Membrane-binding domain of the small G protein G25K contains an S-(all-trans-geranylgeranyl)cysteine methyl ester at its carboxyl terminus.
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 all-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.
Yamane
H K
HK
Jules Stein Eye Institute, University of California, Los Angeles 90024.
Farnsworth
C C
CC
Xie
H Y
HY
Evans
T
T
Howald
W N
WN
Gelb
M H
MH
Glomset
J A
JA
Clarke
S
S
Fung
B K
BK
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis; D004224:Diterpenes / Q000032:analysis*; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis; D010446:Peptide Fragments / Q000302:isolation & purification
D000906:Antibodies; D004224:Diterpenes; D009842:Oligopeptides; D010446:Peptide Fragments; C065109:geranylgeranylcysteine methyl ester; D019204:GTP-Binding Proteins; D003545:Cysteine