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Molecular and Cellular Biology, April 2009, p. 1826-1833, Vol. 29, No. 7
0270-7306/09/$08.00+0     doi:10.1128/MCB.01719-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Latasha P. Wright,1,{dagger} Helen Court,1,{dagger} Adam Mor,1 Ian M. Ahearn,1 Patrick J. Casey,2 and Mark R. Philips1*

NYU Cancer Institute and Departments of Medicine, Cell Biology, and Pharmacology, NYU School of Medicine, New York, New York 10016,1 Department of Pharmacology and Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 277102

Received 8 November 2008/ Returned for modification 19 December 2008/ Accepted 9 January 2009

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.

* Corresponding author. Mailing address: NYU Cancer Institute, Smilow 1205, NYU School of Medicine, 522 First Avenue, New York, NY 10016. Phone (212) 263-7404. Fax: (212) 263-3707. E-mail: philim01{at}med.nyu.edu

{triangledown} Published ahead of print on 21 January 2009.

{dagger} L.P.W. and H.C. are co-first authors.

Molecular and Cellular Biology, April 2009, p. 1826-1833, Vol. 29, No. 7
0270-7306/09/$08.00+0     doi:10.1128/MCB.01719-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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