Richman T J, Johnson D I
Department of Microbiology and Molecular Genetics and Markey Center for Molecular Genetics, University of Vermont Burlington, Vermont 05405, USA.
Mol Cell Biol. 2000 Nov;20(22):8548-59. doi: 10.1128/MCB.20.22.8548-8559.2000.
The Saccharomyces cerevisiae Cdc42p GTPase interacts with multiple regulators and downstream effectors through an approximately 25-amino-acid effector domain. Four effector domain mutations, Y32K, F37A, D38E, and Y40C, were introduced into Cdc42p and characterized for their effects on these interactions. Each mutant protein showed differential interactions with a number of downstream effectors and regulators and various levels of functionality. Specifically, Cdc42(D38E)p showed reduced interactions with the Cla4p p21-activated protein kinase and the Bem3p GTPase-activating protein and cdc42(D38E) was the only mutant allele able to complement the Deltacdc42 null mutant. However, the mutant protein was only partially functional, as indicated by a temperature-dependent multibudded phenotype seen in conjunction with defects in both septin ring localization and activation of the Swe1p-dependent morphogenetic checkpoint. Further analysis of this mutant suggested that the multiple buds emerged consecutively with a premature termination of bud enlargement preceding the appearance of the next bud. Cortical actin, the septin ring, Cla4p-green fluorescent protein (GFP), and GFP-Cdc24p all predominantly localized to one bud at a time per multibudded cell. These data suggest that Cdc42(D38E)p triggers a morphogenetic defect post-bud emergence, leading to cessation of bud growth and reorganization of the budding machinery to another random budding site, indicating that Cdc42p is involved in prevention of the initiation of supernumerary buds during the cell cycle.
酿酒酵母Cdc42p GTP酶通过一个约25个氨基酸的效应结构域与多种调节因子和下游效应物相互作用。将四个效应结构域突变（Y32K、F37A、D38E和Y40C）引入Cdc42p，并对其对这些相互作用的影响进行了表征。每个突变蛋白与许多下游效应物和调节因子表现出不同的相互作用以及不同水平的功能。具体而言，Cdc42(D38E)p与Cla4p p21激活蛋白激酶和Bem3p GTP酶激活蛋白的相互作用减少，并且cdc42(D38E)是唯一能够互补Deltacdc42缺失突变体的突变等位基因。然而，该突变蛋白仅具有部分功能，这表现为在温度依赖性多芽表型中同时出现隔膜环定位缺陷和Swe1p依赖性形态发生检查点的激活缺陷。对该突变体的进一步分析表明，多个芽连续出现，在下一个芽出现之前芽的增大过早终止。在每个多芽细胞中，皮质肌动蛋白、隔膜环、Cla4p-绿色荧光蛋白（GFP）和GFP-Cdc24p一次都主要定位于一个芽。这些数据表明，Cdc42(D38E)p在芽出现后引发形态发生缺陷，导致芽生长停止并将出芽机制重新组织到另一个随机出芽位点，这表明Cdc42p参与了细胞周期中防止额外芽的起始。
