Rho GTP酶在真核生物细胞形态发生中具有重要作用，探索其功能与调控机制具有重要意义。酿酒酵母在出芽繁殖过程中，芽体的极性生长受到包括Rho3在内的多个Rho GTP酶的调控，但是，对于Rho3在发挥功能时的调控因子和下游效应分子了解较少，也不清楚Rho3是否还具有其它功能。在前期研究中，申请人发现Rho3与GTP酶激活蛋白Rga1及蛋白质激酶Kin2之间存在相互作用，推测Rga1可能是新的Rho3负调控因子，而Kin2可能是新的Rho3下游效应分子。本项目将针对Rho3与其潜在负调控因子Rga1和效应分子Kin2及其同源蛋白Kin1之间的功能关系进行鉴定，并通过酵母双杂交方法筛选鉴定新的Rho3调控因子和下游效应分子。此外，还将筛选与rho3缺失突变具有合成致死效应的突变体，鉴定突变体的缺陷功能和突变基因，揭示Rho3的新功能。本项目对于阐明Rho3调控细胞极性生长的机制具有重要意义。

Rho GTPases play crucial roles in cellular morphogenesis in eukaryotes. It is of great importance to investigate the mechanism of Rho GTPase action and their regulation. During bud development in the budding yeast Saccharomyces cerevisiae, polarized growth in the bud is controlled by several Rho GTPases including Rho3. Rho3 controls polarized growth via its regulation on the actin cytoskeleton and vesicular transport. However, not much is known about the regulators and downstream effectors of Rho3 as well as whether Rho3 may have other cellular functions. We previously identified an interaction of Rho3 with Rga1, a RhoGAP, and with Kin2, a serine/threonine protein kinase. This finding raised a possibility that Rga1 could be a novel negative regulator of Rho3 whereas Kin2 could be a novel downstream effector of Rho3. In this proposal, we plan to investigate the functional relationship between Rho3 and the potential Rho3GAP Rga1 as well as the relationship between Rho3 and the potential effector Kin2 and its homologue Kin1. We also plan to identify novel Rho3-interacting regulators and downstream effectors by yeast two-hybrid screen. Moreover, we plan to isolate mutants that are synthetically lethal with rho3 null mutation, followed by characterization of their defective cellular functions and mutated genes, aiming to elucidate novel functions of Rho3. Our proposed investigation will help understand the mechanism of how Rho3 regulates polarized growth.