Myristoylation of an inhibitory GTP-binding protein a subunit is essential for its membrane attachment

We transfected COS cells with cDNAs for the α subunits of stimulatory and inhibitory GTP-binding proteins, α_s and α_i1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [³⁵S]methionine-labeled α_s and α_i were both found primarily in the particulate fraction. [³H]Myristate was incorporated into endogenous and transfected α_i but could not be detected in α_s even when it was overexpressed. We converted the second residue, glycine, of α_i1 into alanine by site-directed mutagenesis. Upon transfection of the mutant α_i1 into COS cells, the [³⁵S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal α_i1, the mutant failed to incorporate [³H]myristate. The unmyristoylated mutant α_i1 could still interact with the β-γ complex, since purified βγ subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant α_i1 subunits. These results indicate that myristoylation is critical for membrane attachment of α_i but not α_s subunits.