Whiteway M, Dignard D, Thomas D Y
Biotechnology Research Institute, National Research Council of Canada, Montréal, Que.
Biochem Cell Biol. 1992 Oct-Nov;70(10-11):1230-7. doi: 10.1139/o92-169.
The yeast STE18 gene product has sequence and functional similarity to the gamma subunits of G proteins. The cloned STE18 gene was subjected to a saturation mutagenesis using doped oligonucleotides. The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STE18 gene (dominant negatives). Three amino acid substitutions that enhanced mating in a specific STE4 (G beta) point mutant background were identified. These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the G beta and G gamma subunits or in the association of the G beta gamma subunit with other components of the signalling pathway. Several dominant negative mutations were also identified, including two C terminal truncations. These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPA1 (G alpha) gene. These mutant proteins may sequester G beta or some other component of the signalling machinery in a nonfunctional complex.
酵母STE18基因产物与G蛋白的γ亚基在序列和功能上具有相似性。利用掺杂的寡核苷酸对克隆的STE18基因进行饱和诱变。对突变基因群体进行筛选，以寻找两类STE18突变：一类是能使含有缺陷STE4基因的菌株交配增加的突变（补偿突变体），另一类是即使在存在功能性STE18基因的情况下仍抑制交配的突变（显性负突变）。鉴定出了三个在特定STE4（Gβ）点突变背景下增强交配的氨基酸取代。这些补偿性突变是等位基因特异性的，且自身没有可检测到的表型；它们可能定义了介导Gβ和Gγ亚基之间关联或Gβγ亚基与信号通路其他组分之间关联的残基。还鉴定出了几个显性负突变，包括两个C末端截短突变。这些突变蛋白自身无法在信号转导中发挥作用，但它们阻止了由信息素介导的信号转导，以及存在于GPA1（Gα）基因缺陷细胞中的组成型信号转导。这些突变蛋白可能将Gβ或信号传导机制的其他组分隔离在无功能的复合物中。
