Intrinsically disordered regions (IDRs) in proteins are often targets of combinatorial posttranslational modifications, which serve to regulate protein structure and function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits, which are essential components of heterotrimeric G proteins, are intrinsically disordered, phosphorylation-dependent determinants of G protein signaling. Here, we found that the yeast Gγ subunit Ste18 underwent combinatorial, multisite phosphorylation events within its N-terminal IDR. G protein–coupled receptor (GPCR) activation and osmotic stress induced phosphorylation at Ser⁷, whereas glucose and acid stress induced phosphorylation at Ser³, which was a quantitative indicator of intracellular pH. Each site was phosphorylated by a distinct set of kinases, and phosphorylation of one site affected phosphorylation of the other, as determined through exposure to serial stimuli and through phosphosite mutagenesis. Last, we showed that phosphorylation resulted in changes in IDR structure and that different combinations of phosphorylation events modulated the activation rate and amplitude of the downstream mitogen-activated protein kinase Fus3. These data place Gγ subunits among intrinsically disordered proteins that undergo combinatorial posttranslational modifications that govern signaling pathway output.

蛋白质中的内在无序区域 (IDR) 通常是组合翻译后修饰的目标，用于调节蛋白质结构和功能。新出现的证据表明，作为异源三聚体 G 蛋白的重要组成部分的 G 蛋白 γ 亚基的 N 末端尾部本质上是无序的、磷酸化依赖性 G 蛋白信号传导的决定因素。在这里，我们发现酵母 Gγ 亚基 Ste18 在其 N 端 IDR 内经历了组合的多位点磷酸化事件。G 蛋白偶联受体 (GPCR) 激活和渗透胁迫诱导 Ser ⁷磷酸化，而葡萄糖和酸胁迫诱导 Ser ^3磷酸化，这是细胞内 pH 值的定量指标。每个位点都被一组不同的激酶磷酸化，一个位点的磷酸化影响另一个位点的磷酸化，这通过暴露于连续刺激和磷酸位点诱变来确定。最后，我们发现磷酸化导致 IDR 结构发生变化，并且磷酸化事件的不同组合调节了下游丝裂原活化蛋白激酶 Fus3 的激活速率和幅度。这些数据将 Gγ 亚基置于本质上无序的蛋白质中，这些蛋白质经历了控制信号通路输出的组合翻译后修饰。