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

Abstract

Distinct phosphorylation patterns in the intrinsically disordered N terminus of Gγ have functional consequences in yeast. Gγ governs signaling The Gγ subunit is the smallest member of the heterotrimeric G protein, and it may have more functional roles beyond its important role in anchoring the Gβγ dimer to the plasma membrane. Nassiri Toosi et al. showed that the N-terminal region of the yeast Gγ subunit Ste18 contains two serine residues within an intrinsically disordered region that were phosphorylated by different kinases in response to distinct stimuli. These combinatorial phosphorylation events likely altered the structure of Gγ and modulated the activation of a downstream effector. Together, these results suggest that distinct phosphorylation events in the Gγ intrinsically disordered region determine functional outcomes. 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.