BRAIN G-PROTEIN-GAMMA SUBUNITS CONTAIN AN ALL-TRANS-GERANYLGERANYL-CYSTEINE METHYL-ESTER AT THEIR CARBOXYL TERMINI

We have shown previously that guanine nucleotide-binding protein (G protein) βγ complexes purified from bovine brain membranes are methyl esterified on a C-terminal cysteine residue of the γ polypeptide. In the present study, 3H-methylated G(βγ) complexes cleaved to their constituent amino acids by exhaustive proteolysis were shown to contain radiolabeled material that coeluted with geranylgeranylcysteine methyl ester on reversed-phase HPLC and two TLC systems. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the prenyl modification occurs on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G(βγ) by treatment with Raney nickel positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. To delineate the distribution of this modification among γ subunits, purified G(βγ) complexes were separated into 5-kDa (γ5) and b-kDa (α6) forms of the γ polypeptide by reversed-phase HPLC. Gas chromatography-coupled mass spectrometry analyses of Raney nickel-treated purified γ5 and γ6 subunits showed that both polypeptides were modified by geranylgeranylation. These results demonstrate that at least two forms of brain γ subunit are posttranslationally modified by geranylgeranylation and carboxyl methylation. These modifications may be important for targeting G(βγ) complexes to membranes.