Coordination of membrane traffic through rab GEF and GAP cascades

描述（由申请人提供）：Rabs代表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，每个通常通过招募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受特异性鸟嘌呤核苷酸交换蛋白（GEF）的调节，GEF催化GDP的置换以及GTP和GTP酶激活蛋白（GAP）的结合，GTP酶激活蛋白刺激GTP水解的缓慢内在速率。我们实验室最近的工作表明，不同的Rabs通过它们的调节器彼此联网。具体来说，我们已经证明，Rab，Ypt 32，在其GTP结合形式招募Sec 2，激活下游Rab，Sec 4的GEF，以及Gyp 1，灭活上游Rab，Ypt 1的差距。净效应是一系列程序化的Rab转换，当其沿胞吐途径沿着流动时，其导致膜功能特性的变化。我们还表明，高尔基体池的磷脂酰肌醇4-磷酸（PI 4P）与Ypt 32协同工作，以招募Sec 2和控制Sec 2功能的调节开关。我们提出了五个具体的目标，以解决Rabs相互联网的分子机制，以及PI 4P的空间分布。1.我们将探讨磷酸化在Sec 2功能调节中的作用。将测试拟磷酸化和不可磷酸化等位基因与Sec 4、Ypt 32、Sec 15和PI 4P的相互作用。2. Sec 4 GEF Sec 2与Sec 4效应子Sec 15结合，导致正反馈环。为了测试这种机制在膜运输中的作用，我们将产生Sec 15结合特异性缺陷的Sec 2等位基因。3.我们已经产生了一个等位基因的Ypt 1，可以激活的Sec 4 GEF，Sec 2。我们将确定这种突变是否会重定向Ypt 1的膜结合，以及这种“短路”对膜交通的影响。4.我们有证据证明Rab-GAP级联的第二个例子，并将根据代表性Rabs筛选所有Rab GAP，以确定更多。我们将测试GAP破坏对Rab结构域重叠的影响。5. PI 4P通常局限于高尔基体，并且不富集在高尔基体衍生的分泌囊泡上。我们将确定PI 4P仅限于高尔基体的机制。

DESCRIPTION (provided by applicant): Rabs represent 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 effectors proteins such as cytoskeletal motors, vesicle tethering proteins and regulators of SNARE complex assembly. Rabs, in turn, are regulated by specific guanine nucleotide exchange proteins (GEFs) that catalyze the displacement of GDP and binding of GTP and GTPase activating proteins (GAPs) that stimulate the slow intrinsic rate of GTP hydrolysis. Recent work from our lab has demonstrated that different Rabs 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 is a programmed series of Rab conversions that lead to changes in the functional identity of the membrane as it flows along the exocytic pathway. We have also shown that the Golgi pool of phosphatidylinositol 4-phosphate (PI4P) works in concert with Ypt32 to recruit Sec2 and to control a regulatory switch in Sec2 function. We propose five specific aims to address the molecular mechanisms by which Rabs are networked to one another and by which the distribution of PI4P is spatially defined. 1. We will explore the role of phosphorylation in the regulation of Sec2 function. Phosphomimetic and non- phosphorylatable alleles will be tested for their interactions with Sec4, Ypt32, Sec15 and PI4P. 2. The Sec4 GEF Sec2 binds to the Sec4 effectors Sec15 leading to a positive feedback loop. To test the role of this mechanism in membrane traffic we will generate Sec2 alleles specifically defective in Sec15 binding. 3. We have generated an allele of Ypt1 that can be activated by the Sec4 GEF, Sec2. We will determine if this mutation redirects the membrane association of Ypt1 and the effects of this "short circuit" on membrane traffic. 4. We have evidence for a second example of a Rab-GAP cascade and will screen all Rab GAPs against representative Rabs to identify more. We will test the effects of GAP disruption on overlap of Rab domains. 5. PI4P is normally restricted to the Golgi and is not enriched on Golgi-derived secretory vesicles. We will determine the mechanism by which PI4P is limited to the Golgi.