Dual lipid modification of the yeast G gamma subunit Ste18p determines membrane localization of G beta gamma

The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein. The G beta gamma subunit (a complex of Ste4p a nd Ste18p) is associated with both internal and plasma membranes, and a por tion is not stably associated with either membrane fraction, Like Ras, Ste1 8p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmito ylation, Mutant Ste18p containing serine at position 106 (mutation sre18-C1 06S) migrated more rapidly than wild-type Ste18p during sodium dodecyl sulf ate-polyacrylamide gel electrophoresis (SDS-PAGE), The electrophoretic mobi lity of wild-type Ste18p (but not the mutant Ste18p) was sensitive to hydro xylamine treatment, consistent with palmitoyl modification at Cys 106, Furt hermore, immunoprecipitation of the G beta gamma complex from cells culture d in the presence of [H-3]palmitic acid resulted in two radioactive species on nonreducing SDS-PAGE gels, with molecular weights corresponding to G ga mma and G beta gamma. Substitution of serine for either Cys 107 or Cys 106 resulted in the failure of G beta gamma to associate with membranes. The Cy s 107 substitution also resulted in reduced steady-state accumulation of St e18p, suggesting that the stability of Ste18p requires modification at Cys 107, All of the mutant forms of Ste18p formed complexes with Ste4p, as asse ssed by coimmunoprecipitation. We conclude that tight membrane attachment o f the wild-type G beta gamma depends on palmitoylation at Cys 106 and preny lation at Cys 107 of Ste18p.