REGULATION OF MEMBRANE AND SUBUNIT INTERACTIONS BY N-MYRISTOYLATION OF A G-PROTEIN ALPHA-SUBUNIT IN YEAST

Initiation of the mating process in yeast Saccharomyces cerevisiae req uires the action of secreted pheromones and G protein-coupled receptor s. As in other eu karyotes, the yeast G protein alpha subunit undergoe s N-myristoylation (GPA1 gene product, Gpa1p). This modification appea rs to be essential for function, since a myristoylation site mutation exhibits the null phenotype in vivo (gpa1(G2A)). Here we examine how m yristoylation affects Gpa1p activity in vitro. We show that the G2A mu tant of Gpa1p, when fused with glutathione S-transferase, can still fo rm a complex with the G protein beta gamma subunits. The complex is st abilized by GDP and is dissociated upon treatment with guanosine 5'-O- (thiotriphosphate). In addition, there is no apparent difference in th e relative binding affinity of G(beta gamma) for mutant and wild-type Gpa1p. Using sucrose density gradient fractionation of cell membranes, Gpa1p associates normally with the plasma membrane whereas Gpa1p(G2A) is mislocalized to a microsomal membrane fraction. A portion of G(bet a gamma) is also mislocalized in these cells, as it is in a gpa1 Delta strain. In contrast, wild-type Gpa1p reaches the plasma membrane in c ells that do not express G(beta gamma) or cell surface receptors. Thes e findings indicate that mislocalization of Gpa1p(G2A) is not caused b y a redistribution of G(beta gamma), nor is it the result of any diffe rence in G(beta gamma) binding affinity. These data suggest that myris toylation is required for specific targeting of Gpa1p to the plasma me mbrane, where it is needed to interact with the receptor and to regula te the release of G(beta gamma).