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J. Biol. Chem., Vol. 278, Issue 38, 36041-36050, September 19, 2003

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Lem3p Is Essential for the Uptake and Potency of Alkylphosphocholine Drugs, Edelfosine and Miltefosine^*

Pamela K. Hanson , Lynn Malone, Jennifer L. Birchmore and J. Wylie Nichols 

From the Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322

The alkylphosphocholine class of drugs, including edelfosine and miltefosine, has recently shown promise in the treatment of protozoal and fungal diseases, most notably, leishmaniasis. One of the major barriers to successful treatment of these infections is the development of drug resistance. To understand better the mechanisms underlying the development of drug resistance, we performed a combined mutant selection and screen in Saccharomyces cerevisiae, designed to identify genes that confer resistance to the alkylphosphocholine drugs by inhibiting their transport across the plasma membrane. Mutagenized cells were first selected for resistance to edelfosine, and the initial collection of mutants was screened a second time for defects in internalization of a short chain, fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD))-labeled phosphatidylcholine reporter. This approach identified mutations in a single gene, YNL323W/LEM3, that conferred resistance to alkylphosphocholine drugs and inhibited internalization of NBD-labeled phosphatidylcholine. Loss of YNL323W/LEM3 does not confer resistance to N-nitroquinilone N-oxide or ketoconazole and actually increases sensitivity to cycloheximide. The defect in internalization is specific to NBD-labeled phosphatidylcholine and phosphatidylethanolamine. Labeled phosphatidylserine is internalized at normal levels in lem3 strains. LEM3 is a member of an evolutionarily conserved family and has two homologues in S. cerevisiae. Single point mutations that produce resistance to alkylphosphocholine drugs and inhibition of NBD-labeled phosphatidylcholine internalization were identified in several highly conserved domains. These data demonstrate a requirement for Lem3p expression for normal phosphatidylcholine and alkylphosphocholine drug transport across the plasma membrane of yeast.

Received for publication, May 20, 2003 , and in revised form, June 27, 2003.

^* This work was supported in part by a National Institutes of Health Grant GM64770 and a grant from the University Research Committee of Emory University (to J. W. N.) and a National Institutes of Health predoctoral training grant (to P. K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Birmingham-Southern College, Box 549022, 900 Arkadelphia Rd., Birmingham, AL 35254. Tel.: 205-226-4881; E-mail: phanson{at}bsc.edu.

To whom correspondence should be addressed: Dept. of Physiology, 605G Whitehead Bldg., 615 Michael St., Emory University School of Medicine, Atlanta, GA 30322. Tel.: 404-727-7422; Fax: 404-727-2648; E-mail: wnichols{at}physio.emory.edu.

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