Topology of Mammalian Isoprenylcysteine Carboxyl Methyltransferase Determined in Live Cells with a Fluorescent Probe

Abstract

Isoprenylcysteine carboxyl methyltransferase (Icmt) is a highly conserved enzyme that methyl esterifies the ␣ 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.

Mutational Analysis of the Integral Membrane Methyltransferase Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Reveals Potential Substrate Binding Sites

“…10 l of streptavidin beads (25 l for reactions using (43) and are also based on hydropathy plots and experimental mapping of Hs ICMT (19), where solid black rectangles represent regions predicted to reside in the membrane and open rectangles indicate helical regions in the cytosol. The Ma MTase helices (gray rectangles) and transmembrane helices (solid gray) were determined from the crystal structure (20).…”

“…2). With the exception of Trp-240 (Trp-241 in Hs ICMT) (19), these residues have not been previously implicated in ICMT function. These mutants were not inhibited by elevated concentrations of AdoMet (50 M) (Fig.…”

“…Studies addressing the topology of the enzyme have suggested that Homo sapiens ICMT (Hs ICMT) contains eight transmembrane helices (TMs) and Saccharomyces cerevisiae ICMT (Sc ICMT) has six TMs ( Fig. 2) (18,19). Plant orthologs (e.g.…”

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

“…Human and beetle ICMT share the same predicted topology 9 and have 58% amino acid sequence identity within the region thought to contain the active site (amino acids 90–281) (Extended Data Fig. 1) 10 .…”

“…The structure can be compared directly to a large body of functional data on ICMT 9,10,12,16 . Plotting residues that disrupt catalytic activity when mutated on the structure identifies the active site (Fig.…”