Davis C R, Richman T J, Deliduka S B, Blaisdell J O, Collins C C, Johnson D I
Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA.
J Biol Chem. 1998 Jan 9;273(2):849-58. doi: 10.1074/jbc.273.2.849.
The Saccharomyces cerevisiae Cdc42p GTPase is localized to the plasma membrane and involved in signal transduction mechanisms controlling cell polarity. The mechanisms of action of the dominant negative cdc42(D118A) mutant and the lethal, gain of function cdc42(G12V) mutant were examined. Cdc42(D118A,C188S)p and its guanine-nucleotide exchange factor Cdc24p displayed a temperature-dependent interaction in the two-hybrid system, which correlated with the temperature dependence of the cdc42(D118A) phenotype and supported a Cdc24p sequestration model for the mechanism of cdc42(D118A) action. Five cdc42 mutations were isolated that led to decreased interactions with Cdc24p. The isolation of one mutation (V44A) correlated with the observations that the T35A effector domain mutation could interfere with Cdc42(D118A, C188S)p-Cdc24p interactions and could suppress the cdc42(D118A) mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain. The cdc42(G12V) mutant phenotypes were suppressed by the intragenic T35A and K183-187Q mutations and in skm1Delta and cla4Delta cells but not ste20Delta cells, suggesting that the mechanism of cdc42(G12V) action is through the Skm1p and Cla4p protein kinases at the plasma membrane. Two intragenic suppressors of cdc42(G12V) were also identified that displayed a dominant negative phenotype at 16 degrees C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.
酿酒酵母Cdc42p GTP酶定位于质膜，参与控制细胞极性的信号转导机制。研究了显性负性cdc42（D118A）突变体和致死性、功能获得性cdc42（G12V）突变体的作用机制。Cdc42（D118A，C188S）p及其鸟嘌呤核苷酸交换因子Cdc24p在双杂交系统中表现出温度依赖性相互作用，这与cdc42（D118A）表型的温度依赖性相关，并支持Cdc24p隔离模型作为cdc42（D118A）作用机制。分离出五个导致与Cdc24p相互作用减少的cdc42突变。一个突变（V44A）的分离与以下观察结果相关：T35A效应结构域突变可干扰Cdc42（D118A, C188S）p-Cdc24p相互作用，并可抑制cdc42（D118A）突变，这表明Cdc24p可能通过其效应结构域与Cdc42p相互作用。cdc42（G12V）突变体表型在skm1Delta和cla4Delta细胞中被基因内T35A和K183-187Q突变抑制，但在ste20Delta细胞中未被抑制，这表明cdc42（G12V）的作用机制是通过质膜上的Skm1p和Cla4p蛋白激酶。还鉴定出两个cdc42（G12V）的基因内抑制子，它们在16℃时表现出显性负性表型，且不受Cdc24p过表达的抑制，这表明这些显性负性突变存在另一种作用机制。
