C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.
smg p25A, also known as the rab3A protein, is a small GTP-binding protein that has been implicated in intracellular vesicle transport and the secretion of neurotransmitters. It has been shown to bind reversibly to membranes, though its cDNA-predicted sequence contains no obvious membrane-binding domains. However, smg p25A does contain a cDNA-predicted C-terminal Cys-Ala-Cys sequence at positions 218 through 220, which suggests that it may be posttranslationally modified. In the present study we used two different approaches to investigate this possibility. First, we incubated pheochromocytoma cells with [3H]mevalonolactone, examined the proteins that became labeled by two-dimensional gel electrophoresis, and demonstrated that two of these proteins exactly corresponded to smg p25A. Second, we purified smg p25A from bovine brain membranes and analyzed both the full-length protein and a proteolytically derived C-terminal peptide by a combination of high performance liquid chromatography and mass spectrometry. This approach revealed that the protein's C-terminal region is methyl-esterified and contains two geranylgeranyl groups linked via thioether bonds to Cys-218 and Cys-220. Since smg p25A is one of several small GTP-binding proteins that share a C-terminal Cys-Xaa-Cys consensus sequence (where Xaa is an unspecified amino acid), our results suggest that these proteins may be similarly geranylgeranylated and methyl-esterified.
Farnsworth
C C
CC
Howard Hughes Medical Institute Laboratory, University of Washington, Seattle, WA 98195.
Kawata
M
M
Yoshida
Y
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Takai
Y
Y
Gelb
M H
MH
Glomset
J A
JA
D000310:Adrenal Gland Neoplasms; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001923:Brain Chemistry; D002417:Cattle; D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis; D004224:Diterpenes / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008401:Gas Chromatography-Mass Spectrometry; D008798:Mevalonic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010673:Pheochromocytoma; D051381:Rats; D016339:Spectrometry, Mass, Fast Atom Bombardment; D020692:rab3 GTP-Binding Proteins
D000906:Antibodies; D004224:Diterpenes; D010455:Peptides; C065109:geranylgeranylcysteine methyl ester; C015367:mevalonolactone; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D003545:Cysteine; D008798:Mevalonic Acid