We have shown previously that guanine nucleotide-binding protein (G protein) beta gamma complexes purified from bovine brain membranes are methyl esterified on a C-terminal cysteine residue of the gamma polypeptide. In the present study, 3H-methylated G beta gamma 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 beta gamma 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 gamma subunits, purified G beta gamma complexes were separated into 5-kDa (gamma 5) and 6-kDa (gamma 6) forms of the gamma polypeptide by reversed-phase HPLC. Gas chromatography-coupled mass spectrometry analyses of Raney nickel-treated purified gamma 5 and gamma 6 subunits showed that both polypeptides were modified by geranylgeranylation. These results demonstrate that at least two forms of brain gamma subunit are posttranslationally modified by geranylgeranylation and carboxyl methylation. These modifications may be important for targeting G beta gamma complexes to membranes.

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We showed previously that a 23-kDa guanine nucleotide-binding protein (G protein) purified from bovine brain membranes is carboxyl methylated and that this modification occurs at or near the membrane-binding domain. In the present study, we identified this small G protein as G25K (formerly termed Gp). We demonstrated that proteolytic digests of 3H-methylated G25K contained radiolabeled material that coeluted with synthetic S-(geranylgeranyl)cysteine methyl ester on reversed-phase HPLC. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the isoprenoid moiety and carboxyl methyl ester are localized on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G25K by Raney nickel treatment positively identified the covalently bound lipid as an afl-trans-geranylgeranyl (C20) isoprenoid moiety. These results suggest that geranylgeranyl modification and perhaps methyl esterification function in the membrane localization of this small G protein.Within the past several years, a family of low molecular weight GTP-binding proteins (G proteins) has been identified by protein purification and molecular cloning techniques (for review, see ref. 1). Members ofthis family of small G proteins are between 20 and 30 kDa, possess 30-70% homology with the ras proteins, and appear to be present in all species and cell types. Although two yeast homologs of these proteins, yptl and sec4 from Saccharomyces cerevisiae, have been implicated in the secretory process (2, 3) and several have been localized to Golgi and secretory vesicles (4-6), no definite function has been ascribed to any. Among the small G proteins for which amino acid sequences have been deduced, a majority possess a cysteine residue at or near the C terminus, and a subgroup contain a CXXX C-terminal sequence. This signature sequence has been implicated as a signal for prenylation of the cysteine sulfhydryl group, followed by proteolytic removal of the three terminal residues and carboxyl methylation of the resulting terminal cysteine (7-9).We showed previously that a 23-kDa small G protein purified from bovine brain membranes is carboxyl methylated (10). Limited tryptic digestion of the 3H-methylated protein generated an -1-kDa peptide containing the [3H]-methyl ester and a 22-kDa major fragment. The 1-kDa carboxyl methylated peptide, but not the 22-kDa fragment, was observed to bind to membranes (10). We have now identified this small G protein as G25K, one of the first small G proteins to be described. G25K (previously known as Gp) was originally purified from human placenta (11) and subsequently from bovine brain (12) and human platelets (13). More recently, cDNA clones of G25K have been obtained from human placenta (14) and human fetal brain (15). The placental and fetal brain G25K cDNA clones encode CVIL and CCIF C-terminal sequences, respectively, both of which are candidates for posttranslational processin...

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