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-+ 转位 ATP 酶 （V-ATP酶）是高度保守的酶，在细胞中发挥核心作用 生理学源于它们酸化细胞内区室的能力， 调节细胞质 p​​H 值和钙浓度，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 不同的细胞环境，因此，V-ATP酶活性与 疾病状态多种多样，如病毒感染、代谢性酸中毒等 肾功能受损，骨质疏松。 Skp1是一种高度保守的蛋白质 在泛素依赖性蛋白水解作用中发挥核心作用 SCF E3 泛素连接酶家族的成员，以及不断增长的 非蛋白水解作用的数量。 SCF 复合物显示出广泛的特异性 磷酸化底物阵列，从而与许多信号相互作用 转导途径。最近，一种新的包含 Skp1 的复合物 RAVE 被 在酵母中鉴定出并显示含有另外两种未表征的蛋白质。 RAVE 复合物似乎不是 SCF 泛素连接酶；相反，它 似乎可以响应翻译后调节 V-ATP 酶活性 通过调节 V-ATP 酶的组装程度来调节细胞外条件 复杂的。在这里提出的项目中，我们将使用酵母菌 酿酒酵母作为模型系统来检查其结构和功能基础 RAVE 复合物调节 V-ATP 酶活性和组装。我们将 检查 RAVE 复合体的三个亚基如何相互作用 与 V-ATP 酶的外周 V1 部分。我们将确定是否 RAVE的蛋白质成分直接受到细胞外物质变化的影响 条件，以及这些变化可能如何影响与 V1 部门的互动。 我们将开发一个系统来监控 RAVE 和 体内 V-ATP 酶，使用两种复合物的荧光衍生物。最后， 我们将通过开发 RAVE 对 V-ATP 酶作用的分子基础 外周和整体依赖 RAVE 组装的体外模型 ATP酶的膜部分并使用该模型系统来测试 假设 RAVE 有助于 V1 亚基之一的功能附着。