Topology of mammalian isoprenylcysteine carboxyl methyltransferase determined in live cells with a fluorescent probe
Wright, L.P Court, H Mor, A Ahearn, I.M Casey, P.J Philips, M.R
Wright, L.P
Court, H
Mor, A
Ahearn, I.M
Philips, M.R
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Alternative Title
Abstract
Isoprenylcysteine carboxyl methyltransferase (Icmt) is a highly conserved enzyme that methyl esterifies the a 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. Copyright © 2009, American Society for Microbiology.
Keywords
amino acid, cyan fluorescent protein, protein s isoprenylcysteine o methyltransferase, asparagine, fluorescent dye, mutant protein, proline, protein methyltransferase, protein s isoprenylcysteine o methyltransferase, protein-S-isoprenylcysteine O-methyltransferase, animal cell, article, controlled study, cytoplasm, cytosol, embryo, endoplasmic reticulum, enzyme activity, enzyme localization, human, human cell, nonhuman, priority journal, protein structure, amino acid substitution, animal, cell strain COS1, cell survival, Cercopithecus, chemistry, enzymology, glycosylation, kinetics, metabolism, nucleotide sequence, protein secondary structure, protein tertiary structure, Mammalia, Saccharomyces cerevisiae, Amino Acid Substitution, Animals, Asparagine, Cell Survival, Cercopithecus aethiops, Conserved Sequence, COS Cells, Cytosol, Endoplasmic Reticulum, Fluorescent Dyes, Glycosylation, Humans, Kinetics, Mutant Proteins, Proline, Protein Methyltransferases, Protein Structure, Secondary, Protein Structure, Tertiary
Source Title
Molecular and Cellular Biology
Publisher
Series/Report No.
Collections
Rights
Attribution 4.0 International
Date
2009
DOI
10.1128/MCB.01719-08
Type
Article