My. Degtyarev et al., THE G-PROTEIN ALPHA(S)-SUBUNIT INCORPORATES [H-3] PALMITIC ACID AND MUTATION OF CYSTEINE-3 PREVENTS THIS MODIFICATION, Biochemistry, 32(32), 1993, pp. 8057-8061
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.