Involvement of N-Myristoylation in Monoclonal Antibody Recognition Sites on Chimeric G Protein Subunits

Monoclonal antibody, LAS-2, directed against the α subunit of transducin (G ), inhibited G -dependent, pertussis toxin-catalyzed ADP ribosylation of G and was specific for G . Immunoblotting studies on proteolytic fragments of G were consistent with an amino-terminal epitope. To define the antibody recognition site, recombinant G was synthesized in Escherichia coli cotransfected with or without yeast N -myristoyltransferase. Amino-terminal fatty acylation of G , verified by use of radiolabeled fatty acid, was required for immunoreactivity. LAS-2 did not react with a chimeric protein consisting of residues 1-9 of G and the remainder G , regardless of its myristoylation. Immunoreactivity was observed when amino acids 1-17 of G were present in a G chimera and the protein was amino-terminally myristoylated; there was no reactivity without myristoylation. It appears that the LAS-2 epitope requires both G -specific sequence in amino acids 10-17 and a fatty acyl group in proximity to these residues. These results are consistent with the hypothesis that the myristoyl group is essential for protein structure; conceivably it “folds back” on and stabilizes the amino-terminal structure of G , as opposed to protruding from an amino-terminal α-helix and serving as an amino-terminal membrane anchor.