Ziman M, O'Brien J M, Ouellette L A, Church W R, Johnson D I
Cell and Molecular Biology Program, University of Vermont, Burlington 05405.
Mol Cell Biol. 1991 Jul;11(7):3537-44. doi: 10.1128/mcb.11.7.3537-3544.1991.
The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.
酿酒酵母CDC42基因产物是低分子量GTP结合蛋白ras超家族的成员，参与细胞极性的调控。我们分析了在假定的GTP结合和水解结构域中的三个CDC42突变（第12位甘氨酸突变为缬氨酸、第61位谷氨酰胺突变为亮氨酸、第118位天冬氨酸突变为丙氨酸）以及在假定的异戊二烯化位点的一个突变（第188位半胱氨酸突变为丝氨酸）的影响。前三个突变存在于单倍体cdc42 - 1ts或野生型菌株的质粒上时，会导致显性致死或剂量依赖性显性致死表型。携带在GAL启动子控制下带有CDC42Val - 12或CDC42Leu - 61等位基因的质粒（pGAL）的野生型和cdc42 - 1ts细胞，均以一种新的表型停滞生长，即细胞变大，带有细长或多个芽。携带pGAL - CDC42Ala - 118的细胞停滞生长为大的、圆形的、无芽的细胞，类似于cdc42 - 1ts停滞生长的细胞。CDC42Ala - 118突变体与CDC42Val - 12和CDC42Leu - 61突变体的不同表型出乎意料，因为所有三个类似的ras突变体的表型彼此相似。这表明Cdc42蛋白的生化特性在某些方面不同于Ras蛋白。cdc42Ser - 188突变基因无法互补cdc42 - 1ts突变，并且对野生型和cdc42 - 1ts均为隐性。在双突变等位基因中，cdc42Ser - 188突变能够抑制与三个假定的GTP结合和水解突变相关的显性致死性，这表明异戊二烯化对于野生型和突变蛋白的活性是必需的。
