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 Noxide 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.

The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C 6 -NBD-PC) and phosphatidylethanolamine (M-C 6 -NBD-PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M-C 6 -NBD-PC was internalized predominantly to the vacuole and degraded. M-C 6 -NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2°C, both were internalized to the nuclear envelope/ ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C 6 -NBD-PC to and degradation in the vacuole was blocked. Internalization of neither phospholipid was reduced in the endocytosis-defective mutant, end4-1. However, following pre-incubation at 37°C, internalization of both phospholipids was inhibited at 2°C and 37°C in sec mutants defective in vesicular traffic. The sec18/ NSF mutation was unique among the sec mutations in further blocking M-C 6 -NBD-PC translocation to the vacuole suggesting a dependence on membrane fusion. Based on these and previous observations, we propose that M-C 6 -NBD-PC and M-C 6 -NBD-PE are transported across the plasma membrane to the cytosolic leaflet by a protein-mediated, energy-dependent mechanism. From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria. Subsequently, M-C 6 -NBD-PC, but not M-C 6 -NBD-PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases. An asymmetric distribution of phospholipids between the two leaflets of the plasma membrane was first established in the early 1970s. Chemical modification (1) and enzymatic reaction (2) of phospholipids exposed to the exoplasmic surface of erythrocytes established that essentially all of the phosphatidylserine (PtdSer) and most of the phosphatidylethanolamine (PtdEtn) reside on the cytoplasmic leaflet, whereas the majority of phosphatidylcholine (PtdCho) and sphingomyelin (SM) are exposed to the exoplasmic surface [reviewed in (3 -5)]. This asymmetry was subsequently shown to be dynamic and regulated. ATP-dependent translocation of PtdSer and PtdEtn from the outer to inner bilayer leaflet (aminophospholipid translocase) was proposed to account for the accumulation of these phospholipids in the cytoplasmic leaflet (6 -9). On the other hand, a Ca 2 + -dependent, non-specific transporter (scramblase in erythrocytes) was proposed to allow phospholipids to equilibrate between the two leaflets resulting in an increased proportion of PtdSer and PtdEtn in the exoplasmic leaflet and PtdCho in the cytoplasmic leaflet (10-12).Quantitative measurement of the phospholipid transbilayer distribution in nucleated cells is confounded by the presence of intracellular membranes. However, the general features of the distribution established in erythrocytes are thought to be present in most eukaryotes [reviewed in (4,5)] based on the presence of aminophospholipid translocase-like activity (13-20) and scramblase-like activity (...

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Ceramide is produced by the condensation of a long chain base with a very long chain fatty acid. In Saccharomyces cerevisiae, one of the two major long chain bases is called phytosphingosine (PHS). PHS has been shown to cause toxicity in tryptophan auxotrophic strains of yeast because this bioactive ceramide precursor causes diversion of the high affinity tryptophan permease Tat2 to the vacuole rather than the plasma membrane. Loss of the integral membrane protein Rsb1 increased PHS sensitivity, which was suggested to be due to this protein acting as an ATP-dependent long chain base efflux protein.More recent experiments demonstrated that loss of the genes encoding the ATP-binding cassette transporter proteins Pdr5 and Yor1 elevated PHS tolerance. This increased resistance was suggested to be due to increased expression of RSB1. Here, we provide an alternative view of PHS resistance influenced by Rsb1 and Pdr5/Yor1. Rsb1 has a seven-transmembrane domain topology more consistent with that of a regulatory protein like a G-protein-coupled receptor rather than a transporter. Importantly, an rsb1⌬ cell does not exhibit higher internal levels of PHS compared with isogenic wild-type cells. However, tryptophan transport is increased in pdr5⌬ yor1 strains and reduced in rsb1⌬ cells. Localization and vacuolar degradation of Tat2 are affected in these genetic backgrounds. Finally, internalization of FM4-64 dye suggests that loss of Pdr5 and Yor1 slows normal endocytic rates. Together, these data argue that Rsb1, Pdr5, and Yor1 regulate the endocytosis of Tat2 and likely other membrane transporter proteins.Sphingolipids represent one of the major components of the lipid fraction of the eukaryotic plasma membrane. Biosynthesis of these lipids proceeds through production of ceramide that is formed from the linkage of a long chain base (LCB) 4 with a very long chain fatty acid. In the yeast Saccharomyces cerevisiae, one of the two LCBs produced in vivo is referred to as phytosphingosine (PHS) (for reviews see Refs.1, 2). PHS is required for sphingolipid production but also has regulatory properties in terms of subcellular localization of proteins. Elevated levels of PHS cause mislocalization of nutrient permeases from the plasma membrane to the vacuole where these proteins are degraded (3, 4). Regulation of PHS levels in the cell is tightly controlled and important to ensure normal metabolism.One of the best described routes of PHS degradation is provided by the LCB-phosphate lyase Dpl1 (5). This enzyme breaks LCB phosphate into an aldehyde and ethanolamine phosphate limiting accumulation of LCBs. Strains lacking Dpl1 are hypersensitive to PHS. This phenotype was exploited to identify an integral membrane protein designated Rsb1 that, when overproduced, suppressed the PHS hypersensitivity of a dpl1⌬ strain (6). Evidence was presented that elevated levels of Rsb1 led to increased LCB efflux. More recent work demonstrated that loss of the multidrug transporters Pdr5 and Yor1 from cells led to a strong increase in PHS tolerance...

The anticancer ruthenium complex trans-[tetrachlorobis(1H-indazole)ruthenate(III)], otherwise known as KP1019, has previously been shown to inhibit proliferation of ovarian tumor cells, induce DNA damage and apoptosis in colon carcinoma cells, and reduce tumor size in animal models. Notably, no doselimiting toxicity was observed in a Phase I clinical trial. Despite these successes, KP1019's precise mechanism of action remains poorly understood. To determine whether Saccharomyces cerevisiae might serve as an effective model for characterizing the cellular response to KP1019, we first confirmed that this drug is internalized by yeast and induces mutations, cell cycle delay, and cell death. We next examined KP1019 sensitivity of strains defective in DNA repair, ultimately showing that rad1D, rev3D, and rad52D yeast are hypersensitive to KP1019, suggesting that nucleotide excision repair (NER), translesion synthesis (TLS), and recombination each play a role in drug tolerance. These data are consistent with published work showing that KP1019 causes interstrand cross-links and bulky DNA adducts in mammalian cell lines. Published research also showed that mammalian cell lines resistant to other chemotherapeutic agents exhibit only modest resistance, and sometimes hypersensitivity, to KP1019. Here we report similar findings for S. cerevisiae. Whereas gain-of-function mutations in the transcription activator-encoding gene PDR1 are known to increase expression of drug pumps, causing resistance to structurally diverse toxins, we now demonstrate that KP1019 retains its potency against yeast carrying the hypermorphic alleles PDR1-11 or PDR1-3. Combined, these data suggest that S. cerevisiae could serve as an effective model system for identifying evolutionarily conserved modulators of KP1019 sensitivity.