The geranylgeranyl moiety but not the methyl moiety of the smg-25A/rab3A protein is essential for the interactions with membrane and its inhibitory GDP/GTP exchange protein.
The smg-25A/rab3A protein (smg p25A), a member of the small GTP-binding protein superfamily, has a C-terminal structure of Cys-Ala-Cys which is post-translationally processed: both cysteine residues are geranylgeranylated followed by the carboxyl methylation of the C-terminal cysteine residue. We reported previously that this posttranslational processing is essential for the interactions of smg p25A with membrane and its inhibitory GDP/GTP exchange protein, named smg p25A GDP dissociation inhibitor (GDI). In this study, we examined which posttranslational modification of smg p25A is necessary for these interactions. The smg p25A which was not posttranslationally processed was produced in Escherichia coli and purified. This protein was then geranylgeranylated at both of the 2 cysteine residues by use of a bovine brain geranylgeranyltransferase in a cell-free system (recombinant smg p25A-GG). By use of this recombinant smg p25A-GG, its membrane-binding activity and its sensitivity to smg p25A GDI were compared with those of the fully posttranslationally processed form of bovine brain smg p25A (smg p25A-GG-Me) and the posttranslationally unprocessed form of bacterial smg p25A (recombinant smg p25A). The membrane-binding activity and sensitivity to smg p25A GDI were similar between the recombinant smg p25A-GG and smg p25A-GG-Me, although recombinant smg p25A lacked both activities. These results indicate that the geranylgeranyl moiety of smg p25A is essential and sufficient for its interactions with membrane and smg p25A GDI and that the methyl moiety is not essential for these interactions.
Musha
T
T
Department of Biochemistry, Kobe University School of Medicine, Japan.
Kawata
M
M
Takai
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004224:Diterpenes / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D020692:rab3 GTP-Binding Proteins
D004224:Diterpenes; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C017338:geranylgeraniol; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins