Coordination of membrane traffic through rab GEF and GAP cascades

描述(申请人提供)：RABS是Ras GTP酶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。它们作为膜交通的主要调节者，每个典型地通过招募不同的效应蛋白来控制膜交通的特定阶段的几个不同方面，如细胞骨架马达、囊泡拴系蛋白和SNARE复合体组装的调节器。进而，RABS受特定的鸟核苷酸交换蛋白(GEF)的调控，这些蛋白催化GDP的置换以及GTP和GTP酶激活蛋白(GAP)的结合，从而刺激GTP的缓慢固有水解率。我们实验室最近的研究表明，不同的RAB通过它们的调节器相互联网。具体地说，我们已经证明了Rab，Ypt32，在其GTP结合的形式中招募了Sec2，即激活下游Rab，Sec4的环境基金，以及Gyp1，这是使上游Rab，Ypt1失活的间隙。净效应是一系列程序化的RAB转化，导致膜在沿着胞外途径流动时功能特性的变化。我们还发现，磷脂酰肌醇4-磷酸高尔基体(PI4P)与Ypt32协同工作，招募Sec2并控制Sec2功能的调节开关。我们提出了五个具体的目标来解决RAB相互联网的分子机制以及PI4P的空间分布。1.探讨磷酸化在Sec2功能调节中的作用。拟磷化和非磷酸化等位基因将测试它们与Sec4、Ypt32、Sec15和PI4P的相互作用。2.Sec4全球环境基金Sec2与Sec4效应器Sec15结合，形成正反馈环。为了测试这一机制在膜运输中的作用，我们将产生在Sec15结合中特异缺陷的Sec2等位基因。3.我们已经产生了一个Ypt1的等位基因，该等位基因可以被Sec4环境基金Sec2激活。我们将确定该突变是否会重定向Ypt1的膜结合，以及这种“短路”对膜交通的影响。4.我们有RAB-GAP级联的第二个例子的证据，并将对照代表性的RAB筛选所有RAB缺口，以确定更多。我们将测试缺口中断对RAB结构域重叠的影响。5.PI4P通常局限于高尔基体，不富含高尔基体源性的分泌囊泡。我们将确定PI4P仅限于高尔基人的机制。

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.