The function of Rho GTPases in vesicular traffic and membrane fusion

Rho GTPases 在囊泡运输和膜融合中的功能

• 批准号: RGPIN-2014-05621
• 负责人: Eitzen, Gary
• 金额: $ 2.55万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=649971
• 关键词: function; Rho; GTPases; vesicular; traffic

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.

我的研究项目主要集中在研究Rho蛋白如何控制整个细胞中货物的运动。Rho蛋白充当膜结合分子开关。当Rho蛋白结合GTP时，该开关打开，当GTP酶活性将GTP转化为GDP时，该开关关闭。 Rho GTP酶开关控制许多细胞过程，例如细胞分裂、细胞形状和货物的移动（a.k.a.囊泡运输）。许多靶向Rho蛋白的病原性毒素清楚地证明了囊泡运输中的作用;这些毒素帮助病原体通过依赖于囊泡运输的方法逃避消除。 ** 虽然Rho GT3蛋白在囊泡运输中的作用已经确定，但对其精确的调节作用知之甚少。 我的工作假设是，Rho GT3激活可以触发许多独特的分子复合物的组装，这些分子复合物在细胞内的不同位点启动过程。 现在的挑战是确定这些独特的信号复合物的组成和功能。 酵母空泡/溶酶体是理想的模型膜，用于进行调节囊泡运输的Rho信号传导复合物的生物化学表征。 可分离出高数量和高纯度的生物活性膜--异戊醇。 我们已经重建膜对接和融合与孤立的空泡，ATP和胞质溶胶。 使用这个系统，我们已经表明，两个Rho GTPases，Rho 1 p和Cdc 42 p，定位于液泡膜和调节不同的子反应。 这个建议的目的是确定这些Rho GTP酶在小泡交通中的作用，使用液泡膜作为范例。 ** 我的研究计划的三个目标提供了一个深入的研究Rho信号的囊泡运输和膜融合;从最初的膜定位Rho蛋白，激活和下游信号。目的1将集中在Rho蛋白如何被传递到膜。 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物。 我们已经表明，增加RhoGDI水平的结果在囊泡运输缺陷，由于在胞质溶胶中的Rho 1 p和Cdc 42 p复合物的水平增加。 子目标1.1提出了检查Rho蛋白从可溶性RhoGDI：：Rho蛋白复合物的置换和膜递送的研究。 子目标1.2研究了使用定位于液泡膜的RhoGDI-Nyv 1 LD嵌合体特异性抑制液泡Rho信号传导的效果。**目标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，生物传感器具有增加灵敏度和实时显示这一过程的额外好处。 使用具有独特荧光特性的探针的新方法将允许同时观察Cdc 42和Rho 1激活。** 在目标3中，我们将鉴定囊泡运输所需的下游Rho效应物复合物。 我们在这里的主要目标是阐明Cdc 42 p和Rho 1 p的功能作用的液泡通过确定其下游效应复合物。子目标3.1检查Cdc 42 p，其可能具有多种作用，因此在液泡上具有多种效应复合物。 子目标3.2描述了Rho 1 p效应复合物的鉴别。 ** 重要性：我们的目标是通过识别在特定运输步骤中操作的Rho信号复合物来定义调节膜运输的Rho信号机制。我们的方法是产生Rho GTP酶，其选择性地仅与调节蛋白或效应蛋白的子集相关联。详细的生化分析中所描述的建议将使我们能够解开下游信号通路的Rho GTP酶特异性激活囊泡交通。