Trypanosoma brucei prenylated-protein carboxyl methyltransferase prefers farnesylated substrates.
Carboxyl methylation of the C-terminal prenylated cysteine, which occurs in most farnesylated and geranylgeranylated proteins, is a reversible step and is implicated in the regulation of membrane binding and cellular functions of prenylated proteins such as GTPases. The gene coding for prenylated-protein carboxyl methyltransferase (PPMT) of the protozoan parasite Trypanosoma brucei has been cloned and expressed in the baculovirus/Sf9 cell system. The protein of 245 amino acids has 24-28% sequence identity to the orthologues from other species including human and Saccharomyces cerevisiae. Methyltransferase activity was detected in the membrane fraction from Sf9 cells infected with the recombinant baculovirus using N -acetyl- S -farnesylcysteine (AFC) and S -adenosyl[ methyl -(3)H]methionine ([(3)H]AdoMet) as substrates. Recombinant T. brucei PPMT prefers AFC to N -acetyl- S -geranylgeranylcysteine (AGGC) by 10-50-fold based on the V (max)/ K (m) values. Native PPMT activity detected in the membrane fraction from T. brucei procyclics displays similar substrate specificity ( approximately 40-fold preference for AFC over AGGC). In contrast, mouse liver PPMT utilizes both AFC and AGGC as substrates with similar catalytic efficiencies. Several cellular proteins of the T. brucei bloodstream form were shown to be carboxyl methylated in a cell-free system. Incorporation of [(3)H]methyl group from [(3)H]AdoMet into most of the proteins was significantly inhibited by AFC but not AGGC at 20 microM, suggesting that T. brucei PPMT acts on farnesylated proteins in the cell. Cells of the T. brucei bloodstream form show higher sensitivity to AFC and AGGC (EC(50)=70-80 microM) compared with mouse 3T3 cells (EC(50)>150 microM).
Department of Medicine, University of Washington, Seattle, WA 98195, USA.
Frederick S
FS
Buckner
David P
DP
Kateete
George W
GW
Lubega
Wesley C
WC
Van Voorhis
Kohei
K
Yokoyama
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011496:Protein Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D017931:DNA Primers; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase