Coordination of membrane traffic through rab GEF and GAP cascades

描述（由申请人提供）：Rabs是Ras GTPase超家族中最大的分支，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要调节剂，通过招募不同的效应蛋白（如细胞骨架马达、囊泡系缚蛋白和SNARE复合物组装调节剂）来控制膜运输特定阶段的几个不同方面。反过来，Rabs受特定的鸟嘌呤核苷酸交换蛋白（gef）的调节，该蛋白催化GDP的位移，并结合GTP和GTP酶激活蛋白（gap），刺激GTP的缓慢内在水解速率。我们实验室最近的工作表明，不同的Rabs通过它们的调节器相互连接。具体来说，我们已经表明，在其gtp结合形式下，Rab, Ypt32，招募Sec2，激活下游Rab， Sec4的GEF，以及Gyp1，使上游Rab， Ypt1失活的GAP。净效应是一系列程序化的Rab转换，导致膜沿着胞外途径流动时功能特性的变化。我们还表明，磷脂酰肌醇4-磷酸（PI4P）的高尔基池与Ypt32协同作用，招募Sec2并控制Sec2功能的调节开关。我们提出了五个具体目标，以解决Rabs相互联网的分子机制，以及PI4P的分布在空间上的定义。1. 我们将探讨磷酸化在Sec2功能调控中的作用。将检测拟磷和非磷酸化等位基因与Sec4、Ypt32、Sec15和PI4P的相互作用。2. Sec4 GEF Sec2与Sec4效应器Sec15结合，形成一个正反馈循环。为了测试这一机制在膜运输中的作用，我们将产生Sec2等位基因在Sec15结合中特异性缺陷。3. 我们已经生成了一个Ypt1的等位基因，它可以被Sec4 GEF、Sec2激活。我们将确定这种突变是否重定向了Ypt1的膜关联，以及这种“短路”对膜交通的影响。4. 我们有第二个lab - gap级联例子的证据，并将对所有lab gap进行筛选，以确定更多具有代表性的lab。我们将测试GAP破坏对Rab结构域重叠的影响。5. PI4P通常局限于高尔基体，在高尔基体衍生的分泌囊泡中不富集。我们将确定PI4P局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.