The function of Rho GTPases in vesicular traffic and membrane fusion

我的研究计划重点是研究Rho蛋白如何控制细胞内货物的移动。Rho蛋白作为膜结合的分子开关。当Rho蛋白与GTP结合时，开关打开，当GTP酶活性将GTP转化为GDP时，开关关闭。Rho GTP酶开关控制许多细胞过程，如细胞分裂、细胞形状和货物的移动(又名。水泡流量)。许多针对Rho蛋白的致病毒素清楚地表明了它在囊泡运输中的作用；这些毒素有助于病原体通过依赖囊泡运输的方法来逃避消除。尽管Rho GTPase蛋白在囊泡运输中的作用已经确定，但其确切的调控作用尚不清楚。我的工作假设是，Rho GTP酶的激活可以触发许多独特的分子复合体的组装，这些复合体在细胞内的不同位置启动过程。现在的挑战是确定这些独特的信号复合体的组成和功能。酵母液泡/溶酶体膜是对Rho信号复合体进行生化表征的理想模型膜，Rho信号复合体调节囊泡运输。液泡可以高质量、高纯度地作为生物活性膜分离出来。我们已经与分离的空泡、三磷酸腺苷和胞浆重组了膜对接和融合。利用这个系统，我们已经证明了两个Rho GTP酶，Rho1p和Cdc42p，定位于液泡膜并调节不同的亚反应。这项提议的目的是以液泡膜为范例，确定这些Rho GTP酶在囊泡运输中的作用。我的研究计划的三个目标是深入研究Rho信号对囊泡运输和膜融合的影响，从Rho蛋白的初始膜定位、激活和下游信号转导。RhoGDI是Rho GTP酶的天然抑制剂，与Rho蛋白形成高亲和力的可溶性复合体。我们已经证明，RhoGDI水平的增加会导致囊泡运输缺陷，这是因为细胞质中Rho1p和Cdc42p复合体的水平增加。次级目标1.1建议进行研究，检查可溶性RhoGDI：：Rho蛋白复合体中Rho蛋白的置换和膜递送。子目标1.2研究了利用定位于液泡膜的RhoGDI-Nyv1LD嵌合体特异性抑制空泡Rho信号的效果。Aim 2将使用新技术--荧光Rho激活生物传感器来检测Rho激活。在任何给定的反应中，只有一部分Rho蛋白被激活，生物传感器具有更高的灵敏度和实时显示这一过程的额外好处。一种使用具有独特荧光性质的探针的新方法将允许同时观察CdC42和Rho1的激活。在目标3中，我们将确定囊泡运输所需的下游Rho效应复合体。我们的主要目标是通过鉴定它们的下游效应复合体来阐明Cdc42p和Rho1p在空泡上的功能作用。子目标3.1检查了可能具有多个角色的Cdc42p，因此，液泡上有多个效应复合体。次级目标3.2描述了Rho1p效应器复合体的识别。意义：我们的目标是通过确定在特定的运输步骤中工作的Rho信号复合体来定义调节膜交通的Rho信号机制。我们的方法是产生Rho GTP酶，选择性地只与调节蛋白或效应蛋白的子集相关联。这项建议中描述的详细的生化分析将使我们能够解开Rho GTP酶的下游信号通路，这些信号通路专为囊泡运输而激活。

My research program is focused on studying how Rho proteins control the movement of cargo throughout the cell. Rho proteins act as membrane bound molecular switches. The switch is turned on when Rho proteins bind GTP, and turned off when the GTPase activity converts GTP to GDP. Rho GTPases switches control numerous cellular processes such as cell division, cell shape and the movement of cargo (a.k.a. vesicle traffic). The role in vesicle traffic is clearly demonstrated by the many pathogenic toxins which target Rho proteins; these help the pathogen to evade eliminate by methods that rely on vesicle traffic. Although a role for Rho GTPase proteins in vesicle trafficking has been established, their precise regulatory role is poorly understood. My working hypothesis is that Rho GTPase activation can trigger the assembly of many unique molecular complexes that initiate processes at distinct sites within the cell. The challenge now is to define the composition and function of these unique signaling complexes. Yeast vacuoles/lysosomes are ideal model membranes to perform a biochemical characterization of Rho signaling complexes that regulate vesicle trafficking. Vacuoles can be isolated as biologically active membranes in high quantity and purity. We have reconstituted membrane docking and fusion with isolated vacuoles, ATP and cytosol. Using this system, we have shown that two Rho GTPases, Rho1p and Cdc42p, localize to the vacuole membrane and regulate distinct sub-reactions. The goal of this proposal is to define the role of these Rho GTPases in vesicle traffic using the vacuole membrane as a paradigm. The three aim of my research program provide a thorough study of Rho signaling for vesicle trafficking and membrane fusion; from initial membrane localization of Rho proteins, activation and downstream signaling.Aim 1 will focus on how Rho proteins are delivered to membranes. RhoGDI, the natural inhibitor of Rho GTPases, forms high-affinity soluble complexes with Rho proteins. We have shown that increased RhoGDI levels results in vesicle trafficking defects due to increased levels of Rho1p and Cdc42p complexes in the cytosol. Sub-Aim 1.1 proposes studies that examine displacement and membrane delivery of Rho proteins from soluble RhoGDI::Rho protein complexes. Sub-Aim 1.2 studies the effect of specific inhibition of vacuolar Rho signaling using a RhoGDI-Nyv1LD chimera that localizes to the vacuole membrane.Aim 2 will examine Rho activation using new technology--fluorescent Rho activation biosensors. Only a portion of Rho proteins are activated in any given reaction, biosensors have the added benefit of increased sensitivity and showing this process in real-time. A novel approach using probes with unique fluorescent properties will allow the simultaneous observation of both Cdc42 and Rho1 activation. In Aim 3 we will identify the downstream Rho effector complexes required for vesicle trafficking. Our primary goal here is to elucidate the functional roles of Cdc42p and Rho1p on vacuoles by identifying their downstream effector complexes. Sub-Aim 3.1 Examines Cdc42p, which might have multiple roles, and therefore, multiple effector complexes on the vacuole. Sub-Aim 3.2 describes the identification of the Rho1p effector complexes. Significance: Our goal is to define the Rho signaling mechanisms that regulate membrane traffic by identifying the Rho signaling complexes that operate at specific trafficking steps. Our approach is to generate Rho GTPases that selectively associate with only a subset of regulator or effector proteins. The detailed biochemical analysis described in this proposal will allow us to unravel the downstream signaling pathway of Rho GTPases specifically activated for vesicular traffic.