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 tra nsfecting COS cells with the cDNA for the wild-type, long form of alph a(s) and metabolically labeling with [H-3]palmitate or [S-35]methionin e. Cells were separated into particulate and soluble fractions and imm unoprecipitated with a specific peptide antibody. [H-3]Palmitate was i ncorporated into both endogenous and transfected alpha(s). Inhibition of protein synthesis with cycloheximide did not block the radiolabelin g of alpha(s) with [H-3]palmitate. Hydroxylamine treatment caused a re lease of the tritium radiolabel, demonstrating that the incorporation was through a thioester bond. The tritium radiolabel was base-labile a nd comigrated with [H-3]palmitate on thin-layer chromatography. The th ird residue of the wild-type alpha(s) was mutated from a cysteine to a n alanine by site-directed mutagenesis. This mutant was expressed in C OS cells and localized to the particulate fraction as determined by im munoprecipitation 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.