Dual Lipid Modification Motifs in G(α) and G(γ) Subunits Are Required for Full Activity of the Pheromone Response Pathway in Saccharomyces cerevisiae

To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide–binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein γ subunit (Ste18p) is unusual among G(γ) subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the G(γ) subunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of G(βγ) after receptor-stimulated release from G(α). The G protein α subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway.

为阐明硫酰化（thioacylation，棕榈酰化palmitoylation）的生物学功能，我们针对酿酒酵母（Saccharomyces cerevisiae）信息素应答通路的异源三聚体鸟苷酸结合蛋白（heterotrimeric guanine nucleotide–binding protein，简称G蛋白）亚基展开了研究。酿酒酵母的G蛋白γ亚基（Gγ亚基）Ste18p在同类Gγ亚基中较为特殊，其在半胱氨酸107位点发生法尼基化，且具备在半胱氨酸106位点发生硫酰化的潜力。对任一上述半胱氨酸位点进行突变替换，均会导致严重的信号通路缺陷。本研究发现，Ste18p在半胱氨酸106位点发生硫酰化修饰，该修饰过程依赖于半胱氨酸107的异戊二烯化。即便缺失异戊二烯化或硫酰化修饰，Ste18p仍可被靶向招募至质膜。但在G蛋白激活后，缺失异戊二烯化或硫酰化修饰的Ste18p会被释放至细胞质中。由此可见，Gγ亚基的脂质修饰对于受体介导的G蛋白激活并非必需，但在受体刺激下Gα亚基释放Gβγ二聚体后，却对于维持Gβγ二聚体与质膜的结合至关重要。G蛋白α亚基（Gα亚基）Gpa1p的N端存在串联式脂肪酸修饰，该修饰同时通过酰胺键与硫酯键连接。本研究证实，Gpa1p在活体内可结合放射性标记的肉豆蔻酸与棕榈酸混合物，发生硫酰化修饰。对Gpa1p的硫酰化位点进行突变后，酵母细胞会在未暴露于信息素的情况下，出现通路部分激活的表型。综上，Gpa1p与Ste18p上的双重脂质修饰基序，是信息素应答通路实现完整功能所必需的。