Topology of mammalian isoprenylcysteine carboxyl methyltransferase determined in live cells with a fluorescent probe.
Isoprenylcysteine carboxyl methyltransferase (Icmt) is a highly conserved enzyme that methyl esterifies the alpha carboxyl group of prenylated proteins including Ras and related GTPases. Methyl esterification neutralizes the negative charge of the prenylcysteine and thereby increases membrane affinity. Icmt is an integral membrane protein restricted to the endoplasmic reticulum (ER). The Saccharomyces cerevisiae ortholog, Ste14p, traverses the ER membrane six times. We used a novel fluorescent reporter to map the topology of human Icmt in living cells. Our results indicate that Icmt traverses the ER membrane eight times, with both N and C termini disposed toward the cytosol and with a helix-turn-helix structure comprising transmembrane (TM) segments 7 and 8. Several conserved amino acids that map to cytoplasmic portions of the enzyme are critical for full enzymatic activity. Mammalian Icmt has an N-terminal extension consisting of two TM segments not found in Ste14p and therefore likely to be regulatory. Icmt is a target for anticancer drug discovery, and these data may facilitate efforts to develop small-molecule inhibitors.
Wright
Latasha P
LP
NYU Cancer Institute and Departments of Medicine, Cell Biology, and Pharmacology, NYU School of Medicine, New York, New York 10016, USA.
Court
Helen
H
Mor
Adam
A
Ahearn
Ian M
IM
Casey
Patrick J
PJ
Philips
Mark R
MR
D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D003600:Cytosol / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005456:Fluorescent Dyes / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D011496:Protein Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D005456:Fluorescent Dyes; D050505:Mutant Proteins; D001216:Asparagine; D011392:Proline; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase