Functional analysis of plp1 and plp2, two homologues of phosducin in yeast
Functional analysis of plp1 and plp2, two homologues of phosducin in yeast
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DOI：
10.1074/jbc.m002163200
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发表时间：
2000-06-16
影响因子：
4.8
通讯作者：
Dohlman, HG
中科院分区：
文献类型：
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作者：
Flanary, PL;DiBello, PR;Dohlman, HG
哺乳动物的光导蛋白在体外与G蛋白β-伽马亚基结合，并被认为在体内调节其信号功能。在这里，我们描述了酵母中的两种硫代蛋白同源物，称为PLP1和PLP2。两种基因产物均被克隆、表达和纯化为谷胱甘肽S-转移酶融合蛋白。在这两种异构体中，Plp1最优先与Gβ-γ结合。通过信息素刺激和添加GTP-伽马S，有利于G-β-伽马与G-α解离的条件增强了结合。制备基因中断突变体和基因过表达载体，分析信号和非信号表型的变化。携带plp2 Delta突变体的单倍体孢子产物不能生长，表明plp2是一个必需基因。细胞活力没有恢复，BG是STE7的一个突变，它阻断了G蛋白下游的信号传递。携带plp1 Delta突变体的单倍体产物是活的，并且在信息素介导的基因诱导中显示出6-7%的增加。过量表达PLP1或PLP2的细胞在基因诱导方面下降了70%-80%，但在信息素介导的生长停滞方面没有变化。这些数据表明，光导蛋白可以选择性地调节信息素刺激后的早期信号事件，并在细胞生长中发挥重要作用，而不是依赖于它在细胞信号中的调节作用。
Mammalian phosducins are known to bind G protein beta gamma subunits in vitro, and are postulated to regulate their signaling function in vivo. Here we describe two homologues of phosducin in yeast, called PLP1 and PLP2. Both gene products were cloned, expressed, and purified as glutathione S-transferase fusions. Of the two isoforms, Plp1 bound most preferentially to G beta gamma. Binding was enhanced by pheromone stimulation and by the addition of GTP gamma S, conditions that favor dissociation of G beta gamma from G alpha. Gene disruption mutants and gene over expression plasmids were prepared and analyzed for changes in signaling and non-signaling phenotypes. Haploid spore products bearing the plp2 Delta mutant failed to grow, suggesting that PLP2 is an essential gene. Cell viability was not restored bg a mutation in STE7 that blocks signaling downstream of the G protein. Haploid products bearing the plp1 Delta mutant were viable and exhibited a 6-7% increase in pheromone-mediated gene induction. Cells overexpressing PLP1 or PLP2 exhibited a 70-80% decrease in gene induction but no change in pheromone-mediated growth arrest. These data indicate that phosducin can selectively regulate early signaling events following pheromone stimulation and has an essential role in cell growth independent of its regulatory role in cell signaling.