Lem3p is essential for the uptake and potency of alkylphosphocholine drugs, edelfosine and miltefosine.
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.
Hanson
Pamela K
PK
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. phanson@bsc.edu
Malone
Lynn
L
Birchmore
Jennifer L
JL
Nichols
J Wylie
JW
D000483:Alleles; D000981:Antiprotozoal Agents / Q000494:pharmacology; D001692:Biological Transport; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D018432:Drug Resistance, Multiple; D004705:Endocytosis; D017353:Gene Deletion; D007654:Ketoconazole / Q000494:pharmacology; D007896:Leishmaniasis / Q000188:drug therapy; D050356:Lipid Metabolism; D008244:Lysophosphatidylcholines / Q000493:pharmacokinetics*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010742:Phospholipid Ethers / Q000493:pharmacokinetics*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D013997:Time Factors
D000981:Antiprotozoal Agents; C478771:Lem3 protein, S cerevisiae; D008244:Lysophosphatidylcholines; D026901:Membrane Transport Proteins; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D010726:Phosphodiesterase Inhibitors; D010742:Phospholipid Ethers; D029701:Saccharomyces cerevisiae Proteins; D010767:Phosphorylcholine; C026659:edelfosine; C039128:miltefosine; D003513:Cycloheximide; D007654:Ketoconazole