N-myristoylation and βγ play roles beyond Anchorage in the palmitoylation of the G protein αo subunit

Many of the alpha subunits of heterotrimeric GTP-binding regulatory proteins (G proteins) are palmitoylated, a modification proposed to play a key role in the stable anchorage of the subunits to the plasma membrane. Palmitoylation of alpha subunits from the G(i) family is preceded by N-myristoylation, which alone or together with beta gamma probably supports a reversible interaction of the alpha subunit with membrane as a prerequisite to the eventual incorporation of palmitate, Previous studies have not addressed, however, the question of whether membrane association alone, carried out through N-myristoylation, interaction with beta gamma, or other events, is sufficient for palmitoylation, We report here for alpha(o) that it is not. We found that N-myristoylation is required for palmitoylation at least in part because it supports events subsequent to membrane attachment. Mutants of alpha(o) designed to target the subunit to membrane without an N-myristoyl group are unable to be palmitoylated as evaluated by incorporation of [H-3]palmitate. Mutants of alpha(o) unable to interact normally with beta gamma yet still attach to membrane demonstrate that beta gamma, in contrast, is not required for palmitoylation. beta gamma becomes necessary, however, when the N-myristoyl group is absent. Our results suggest that N-myristoylation and beta gamma, while almost certainly relevant to the reversible interaction of alpha(o) with membrane, also play at least partly overlapping, post-anchorage roles in palmitoylation.