PARTIAL CONSTITUTIVE ACTIVATION OF PHEROMONE RESPONSES BY A PALMITOYLATION-SITE MUTANT OF A G-PROTEIN ALPHA-SUBUNIT IN YEAST

G protein alpha subunits are often myristoylated and/or palmitoylated near their amino terminus. The G protein a subunit in the yeast Saccha romyces cerevisiae (GPA1 gene product, Gpa1p) is known to be myristoyl ated. and this modification is essential for G protein activity in viv o. Here we examined whether Gpa1p is palmitoylated and determined the functional consequences of this modification. [H-3]-Palmitic acid was incorporated into Gpa1p in cells expressing myc-tagged Gpa1p or Gpa1p- Gst. The label was released upon hydroxylamine treatment. Substitution of the conserved Cys 3 for Ser blocked incorporation of the label (Gp a1p(C3S)). Palmitoylation was also blocked by a mutation that prevents myristoylation (Gly2Ala), whereas the palmitoylation-site mutation ha d no effect on myristoylation of Gpa1p. Gpa1p(C3S) complemented the gp al Delta mutation in vivo and formed a complex with G(beta gamma) that was able to undergo nucleotide exchange in vitro. However, basal and pheromone-induced FUS1-lacZ transcription were 2-5-fold higher in the C3S mutant. Pheromone-induced growth arrest was also enhanced by the m utation, but recovery from arrest was not affected. Like wild-type Gpa 1p. the C3S mutant was predominantly membrane-associated. Upon Triton X-114 partitioning or high pH treatment, no difference in the membrane -binding properties of the wild-type Gpa1p and the C3S mutant was dete cted. By sucrose density gradient centrifugation of membranes, however , most of the mutant protein was mislocalized to a non-plasma membrane compartment, whereas G(beta gamma) localization was unaltered. Taken together, our data suggest that Gpa1p is palmitoylated via a thioester bond at Cys 3 and that palmitoylation plays a role in modulating Gpa1 p signaling and membrane localization.