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

The authors transfected COS cells with cDNAs for the α subunits of stimulatory and inhibitory GTP-binding proteins, αs and αi1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled αs and αi were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected αi but could not be detected in αs even when it was overexpressed. They converted the second residue, glycine, of αi1 into alanine by site-directed mutagenesis. Upon transfection of the mutant αi1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal αi1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant αi1 could still interact with the β-γ complex, since purified βγ subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant αi1 subunits. These results indicate that myristoylation is critical for membrane attachment of αi but not αs subunits