Open AccessTopology of Mammalian Isoprenylcysteine Carboxyl Methyltransferase Determined in Live Cells with a Fluorescent Probe
Author(s) -
Latasha Wright,
Helen Court,
Adam Mor,
Ian M. Ahearn,
Patrick J. Casey,
Mark R. Philips
Publication year - 2009
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.01719-08
Subject(s) - biology , endoplasmic reticulum , methyltransferase , transmembrane domain , prenylation , biochemistry , transmembrane protein , cytoplasm , enzyme , saccharomyces cerevisiae , cytosol , transferase , membrane protein , microbiology and biotechnology , amino acid , membrane , yeast , gene , receptor , methylation
Isoprenylcysteine carboxyl methyltransferase (Icmt) is a highly conserved enzyme that methyl esterifies the α 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). TheSaccharomyces 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.