制提供重要的见解。这些见解反过来将促进调节V-ATP酶活性的疗法的开发，所述疗法可以证明在治疗V-ATP酶参与的疾病如病毒感染、骨质疏松症和癌症中是有效的。

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.