THE G-PROTEIN ALPHA(S)-SUBUNIT INCORPORATES [H-3] PALMITIC ACID AND MUTATION OF CYSTEINE-3 PREVENTS THIS MODIFICATION

We investigated whether alpha(s) could be acylated by palmitate by transfecting COS cells with the cDNA for the wild-type, long form of alpha(s) and metabolically labeling with [H-3]palmitate or [S-35]methionine. Cells were separated into particulate and soluble fractions and immunoprecipitated with a specific peptide antibody. [H-3]Palmitate was incorporated into both endogenous and transfected alpha(s). Inhibition of protein synthesis with cycloheximide did not block the radiolabeling of alpha(s) with [H-3]palmitate. Hydroxylamine treatment caused a release of the tritium radiolabel, demonstrating that the incorporation was through a thioester bond. The tritium radiolabel was base-labile and comigrated with [H-3]palmitate on thin-layer chromatography. The third residue of the wild-type alpha(s) was mutated from a cysteine to an alanine by site-directed mutagenesis. This mutant was expressed in COS cells and localized to the particulate fraction as determined by immunoprecipitation of the [S-35]methionine-labeled cells. The cysteine-3 mutant did not undergo radiolabeling with [H-3]palmitate, indicating that this residue is crucial for the modification.