Characterization of the potential Cdc42 regulators Dop1 and Rdi1 in budding yeast

芽殖酵母中潜在 Cdc42 调节因子 Dop1 和 Rdi1 的表征

• 批准号: 5448950
• 负责人: Dr. Thomas Höfken
• 金额: --
• 依托单位: Division of Biosciences
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2005
• 资助国家: 德国
• 起止时间: 2004-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5448950?language=en
• 关键词: Characterization; potential; Cdc42; regulators; Dop1

The small GTPase Cdc42 is a key regulator of cell polarity. In the budding yeast Saccharomyces cerevisiae it is required at different stages of the cell cycle during vegetative growth but also during mating and filamentous growth. Cdc42 interacts in the active state with numerous effectors which mediate various functions such a bud growth, exit from mitosis and cytokinesis. It seems very likely that this specific activation of Cdc42 effectors is controlled by Cdc42 regulators. Among these regulators are the GEF Cdc42, the GAPs Bem3, Rga1 and Rga2 and the GDI Rdi1. Research has mainly focused on Cdc24, whereas less is known about the other proteins. The conserved polarity protein Dop1 was identified as an interactor of Bem3 but has not been further analyzed to date. We plan to characterize the proteins Dop1 and Rdi1 and examine how they contribute to the regulation of Cdc42 activity and cell polarity in general. This includes the analysis of genetic interactions between cell polarity proteins and Dop1 and Rdi1, respectively. Protein interactions will be examined using the yeast two-hybrid system, co-immunoprecipitations and in vitro binding assays. Furthermore, the phenotypes of overexpression and inactivation of Dop1 and Rdi1 will be analyzed in detail. Assays for Cdc42 activity will be used to examine the relationship between other cell polarity proteins and Dop1 and Rdi1, respectively. Finally, we will screen for novel factors that regulate Cdc42 and characterize these interactors in a similar way.

小的GTP酶CDC42是细胞极性的关键调节因子。在芽生酵母中，营养生长的细胞周期的不同阶段都需要它，交配和丝状生长也需要它。在活跃状态下，CDC42与许多效应器相互作用，这些效应器调节各种功能，如芽的生长、退出有丝分裂和胞质分裂。CDC42效应器的这种特异性激活很可能是由CDC42调节因子控制的。其中包括全球环境基金CDC42、缺口Bem3、RGa1和RGa2以及GDI Rdi1。研究主要集中在cdc24上，而对其他蛋白质知之甚少。保守的极性蛋白DOP1被鉴定为Bem3的相互作用因子，但到目前为止还没有进一步的分析。我们计划表征蛋白质DOP1和Rdi1，并研究它们如何在总体上调节CDC42的活性和细胞极性。这包括分析细胞极性蛋白与DOP1和Rdi1之间的遗传相互作用。蛋白质的相互作用将使用酵母双杂交系统、免疫共沉淀和体外结合试验进行检测。此外，还将详细分析DOP1和Rdi1过表达和失活的表型。对CDC42活性的分析将分别用于检查其他细胞极性蛋白与多普利和Rdi1之间的关系。最后，我们将筛选调控CDC42的新因素，并以类似的方式表征这些相互作用。