The posttranslationally modified C-terminal structure of bovine aortic smooth muscle rhoA p21.
rhoA p21, a ras p21-like small GTP-binding protein, has the same C-terminal consensus motif of Cys-A-A-X (A is an aliphatic amino acid and X is any amino acid) as ras p21s, which is posttranslationally processed. We here determine the posttranslationally processed C-terminal structure of the rhoA p21 purified from bovine aortic smooth muscle. Incubation of rhoA p21-expressing insect cells with exogenous [3H]mevalonolactone caused the labeling of rhoA p21, suggesting that rhoA p21 is prenylated. Consistently, Raney nickel treatment of rhoA p21 released a geranylgeranyl moiety as estimated by gas chromatography/mass spectrometry. No lipid moiety was released by KOH or NH2OH treatment. Extensive digestion of rhoA p21 with Achromobacter protease I yielded a C-terminal peptide, Ser-Gly-Cys190, that lacked the three C-terminal amino acids predicted from the cDNA but was geranylgeranylated and carboxyl methylated at the cysteine residue. Bovine brain cytosol geranylgeranylated the bacterial rhoA p21 having the three C-terminal amino acids predicted from the cDNA but not the protein lacking the three C-terminal amino acids. Bovine brain membranes methylated the synthetic C-terminal peptide with 10 amino acids of rhoA p21 which was geranylgeranylated at its C-terminal cysteine residue but not the peptide which was not geranylgeranylated. These results suggest that rhoA p21 is first geranylgeranylated followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the exposed cysteine residue.
Katayama
M
M
Department of Biochemistry, Kobe University School of Medicine, Japan.
Kawata
M
M
Yoshida
Y
Y
Horiuchi
H
H
Yamamoto
T
T
Matsuura
Y
Y
Takai
Y
Y
D000421:Alcaligenes / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004247:DNA / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000235:genetics*; D008401:Gas Chromatography-Mass Spectrometry; D005786:Gene Expression Regulation; D007313:Insecta / Q000235:genetics; D008745:Methylation; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020742:rhoA GTP-Binding Protein
D004247:DNA; D010450:Endopeptidases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein