We investigated whether αs could be acylated by palmitate by transfecting COS cells with the cDNA for the wild-type, long form of αs 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 αs. Inhibition of protein synthesis with cycloheximide did not block the radiolabeling of αs 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 αs 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.
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