The G protein alpha s subunit incorporates [3H]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 [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 alpha s. Inhibition of protein synthesis with cycloheximide did not block the radiolabeling of alpha 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 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 [35S]methionine-labeled cells. The cysteine-3 mutant did not undergo radiolabeling with [3H]palmitate, indicating that this residue is crucial for the modification.
Degtyarev
M Y
MY
Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Spiegel
A M
AM
Jones
T L
TL
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D014316:Tritium
D010169:Palmitic Acids; D014316:Tritium; D019308:Palmitic Acid; D003513:Cycloheximide; D008715:Methionine; D019204:GTP-Binding Proteins; D003545:Cysteine