The function of Rho GTPases in vesicular traffic and membrane fusion

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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.