The putative aminophospholipid translocases, DNF1 and DNF2, are not required for 7-nitrobenz-2-oxa-1,3-diazol-4-yl-phosphatidylserine flip across the plasma membrane of Saccharomyces cerevisiae.

The regulation of phosphatidylserine (PS) distribution across the plasma membrane of eukaryotic cells has been implicated in numerous cell functions (e.g. apoptosis and coagulation). In a recent study, fluorescent phospholipids labeled in the acyl chain with 7-nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD) were used to identify two members of the P4 subfamily of P-type ATPases, Dnf1p and Dnf2p, that are necessary for the inward-directed transport of phospholipids across the plasma membrane (flip) of yeast ( Pomorski, T., Lombardi, R., Riezman, H., Devaux, P. F., Van Meer, G., and Holthuis, J. C. (2003) Mol. Biol. Cell 14, 1240-1254 ). Herein, we present evidence that the flip of NBD-labeled PS (NBD-PS) across the plasma membrane does not require the expression of Dnf1p or Dnf2p. In strains in which DNF1 and DNF2 are both deleted, the flip of NBD-PS is increased approximately 2-fold over that of the isogenic parent strain, whereas the flip of NBD-labeled phosphatidylcholine and NBD-labeled phosphatidylethanolamine are reduced to approximately 20 and approximately 50%, respectively. The mechanism responsible for NBD-PS flip is similar to that for NBD-labeled phosphatidylcholine and NBD-labeled phosphatidylethanolamine in its dependence on cellular ATP and the plasma membrane proton electrochemical gradient, as well as its regulation by the transcription factors Pdr1p and Pdr3p. Based on the observation that deletion or inactivation of all four members of the DRS2/DNF essential subfamily of P-type ATPases does not affect NBD-PS flip, we conclude that the activity reflected by NBD-PS internalization is not the essential function of the DRS2/DNF subfamily of P-type ATPases.

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.