Coordination of membrane traffic through rab GEF and GAP cascades

描述（由申请人提供）：Rab 代表 Ras GTP 酶超家族的最大分支，在酵母中有 10 个成员，在哺乳动物细胞中有 60 多个成员。它们作为膜交通的主调节器，通常通过招募不同的效应蛋白（例如细胞骨架马达、囊泡束缚蛋白和 SNARE 复合体组装的调节器）来控制膜交通特定阶段的几个不同方面。反过来，Rab 受到特定鸟嘌呤核苷酸交换蛋白 (GEF) 的调节，该蛋白催化 GDP 的置换以及 GTP 和 GTP 酶激活蛋白 (GAP) 的结合，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 Rab 通过其监管者相互联网。具体来说，我们已经表明，GTP 结合形式的 Rab Ypt32 会募集 Sec2（激活下游 Rab Sec4 的 GEF）以及 Gyp1（失活上游 Rab Ypt1 的 GAP）。最终效果是一系列程序化的 Rab 转换，当膜沿着胞吐途径流动时，会导致膜的功能特性发生变化。我们还表明，高尔基体 4-磷酸磷脂酰肌醇 (PI4P) 库与 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来解决 Rab 相互联网以及 PI4P 分布在空间上定义的分子机制。 1. 我们将探讨磷酸化在Sec2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 Sec4、Ypt32、Sec15 和 PI4P 的相互作用。 2. Sec4 GEF Sec2 与 Sec4 效应器 Sec15 结合，形成正反馈循环。为了测试这种机制在膜运输中的作用，我们将生成在 Sec15 结合中特别有缺陷的 Sec2 等位基因。 3. 我们生成了可以被 Sec4 GEF 激活的 Ypt1 等位基因，Sec2。我们将确定这种突变是否会重定向 Ypt1 的膜关联以及这种“短路”对膜流量的影响。 4. 我们有 Rab-GAP 级联的第二个例子的证据，并将针对代表性 Rab 筛选所有 Rab GAP，以识别更多。我们将测试 GAP 破坏对 Rab 结构域重叠的影响。 5. PI4P 通常仅限于高尔基体，并且在高尔基体衍生的分泌囊泡中不富集。我们将确定 PI4P 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.