Coordination of membrane traffic through rab GEF and GAP cascades

 描述（由申请人提供）：Rabs构成Ras GT3超家族的最大分支，在酵母中有10个成员，在哺乳动物细胞中有60多个成员。它们作为膜运输的主调节剂，每种通常通过募集不同的效应蛋白如细胞骨架马达、囊泡束缚蛋白和SNARE复合物组装的调节剂来控制膜运输的特定阶段的几个不同方面。反过来，Rabs又受到特异性鸟嘌呤核苷酸交换因子（GEF）的调节，GEF催化GDP的置换和GTP的结合，以及刺激GTP水解的缓慢内在速率的GTP酶激活蛋白（GAP）。我们已经提出，一条通路上相邻的Rab通过它们的调节器相互联网;具体地说，我们已经证明Rab，Ypt 32，以其GTP结合的形式招募Sec 2，激活下游Rab的GEF，Sec 4，以及Gyp 1，使上游Rab失活的差距，Ypt 1。这些逆流级联的净效应是一系列程序化的Rab转变，当膜沿着胞吐途径流动时，会导致膜功能特性的关键变化。磷脂酰肌醇在膜运输的时间和空间调节中也起关键作用。磷脂酰肌醇4-磷酸（PI（4）P）的高尔基体池与Ypt 32协同工作，最初招募Sec 2，但随后PI（4）P水平的下降指导Sec 2功能的调节开关，其中它在正反馈回路中与Sec 4效应器Sec 15结合。PI（4）P分布与Sec 2磷酸化一起决定何时使用每个调节回路。我们提出三个目标：1。我们将确定两种蛋白激酶在Sec 2和分泌机制的其他组分的调节中的作用。2.我们将测试rab调节回路“重新布线”的效果，以评估几种关于rab在 膜交通的控制。3.我们将确定分泌囊泡成熟的几个阶段的分子机制。

 DESCRIPTION (provided by applicant): Rabs constitute the largest branch of the Ras GTPase superfamily, with ten members in yeast and more than 60 in mammalian cells. They serve as master regulators of membrane traffic, each typically controlling several different aspects of a specific stage of membrane traffic by recruiting diverse effector proteins such as cytoskeletal motors, vesicle tethering proteins and regulators of SNARE complex assembly. Rabs, in turn, are regulated by specific guanine nucleotide exchange factors (GEFs) that catalyze the displacement of GDP and binding of GTP as well as GTPase activating proteins (GAPs) that stimulate the slow intrinsic rate of GTP hydrolysis. We have proposed that adjacent Rabs on a pathway are networked to one another through their regulators; specifically we have shown that the Rab, Ypt32, in its GTP-bound form recruits Sec2, the GEF that activates the downstream Rab, Sec4, as well as Gyp1, the GAP that inactivates the upstream Rab, Ypt1. The net effect of these counter-current cascades is a programmed series of Rab transitions that lead to critical changes in the functional identity of the membrane as it flows along the exocytic pathway. Phosphoinositide also play key roles in the temporal and spatial regulation of membrane traffic. The Golgi pool of phosphatidylinositol 4-phosphate (PI(4)P) works in concert with Ypt32 to initially recruit Sec2, yet a subsequent drop in PI(4)P levels directs a regulatory switch in Sec2 function in which it binds to the Sec4 effector Sec15 in a positive feedback loop. PI (4) P distribution together with Sec2 phosphorylation determine when each regulatory circuit is used. We propose three aims: 1. we will define the role of two protein kinases in the regulation of Sec2 and other components of the secretory machinery. 2. We will test the effects of "rewiring" the rab regulatory circuits to evaluate several models concerning the role of rabs in the control of membrane traffic. 3. We will determine the molecular mechanisms underlying several stages of secretory vesicle maturation.