Brain G protein gamma subunits contain an all-trans-geranylgeranylcysteine methyl ester at their carboxyl termini.
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.
Yamane
H K
HK
Jules Stein Eye Institute, Los Angeles, CA.
Farnsworth
C C
CC
Xie
H Y
HY
Howald
W
W
Fung
B K
BK
Clarke
S
S
Gelb
M H
MH
Glomset
J A
JA
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis; D004224:Diterpenes / Q000032:analysis* / Q000138:chemical synthesis; D019204:GTP-Binding Proteins / Q000302:isolation & purification*; D008401:Gas Chromatography-Mass Spectrometry; D046911:Macromolecular Substances; D008970:Molecular Weight; D011865:Radioisotope Dilution Technique; D013729:Terpenes / Q000138:chemical synthesis; D014316:Tritium
D004224:Diterpenes; D046911:Macromolecular Substances; D013729:Terpenes; D014316:Tritium; C065109:geranylgeranylcysteine methyl ester; D019204:GTP-Binding Proteins; D003545:Cysteine