A Skpl-containing Complex Regulating V-ATPase Activity

含 Skpl 的调节 V-ATP 酶活性的复合物

• 批准号: 6927940
• 负责人: PATRICIA M KANE
• 金额: $ 20.22万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6927940
• 关键词: SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; biological signal transduction; biological transport; enzyme activity; fluorescence resonance energy transfer; gene deletion mutation; hydrogen transport; immunoprecipitation; membrane transport proteins; oxidative phosphorylation; point mutation; protein binding; protein protein interaction; protein structure function; ubiquitin; western blottings; yeast two hybrid system

DESCRIPTION: (provided by applicant) Vacuolar H-+ translocating ATPases (V-ATPases) are highly conserved enzymes that play a central role in cell physiology stemming from their ability to acidify intracellular compartments, regulate cytosolic pH and calcium concentrations, and establish proton gradients that drive other transporters. These functions can be adapted to many different cellular contexts, and as a result, V-ATPase activity is linked to disease states as diverse as viral infection, metabolic acidosis due to impaired kidney function, and osteoporosis. Skp1 is a highly conserved protein that plays a central role in ubiquitin-dependent proteolysis as an essential member of the family of SCF E3 ubiquitin ligases in addition to a growing number of non-proteolytic roles. SCF complexes show specificity for a wide array of phosphorylated substrates and thus interact with many signal transductional pathways. Recently a new Skp1-containing complex, RAVE, was identified in yeast and shown to contain two other uncharacterized proteins. The RAVE complex does not appear to be an SCF ubiquitin ligase; instead, it appears to post-translationally regulate V-ATPase activity in response to extracellular conditions by modulating the extent of assembly of the V-ATPase complex. In the project proposed here, we will use the yeast Saccharomyces cerevisiae as a model system to examine the structural and functional basis for regulation of V-ATPase activity and assembly by the RAVE complex. We will examine how the three subunits of the RAVE complex interact with each other and with the peripheral V1 sector of the V-ATPase. We will determine whether protein components of RAVE are directly affected by changes in extracellular conditions, and how these changes might affect interaction with the V1 sector. We will develop a system for monitoring interactions between RAVE and the V-ATPase in vivo, using fluorescent derivatives of the two complexes. Finally, we will probe the molecular basis of RAVE action on the V-ATPase by developing an in vitro model for RAVE-dependent assembly of the peripheral and integral membrane sectors of the ATPase and using this model system to test the hypothesis that RAVE assists functional attachment of one of the V1 subunits.

描述：（由申请人提供）液泡H-+转移ATPase （V-ATPases）是高度保守的酶，在细胞中起着中心作用 生理学源于它们酸化细胞内室的能力， 调节胞质pH和钙浓度，并建立质子 驱动其他转运蛋白的梯度。这些功能可以适应许多 不同的蜂窝上下文，结果，V-ATPase活性链接到 疾病状态与病毒感染一样多样化，由于 肾功能受损和骨质疏松症。 SKP1是一种高度保守的蛋白质 这在泛素依赖性蛋白水解中起着核心作用作为必不可少的 SCF E3泛素连接酶家族的成员除了增长 非蛋白水解作用的数量。 SCF复合物显示出特异性的特异性 磷酸化的底物阵列，因此与许多信号相互作用 横向途径。最近，一个新的含SKP1的综合大楼是 在酵母中鉴定，并显示出其他两种未表征的蛋白质。 狂欢复合物似乎不是SCF泛素连接酶。相反，它 似乎在翻译后调节了V-ATPase活性。 细胞外条件通过调节V-ATPase的组装程度 复杂的。在此处提出的项目中，我们将使用酵母糖疗法 酿酒酵母作为模型系统，以检查结构和功能基础 通过Rave复合物调节V-ATPase活性和组装。我们将 检查狂欢复合物的三个亚基如何相互作用 带有V-ATPase的外围V1扇区。我们将确定是否 狂欢的蛋白质成分直接受细胞外变化的影响 条件以及这些变化如何影响与V1部门的相互作用。 我们将开发一个系统，以监视Rave和 V-ATPase在体内，使用两个复合物的荧光衍生物。最后， 我们将通过发展对V-ATPase的狂欢行动的分子基础 一个体外模型，用于依赖外围和积分的狂欢依赖性组装 ATPase的膜扇区并使用此模型系统测试 Rave有助于一个V1亚基的功能附着的假设。