The G Protein α_s Subunit Incorporates [³H]Palmitic Acid and Mutation of Cysteine-3 Prevents This Modification

We investigated whether α_s could be acylated by palmitate by transfecting COS cells with the cDNA for the wild-type, long form of α_s and metabolically labeling with [³H]palmitate or [³⁵S]methionine. Cells were separated into particulate and soluble fractions and immunoprecipitated with a specific peptide antibody. [³H]Palmitate was incorporated into both endogenous and transfected α_s. Inhibition of protein synthesis with cycloheximide did not block the radiolabeling of α_s with [³H]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 [³H]palmitate on thin-layer chromatography. The third residue of the wild-type α_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 [³⁵S]methionine-labeled cells. The cysteine-3 mutant did not undergo radiolabeling with [³H]palmitate, indicating that this residue is crucial for the modification.