Negative Feedback Phosphorylation of Gγ Subunit Ste18 and the Ste5 Scaffold Synergistically Regulates MAPK Activation in Yeast

Heterotrimeric G proteins (Gαβγ) are essential transducers in G protein signaling systems in all eukaryotes. In yeast, G protein signaling differentially activates mitogen-activated protein kinases (MAPKs)—Fus3 and Kss1—a phenomenon controlled by plasma membrane (PM) association of the scaffold protein Ste5. Here, we show that phosphorylation of the yeast Gγ subunit (Ste18), together with Fus3 docking on Ste5, controls the rate and stability of Ste5/PM association. Disruption of either element alone by point mutation has mild but reciprocal effects on MAPK activation. Disabling both elements results in ultra-fast and stable bulk Ste5/PM localization and Fus3 activation that is 6 times faster and 4 times more amplified compared to wild-type cells. These results further resolve the mechanism by which MAPK negative feedback phosphorylation controls pathway activation and provides compelling evidence that Gγ subunits can serve as intrinsic regulators of G protein signaling.