Geranylgeranylated Rab proteins terminating in Cys-Ala-Cys, but not Cys-Cys, are carboxyl-methylated by bovine brain membranes in vitro.
Geranylgeranylated Rab proteins usually terminate in either Cys-Cys or Cys-Xaa-Cys, where Xaa is Ala, Ser, or Gly. In both classes of proteins, the two cysteines are geranylgeranylated, but only the Cys-Xaa-Cys class has been shown to be carboxyl-methylated on the terminal cysteine in vivo. In the current study, we used recombinant Rab geranylgeranyltransferase and a Rab escort protein (REP-1) to attach geranylgeranyl residues to the two cysteines at the carboxyl terminus of Rab3A (Cys-Ala-Cys) and Rab1A (Cys-Cys). The geranylgeranylated proteins were then incubated with bovine cerebellar membranes that contain an enzyme that transfers [3H]methyl from S-[methyl-3H]adenosyl-L-methionine to geranylgeranylated cysteine. The enzyme transferred [3H]methyl to geranylgeranylated Rab3A but not to geranylgeranylated Rab1A. Replacement of the Cys-Ala-Cys terminus of Rab3A with Cys-Cys abolished methylation, and the opposite result was obtained when the Cys-Cys of Rab1A was replaced with Cys-Ala-Cys. When the Cys-Cys terminus of Rab1A was changed to Ser-Cys, methylation was restored. These studies suggest that the carboxyl-terminal cysteine of Rab proteins terminating in Cys-Xaa-Cys but not Cys-Cys is methylated and that the resistance of Cys-Cys proteins to methylation is attributable to the vicinal geranylgeranylated cysteines.
Smeland
T E
TE
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
Seabra
M C
MC
Goldstein
J L
JL
Brown
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002531:Cerebellum / Q000378:metabolism*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D017931:DNA Primers; D004224:Diterpenes / Q000032:analysis / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D008861:Microsomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013329:Structure-Activity Relationship; D014316:Tritium; D020761:rab1 GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins
D017931:DNA Primers; D004224:Diterpenes; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D014316:Tritium; C065977:geranylgeranylcysteine; D012436:S-Adenosylmethionine; D019204:GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D003545:Cysteine