Combinatorial phosphorylation modulates the structure and function of the G protein gamma subunit in yeast

Protein intrinsically disordered regions (IDRs) are often targets of combinatorial post-translational modifications (PTMs) that serve to regulate protein structure and/or function. Emerging evidence suggests that the N-terminal tails of G protein {gamma} subunits - essential components of heterotrimeric G protein complexes - are intrinsically disordered, highly phosphorylated governors of G protein signaling. Here, we demonstrate that the yeast G{gamma} Ste18 undergoes combinatorial, multi-site phosphorylation within its N-terminal IDR. Phosphorylation at S7 is responsive to GPCR activation and osmotic stress while phosphorylation at S3 is responsive to glucose stress and is a quantitative indicator of intracellular pH. Each site is phosphorylated by a distinct set of kinases and both are also interactive, such that phosphomimicry at one site affects phosphorylation on the other. Lastly, we show that phosphorylation produces subtle yet clear changes in IDR structure and that different combinations of phosphorylation modulate the activation rate and amplitude of the scaffolded MAPK Fus3. These data place G{gamma} subunits among the growing list of intrinsically disordered proteins that exploit combinatorial post-translational modification to govern signaling pathway output.