A Skpl-containing Complex Regulating V-ATPase Activity

描述：(申请人提供)空泡H-+转位ATPase (v-ATPase)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隔间的能力， 调节胞浆pH和钙浓度，建立质子 驱动其他转运体的梯度。这些函数可以适用于许多 不同的细胞环境，因此，V-ATPase活性与 疾病状态多样，如病毒感染，代谢性酸中毒 肾功能受损，骨质疏松症。Skp1是一种高度保守的蛋白质 这在泛素依赖的蛋白分解中起着核心作用，是一种必不可少的 SCF E3泛素连接酶家族的成员 非蛋白水解性角色的数量。SCF复合体表现出广泛的特异性 磷酸化底物阵列，并因此与许多信号相互作用 传导通路。最近，一种新的含有Skp1的复合体RAVE 在酵母中被鉴定，并被证明含有另外两种未鉴定的蛋白质。 RAVE复合体似乎不是SCF泛素连接酶；相反，它 似乎在翻译后调节V-ATPase活性 调节V-ATPase组装程度的胞外条件 很复杂。在这里提出的项目中，我们将使用酵母菌 作为模式系统来考察酿酒酵母的结构和功能基础 RAVE复合体对V-ATPase活性和组装的调节。我们会 研究RAVE复合体的三个亚单位是如何相互作用的 与V-ATPase的外设V1扇区连接。我们将确定是否 RAVE的蛋白质组分直接受到胞外变化的影响 条件，以及这些变化可能如何影响与V1扇区的互动。 我们将开发一个系统来监控RAVE和 V-ATPase在体内，使用荧光衍生物的两个络合物。最后， 我们将通过研究RAVE对V-ATPase作用的分子基础 一种依赖RAVE的外周和整体组装的体外模型 ATPase的膜扇区，并利用该模型系统测试 RAVE有助于V1亚基之一的功能附着的假说。

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.