Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:Protein N-myristoyltransferase by co-expressing it with mammalian G protein α subunits in Escherichia coli

A dual plasmid system was used to examine the protein and acyl-CoA specificities of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by co-expressing it in Escherichia coli with each of four homologous a subunits of the signal-transducing, heterotrimeric G proteins. Exogenous [³H]myristate was incorporated into rat G_iα1 and rat G_oα but not into bovine G_sα or human G_zα. Oxygen for methylene group substitutions in myristate result in analogs with comparable chain length and stereochemistry but marked reductions in hydrophobicity. Metabolic labeling studies with 6-, 11-, or 13-[³H]oxatetradecanoic acid indicated that they were incorporated into rat G_iα1 and G_oα with an efficiency that could be correlated with their accumulation into E. coli and their interactions with purified NMT in vitro. Octapeptides derived from the NH₂-terminal sequences of these four G_α polypeptides were tested as substrates for purified S. cerevisiae NMT. None were bound by the enzyme. Acidic residues at positions 7 and 8 appear to contribute to this effect; deletion of these two amino acids or addition of the next 9 residues of rat G_oα produced active substrates. These results imply that productive interactions between NMT and G_α protein substrates in vivo require structural features that are not fully represented within their NH₂-terminal 8 residues.