Coordination of membrane traffic through rab GEF and GAP cascades

项目摘要 Rabs是Ras GT3超家族中最大的分支，在酵母中有10个成员， 60在哺乳动物细胞中。它们是调节膜交通的关键节点，每个节点通常 通过募集不同的效应物来控制膜运输的特定阶段的几个不同方面 蛋白质如细胞骨架马达、囊泡栓系蛋白和SNARE复合物组装的调节剂。 Rabs被特异性鸟嘌呤核苷酸交换因子（GEF）激活，GEF催化Rabs的置换， GDP和GTP的结合，并被GTP酶激活蛋白（GAP）灭活，GAP刺激缓慢的 GTP水解的固有速率。我们已经提出，交通路径上的相邻Rabs联网， 通过他们的监管机构。这些逆流级联的假设效应是一个程序化的 一系列突然的Rab转换，导致膜流动时功能特性的变化 沿着一条小路。Rabs也被认为可以定义囊泡的顺行与逆行方向性。 运输此外，据信Rab GEF-效应物相互作用产生正反馈回路， 促进囊泡成熟。虽然监管网络的各个要素的作用一直是 这些建议尚未在更加全球性的综合层面上得到检验。我们提出了三 具体目标，将解决Rab监管网络的重要方面： 1.我们将测试重组Rab调节回路的效果，以评估其在建立细胞器中的作用。 鉴定和追求从这些研究中出现卷曲螺旋囊泡系链的新的抑制作用 2.我们将直接测试Rabs在控制膜交通方向性中的作用 3.我们将确定Rab GEF-效应物相互作用在分泌囊泡成熟中的作用

Project Summary Rabs constitute the largest branch of the Ras GTPase superfamily, with ten members in yeast and more then 60 in mammalian cells. They serve as key nodes in the regulation 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 are activated by specific guanine nucleotide exchange factors (GEFs) that catalyze the displacement of GDP and binding of GTP and are inactivated by GTPase activating proteins (GAPs) that stimulate the slow intrinsic rate of GTP hydrolysis. We have proposed that adjacent Rabs on a traffic pathway are networked to one another through their regulators. The postulated effect of these counter-current cascades is a programmed series of abrupt Rab conversions that lead to changes in the functional identity of the membrane as it flows along a pathway. Rabs are also thought to define the anterograde versus retrograde directionality of vesicular transport. In addition, Rab GEF-effector interactions are believed to generate positive feedback loops that promote vesicle maturation. While the roles of individual elements of the regulatory network have been examined, these proposals have not yet been tested at a more global, integrated level. We propose three specific aims that will address important aspects of the Rab regulatory network: 1. We will test the effects of rewiring a Rab regulatory circuit to evaluate its role in establishing organelle identity and pursue a novel inhibitory role for a coiled-coil vesicle tether that has emerged from these studies 2. We will directly test the role of Rabs in the control of the directionality of membrane traffic 3. We will determine the role of a Rab GEF-effector interaction in secretory vesicle maturation