酿酒酵母极性生长关键调控分子Rho3的新调控因子、下游效应分子及功能的鉴定

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.

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。

酿酒酵母Rho家族GTP酶Rho3调控肌动蛋白细胞骨架的组织和囊泡运输与融合过程，在细胞极性生长过程中发挥着重要作用，然而，人们对于Rho3如何在胞内被调控了解非常少，对于Rho3发挥功能所需的下游效应分子了解也不多。在本项目资助下，我们对Rho3的新调控因子和效应分子进行了筛选鉴定。我们通过酵母双杂交筛文库筛选得到11个新的，除Myo2、Exo70和Bni1三个已知分子之外的，能够与Rho3相互作用的蛋白质分子，从中鉴定出一个新的Rho3调控因子Rga1和一个潜在的Rho3调控因子Bem3，以及一个新的Rho3效应分子Kin2，并对Rga1、Kin2和Bem3的功能与调控进行了研究。Rga1和Bem3均为Rho GTP酶激活蛋白（RhoGAP），它们之前被认为特异性作用于Rho GTP酶Cdc42，我们发现Rga1能够在极性生长中负调控Rho3，揭示了Rga1能够调控两个Rho GTP酶。我们推测Bem3也可能在胞内负调控Rho3，可是目前还没有获得足够的证据支持。我们发现Rga1的表达水平和磷酸化水平在细胞周期中波动较大，且Rga1活性受到磷酸化负调控，而Bem3在极性生长位点的定位依赖于其与极性小体成员Epo1之间的相互作用，这些结果表明Rga1和Bem3两个RhoGAP自身在胞内也受到严格的调控。Kin2是MARK/PAR-1家族蛋白质激酶，它能够调控囊泡分泌过程，我们的实验结果显示Kin2与Rho3在体内相互作用，是Rho3的一个新的下游效应分子。Kin2还能够与septin亚基Cdc11和极性小体成员Pea2相互作用，在septin细胞骨架的组织与细胞壁组织过程中有重要作用。我们的研究结果揭示了Rho GTP酶Rho3的新调控因子和下游效应分子，增进了对于Rho3功能调控与功能机制的认识。