N-terminal intrinsic disorder is an ancestral feature of G subunits that influences the balance between different G¦ signaling axes in yeast

Activated G protein-coupled receptors promote the dissociation of heterotrimeric G proteins into G alpha and Gtl gamma subunits that bind to effector proteins to drive intracellular signaling responses. In yeast, Gtl gamma subunits coordinate the simultaneous activation of multiple signaling axes in response to mating pheromones, including MAP kinase (MAPK)-dependent transcription, cell polarization, and cell cycle arrest responses. The G gamma subunit in this complex contains an N-terminal intrinsically disordered region that governs Gtl gamma-dependent signal transduction in yeast and mammals. Here, we demonstrate that N-terminal intrinsic disorder is likely an ancestral feature that has been conserved across different G gamma subtypes and organisms. To understand the functional contribution of structural disorder in this region, we introduced precise point mutations that produce a stepwise disorder-to-order transition in the N-terminal tail of the canonical yeast G gamma subunit, Ste18. Mutant tail structures were confirmed using circular dichroism and molecular dynamics and then substituted for the wildtype gene in yeast. We find that increasing the number of helix-stabilizing mutations, but not isometric mutation controls, has a negative and proteasome-independent effect on Ste18 protein levels as well as a differential effect on pheromone-induced levels of active MAPK/Fus3, but not MAPK/Kss1. When expressed at wildtype levels, we further show that mutants with an alphahelical N terminus exhibit a counterintuitive shift in Gtl gamma increasing cell polarization and cell cycle arrest. These data reveal a role for G gamma subunit intrinsically disordered regions in governing the balance between multiple Gtl gamma signaling axes.