The membrane-binding domain of a 23-kDa G-protein is carboxyl methylated.
We have purified to homogeneity a 23-kDa protein from bovine brain membranes using [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding as an assay. GTP gamma S binding to the purified protein is inhibited by GDP, GTP, and GTP analogs but not by cGMP, GMP, or adenine nucleotides, consistent with the nucleotide-binding behavior of members of the family of GTP-binding regulatory proteins. On addition of the methyl donor S-adenosyl-L-methionine and a methyltransferase present in bovine brain membranes, the purified 23-kDa G-protein is carboxyl methylated. When subjected to limited tryptic proteolysis, the 23-kDa protein is converted to a 22-kDa major fragment with concomitant release of a carboxyl methylated protein fragment of 1 kDa. Furthermore, when the cleaved protein is reconstituted with stripped bovine brain membranes, the small carboxyl-methylated fragment but not the 22-kDa major fragment is found to reassociate with the membranes. These results indicate that the site of carboxyl methylation and the region responsible for membrane anchoring, most likely, are localized to a small region at the carboxyl terminus. It is attractive to speculate that carboxyl methylation and membrane anchoring are interrelated processes and play key roles in the function of this small G-protein.
Yamane
H K
HK
Jules Stein Eye Institute, University of California School of Medicine, Los Angeles 90024.
Fung
B K
BK
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008745:Methylation; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D012265:Ribonucleotides / Q000494:pharmacology; D013873:Thionucleotides / Q000378:metabolism / Q000494:pharmacology; D014357:Trypsin
D010446:Peptide Fragments; D012265:Ribonucleotides; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D014357:Trypsin; D019204:GTP-Binding Proteins