The development of MDR (multidrug resistance) in yeast is due to a number of mechanisms. The most documented mechanism is enhanced extrusion of drugs mediated by efflux pump proteins belonging to either the ABC (ATP-binding cassette) superfamily or MFS (major facilitator superfamily). These drug-efflux pump proteins are localized on the plasma membrane, and the milieu therein affects their proper functioning. Several recent studies demonstrate that fluctuations in membrane lipid composition affect the localization and proper functioning of the MDR efflux pump proteins. Interestingly, the efflux pumps of the ABC superfamily are particularly susceptible to imbalances in membrane-raft lipid constituents. This review focuses on the importance of the membrane environment in functioning of the drug-efflux pumps and explores a correlation between MDR and membrane lipid homoeostasis.

INTRODUCTION

In both pathogenic (Candida sp., Aspergillus and Cryptococcus) and non-pathogenic (Saccharomyces cerevisiae) yeast, several mechanisms can contribute to the development of MDR (multidrug resistance). Some of the most common strategies employed by drug-resistant yeast include mutation or overexpression of the drug target, decrease in the import of drugs and enhanced efflux of drugs etc. [13]. Extrusion of noxious compounds from the cell by efflux pumps is one of the most frequently used strategies for the development of drug resistance in yeast, and it holds true for several others [46].

Efflux pump proteins of the ABC (ATP-binding cassette) superfamily and MFS (major facilitator superfamily) of transporters are common exporters of structurally unrelated drugs. The ABC transporters use the energy derived from ATP hydrolysis to power the efflux, whereas the MFS transporters make use of proton gradient across the plasma membrane for the extrusion of drugs. Genomic analyses of S. cerevisiae and of the pathogenic yeast Candida albicans reveal the existence of 30 and 28 putative ABC transporters respectively, of which only a few have been found to function as drug transporters [7,8].

In cancer cell lines, the overexpression of human Pgp (P-glycoprotein)/MDR1, a homologue of yeast ABC proteins, is invariably associated with the development of drug resistance, which has been the major cause of failure of cancer chemotherapy [4]. Interestingly, human Pgp/MDR1 is predominantly localized in plasma-membrane domains enriched in cholesterol (mammalian sterol), and the depletion of cholesterol impairs drug transport in a substrate- and cell-type-specific manner [9]. It has also been observed that human Pgp/MDR1 contributes to stabilizing the cholesterol-rich microdomains by mediating cholesterol redistribution within the cell membrane [10]. It has been suggested that the activities of the yeast efflux pumps, particularly those that belong to the ABC superfamily, are also influenced by subtle modifications in membrane lipid composition [1113]. For example, fluctuations in sterol level, particularly, result in destabilization of the membrane and a decrease in drug resistance of yeast cells [1113]. These observations acquire significance with the reported existence, within the lipid bilayer, of discrete microdomains in yeast membranes (lipid rafts), predominantly composed of sphingolipids and sterols [14,15]. Of note, the acquisition of the MDR phenotype in certain mammalian cell lines also is accompanied by an up-regulation of genes encoding proteins and the metabolism of lipids that constitute membrane rafts and caveolar membranes [16]. Coupled together, it appears that membrane sphingolipids and sterols, both individually and through their mutual interactions, can critically affect the functioning of drug-efflux pump proteins. This review focuses on the roles of these important membrane microdomain lipids in influencing the function of drug-efflux pumps in yeast. The discussion on the role of raft lipids is preceded by a short account of ABC and MFS drug-transporter proteins in yeast.

YEAST MULTIDRUG TRANSPORTERS

ABC multidrug-efflux proteins

Yeast ABC proteins are generally made up of two TMDs (transmembrane domains) and two cytoplasmically located NBDs (nucleotide-binding domains), although putative ‘half-proteins’, which probably dimerize to become fully functional, are also known to exist in yeast [7,8]. These half-protein transporters in some prokaryotes are known to function as MDR pumps (Figure 1) [17,18]. Pdr5p, Snq2p (sensitivity to 4-nitroquinoline-N-oxide 2) and Yor1p (yeast oligomycin resistance 1) are the major full drug transporters in S. cerevisiae, whereas Cdr1p and Cdr2p (Pdr5p homologues) contribute to drug resistance in C. albicans [1920d]. Typically, each of the two TMDs of full ABC proteins comprise six transmembrane-spanning segments (TMSs) that are preceded or followed by the NBDs (NBD–TMS2) or TMS–NBD2 (Figure 1). Although it appears that several TMSs come together to form the substrate-binding site(s), this alone does not appear to be sufficient for drug transport across the membrane bilayer. Given their varied roles and the different structural characteristics of substrates that members of ABC superfamily appear to efflux, it is hardly surprising that, despite an overall conservation of the domain architecture of TMDs, their primary sequences are significantly different. On the other hand, the NBDs of ABC transporters, which power drug transport, are highly conserved, both in terms of primary structure and architecture (Figure 2) [21]. TMDs and NBDs, together, form the minimal functional unit necessary for substrate transport. Unlike most other ABC transporters, the NBDs of fungal transporters have unique positioning of typical, but critical, amino-acid residues within the conserved N-terminal NBD domain, such as in the Walker A and Walker B motifs (Figure 2). On the other hand, the C-terminal NBD of fungal ABC transporters have fully conserved motifs, which are essentially identical to motifs in non-fungal transporters [22]. The structural and functional analyses of human Pgp/MDR1 and its other homologues have demonstrated the importance of NBDs and TMDs in drug extrusion [23]. In comparison, studies pertaining to the identification of the molecular determinants of yeast ABC drug transporters have only been initiated recently [22,24].

Predicted topology of the yeast ABC proteins

Figure 1
Predicted topology of the yeast ABC proteins

The various ABC proteins are divided into subfamilies. Full transporters have 12 TMSs and two NBDs. Half transporters have six TMSs and a single NBD. The NBDs are the ATP-binding domains comprising Walker A, signature sequence and Walker B motifs, in the order mentioned. Key proteins from both S. cerevisiae and C. albicans are listed in respective panels (for more details, see references [7,8]). ALDP, adrenoleukodystrophy protein; CFTR, cystic fibrosis transmembrane conductance regulator; EF3/RL1, elongation factor 3/RNase L inhibitor.

Figure 1
Predicted topology of the yeast ABC proteins

The various ABC proteins are divided into subfamilies. Full transporters have 12 TMSs and two NBDs. Half transporters have six TMSs and a single NBD. The NBDs are the ATP-binding domains comprising Walker A, signature sequence and Walker B motifs, in the order mentioned. Key proteins from both S. cerevisiae and C. albicans are listed in respective panels (for more details, see references [7,8]). ALDP, adrenoleukodystrophy protein; CFTR, cystic fibrosis transmembrane conductance regulator; EF3/RL1, elongation factor 3/RNase L inhibitor.

Structural organization of the CaCdr1p of ABC superfamily in C. albicans

Figure 2
Structural organization of the CaCdr1p of ABC superfamily in C. albicans

The cartoon (top) shows the presence of 12 TMSs and two NBDs for Cdr1p. The conserved Walker A, Walker B and signature sequences are enlarged and aligned with conserved sequences of other fungal and non-fungal ABC transporters to show the degree of similarity and unique placement of certain amino acids. CFTR, cystic fibrosis transmembrane conductance regulator; N, N-terminal. A. Fumigatus, Aspergillus fumigatus; A. Nidulans, Aspergillus nidulans; A. Radiobacter, Agrobacterium radiobacter; C. Galbrata, Candida glabrata; C. Neoformans, Cryptococcus neoformans; E. coli, Escherichia coli; H. Sapiens, Homo sapiens; S. Typhimurium, Salmonella typhimurium.

Figure 2
Structural organization of the CaCdr1p of ABC superfamily in C. albicans

The cartoon (top) shows the presence of 12 TMSs and two NBDs for Cdr1p. The conserved Walker A, Walker B and signature sequences are enlarged and aligned with conserved sequences of other fungal and non-fungal ABC transporters to show the degree of similarity and unique placement of certain amino acids. CFTR, cystic fibrosis transmembrane conductance regulator; N, N-terminal. A. Fumigatus, Aspergillus fumigatus; A. Nidulans, Aspergillus nidulans; A. Radiobacter, Agrobacterium radiobacter; C. Galbrata, Candida glabrata; C. Neoformans, Cryptococcus neoformans; E. coli, Escherichia coli; H. Sapiens, Homo sapiens; S. Typhimurium, Salmonella typhimurium.

MFS multidrug-efflux proteins

The MFS was originally defined as a superfamily of permeases that are characterized by two structural units of six TMS–α-helical segments, which are linked by a cytoplasmic loop. It consists of evolutionary conserved membrane transport proteins involved in the symport, antiport or uniport of various substrates [2529]. One major cluster of this family consists of the PMF (proton motive force)-dependent drug-efflux proteins and some substrate-specific drug-efflux proteins, such as the well-studied tetracycline exporter, TetB [3033]. The phylogenic analyses of MFS drug exporters show that its members possess either 12 or 14 TMSs [34].

MFS proteins also function as major drug transporters, which are involved in drug efflux, thus contributing to MDR in yeast. Similar to the ABC protein superfamily, very few members of the MFS family are drug exporters. For example, in a total of 62 proteins, only FLR1 in S. cerevisiae (fluconazole resistance) has been shown to confer resistance to fluconazole, 4-NQO (4-nitroquinoline-N-oxide), cycloheximide, benomyl, methotrexate, cerulenin and diazaborine etc. [35]. Also, in the pathogenic C. albicans, from a total of 71 MFS proteins, only CaMDR1 (C. albicans MDR1) is known to extrude drugs, where its overexpression has been linked to azole resistance in clinical isolates [3639]. FLU1 (fluconazole resistance), another member of the MFS of C. albicans, was cloned by functional complementation of a fluconazole-sensitive strain of S. cerevisiae. However, FLU1 is not involved in the development of fluconazole resistance in clinical isolates of C. albicans. Interestingly, studies revealed that the preferred substrate of Flu1p is mycophenolic acid, rather than fluconazole [40].

The antiporter motif in the predicted TMS5 of MFS transporters is well conserved in all of the functionally related subgroups of bacteria and plants [29,34,41]. MFS drug exporters in yeast also possess this motif. Alanine scanning of all the 21 amino acids of TMS5 of CaMdr1p has recently highlighted the residues that probably contribute to the efflux activity of CaMdr1p [38] (Figure 3). Additionally, there are many conserved motifs present within TMDs of MFS proteins which may contribute to drug specificities [34]. Multiple-sequence analyses of the MFS proteins suggest that proteins within this family share greater similarity between their N-terminal domains than in their C-terminal domains, which is consistent with the hypothesis that the latter domain is responsible for substrate recognition [34].

Structural organization of the CaMdr1p of MFS superfamily in C. albicans

Figure 3
Structural organization of the CaMdr1p of MFS superfamily in C. albicans

The ‘antiporter motif’ or ‘motif C’ in the transmembrane helix V is enlarged and aligned with other drug transporters to show the degrees of similarity. Right-hand panel: antiporter activity of CaMdr1p. B. subtilis, Bacillus subtilis; C. dubliensis, Candida dubliensis; C. maltosa, Candida maltosa; C. tropicalis, Candida tropicalis; E. coil, Escherichia coli; S. aureus, Staphylococcus aureus.

Figure 3
Structural organization of the CaMdr1p of MFS superfamily in C. albicans

The ‘antiporter motif’ or ‘motif C’ in the transmembrane helix V is enlarged and aligned with other drug transporters to show the degrees of similarity. Right-hand panel: antiporter activity of CaMdr1p. B. subtilis, Bacillus subtilis; C. dubliensis, Candida dubliensis; C. maltosa, Candida maltosa; C. tropicalis, Candida tropicalis; E. coil, Escherichia coli; S. aureus, Staphylococcus aureus.

MDR AND LIPIDS

On the basis of several studies, a close interaction between membrane lipids and drug-extrusion pump proteins has been recognized [1113,42]. On the one hand, it has been observed that the ABC drug-efflux proteins in human (Pgp/MDR1) and yeast (Pdr5p and Yor1p in S. cerevisiae, and CaCdr1p and CaCdr2p in C. albicans) can translocate phospholipids between the two monolayers of the plasma membrane, whereas, on the other hand, human Pgp/MDR1 is also known to participate in sterol homoeostasis [43,44]. Additionally, these drug-extrusion pumps are found to be particularly sensitive to the nature and the physical state of the surrounding lipids [1113,42]. For example, both CaCdr1p and Pdr5p are sensitive to fluctuations in the lipid environment where selective functions mediated by these drug-extrusion pumps are affected [11,13,34].

Ergosterol levels affect drug susceptibilities

Various studies employing erg (enzymes involved in ergosterol biosynthesis) mutants of S. cerevisiae and C. albicans have revealed a close relationship between MDR and sterol levels of the plasma membrane [13,42]. In azole-resistant yeast, a decreased level of ergosterol and increased membrane fluidity is commonly observed [45]. Further investigations confirmed a correlation between drug susceptibility and an altered membrane composition [11,12,42]. The increased drug susceptibility of the erg mutants was attributed to an increased membrane fluidity, which leads to an enhanced passive diffusion of drug molecules across the plasma membrane [11,42,47]. The lack of ergosterol in the erg mutants could also destabilize or disrupt the ergosterol-rich microdomains, thus affecting membrane organization and function of drug-export proteins that are preferentially localized within these domains [11,12].

Sphingolipid levels affect drug susceptibilities

The early steps in mammalian and fungal sphingolipid synthesis are conserved, but they diverge later in the pathway to produce structurally and chemically different types of sphingoid bases, ceramides and complex sphingolipids [49,50]. Therefore, over the years, the sphingolipid biosynthetic pathway has been exploited as an antifungal drug target in pathogenic yeast [51,52]. Unlike mammals, fungi do not have phosphatidylcholine as part of their polar head group in sphingolipids; instead they have phosphoinositol which is transferred on to ceramide to make IPC (inositol phosphoceramide) [53,54]. IPC is further modified by the addition of mannose to produce MIPC (mannosyl IPC), and the addition of a second inositol phosphate to make M(IP)2C (mannosyl bi-inositol diphosphoceramide) [53,55]. Although the synthesis of these sphingolipids is critical to maintain normal plasma-membrane function, synthesis of M(IP)2C is not critical for viability [55]. In S. cerevisiae, the IPT1 (inositol phosphotransferase 1) deletion mutant grows normally, but displays sensitivity to calcium and increased resistance to the polyene antibiotic nystatin [55]. Hallstrom et al. [56] observed that loss of IPT1 has complex effects on drug resistance in S. cerevisiae, mediated through Pdr1p and Pdr3p TFs (transcription factors), which regulate MDR genes in S. cerevisiae (see below). In order to explore the role of sphingolipids, a specific inhibitor fumonisin B1, which blocks the synthesis of phytoceramide, a precursor for the three major sphingolipid species was employed [11]. The study revealed a close interaction between plasma membrane ergosterol and sphingolipids in C. albicans cells [11]. The depletion of either of the two impaired the function of a major drug-efflux pump Cdr1p, which resulted in the Candida cells becoming hypersensitive to several drugs. In another study, when sphingolipid synthesis was specifically blocked by homozygous disruption of the CaIPT1 gene, it led to decrease in drug resistance due to altered sphingolipid composition [11]. Besides this, ipt1 mutants of C. albicans were unable to form hyphae. The effect of MIPC accumulation and absence of M(IP)2C in ipt1 mutants on efflux of drug substrates was very selective in terms of the effects on different efflux pump proteins. For example, in comparison with the efflux of fluconazole, a substrate of CaCdr1p, the efflux of methotrexate, a specific substrate of MFS CaMdr1p, remained unaffected in ipt1 mutant cells. Taken together, it appears that similar to ergosterol levels, altered sphingolipid composition, both of which are among the major constituents of membrane rafts, affect drug susceptibilities and morphogenesis in C. albicans.

ERGOSTEROL AND SPHINGOLIPIDS ARE MEMBRANE-RAFT CONSTITUENTS

Yeast sphingolipids and ergosterol are important components of distinct membrane lipid domains known as rafts [14,57,58]. In several organisms, various proteins with diverse functions in cellular processes, such as signal transduction, membrane trafficking, lipid and protein sorting, and even receptors for certain pathogens, ‘home’ to the rafts [60,61]. The presence of rafts offers a platform for the cell to allow interactions between different partners of metabolic cascades, ensuring their efficiency. The existence of rafts acquired significance in the area of MDR due to the finding that 24–40% of human Pgp/MDR1 is present in these detergent-resistant plasma-membrane domains [62]. The presence of lipid rafts has been associated with hyphal growth in C. albicans [14]. In yeast, the mating tips projection ‘shmoos’ are enriched in these microdomains [63,64]. The presence of these domains in the shmoos is probably required for specific interactions between various mating partners or with any other process where cell–cell fusion is necessary [64]. The following part of this review highlights evidence that supports a direct relationship between membrane-raft constituents and MDR, preceded by a short overview of regulation of MDR genes in yeast.

REGULATION OF MDR

The transcriptional activation of MDR genes, leading to overexpression of drug-efflux pumps in the development of azole resistance, is well known in pathogenic yeast [65,66]. However, the mechanisms by which the expression of the MDR genes is altered in clinical azole-resistant Candida isolates are not fully understood. On the other hand, the mechanisms underlying the up-regulation of MDR genes in the development of MDR are relatively well described in S. cerevisiae, wherein three networks of trans-acting factors, such as PDR (pleiotropic drug resistance), YAP1 (yeast activator protein 1)-like factor and YRR (yeast reveromycin resistance), are mainly involved in controlling the expression of MDR genes [2,67,68].

The TFs PDR1 and PDR3 of the zinc-cluster protein family regulate the transcription of genes encoding ABC drug transportes, such as PDR5 and SNQ2 [2,67,68]. YAP-like factor of the bZip family of TFs confers resistance to a variety of toxicants. YAP-like factor 1 targets include an MFS-type drug extrusion pump FLR1 [69] and a glutathione reductase gene, GLR1, involved in conferring oxidative tolerance to yeast cells [70]. Additionally, YAP1 also activates PDR5 expression under stress conditions, such as heat shock [71]. A link between the YAP and PDR networks has also been established in S. cerevisiae [72]. Another zinc-finger-containing TF, YRR1 is involved in complex PDR network regulation by directly activating SNQ2 and YOR1 [73], both of which are also common targets of Pdr1p, Pdr3p and Yap1p [74]. In addition to PDR1, PDR3, YAP1 and YRR1, a transcriptional repressor of PDR, called RDR1 (repressor of drug resistance) [75,76], has been shown to bind to the PDRE (pleiotropic drug-response element), a cis-acting regulatory element on the PDR5 promoter [75]. PDREs are also required for the regulatory actions of Pdr1p and Pdr3p. Furthermore, Pdr1p and Pdr3p have been shown to cross-talk with another TF, Stb5p. Yrr1p, on the other hand, mainly exists as a homodimer. This indicates a complex regulatory circuit that is required for the expression of PDR genes in S. cerevisiae [77].

As mentioned above, the up-regulation of genes encoding drug-extrusion pumps of either the ABC (CaCDR1 and CaCDR2) or MFS (CaMDR1) superfamilies represents one of the most prevalent mechanisms of drug resistance in Candida [78,79]. The various possibilities that may affect the expression of CaCDR1 in azole-resistant clinical isolates of C. albicans include mutations in the promoter region (cis element) of the gene, altered regulation by trans-regulatory factors controlling expression of these genes or molecular changes taking place during mRNA processing [8083]. In one study, the molecular changes responsible for CaMDR1 activation in matched fluconazole-sensitive and -resistant isolates were examined by Wirsching et al. [81]. Since sequence analyses of the CaMDR1 promoter region did not reveal any mutation in the matched pair of fluconazole-sensitive/resistant isolates, it was proposed that the CaMDR1 promoter was probably activated by a trans-regulatory factor(s) that might be mutated in fluconazole-resistant isolates [81]. A mutation in the trans-regulatory factor has been reported as the cause for the activation of the PDR16 gene (phosphatidylinositol inositol transfer protein), which is co-ordinately regulated with CaCDR1 and CaCDR2 in clinical isolates of C. albicans [80]. A search for homologues of Pdr1p/Pdr3p in C. albicans identified regulators that turned out to be negative regulators of genes encoding CaCDR1 and CaCDR2. One such TF, FCR1 (fluconazole resistance 1), was isolated using the strategy of functional complementation in a pdr1/pdr3 mutant strain of S. cerevisiae [84]. Although Fcr1p was able to up-regulate the expression of Pdr5p in S. cerevisiae in a manner similar to Pdr1p/Pdr3p, it acted as a negative regulator of CaCdr1p in C. albicans. Consistent with this result, its deletion made C. albicans cells resistant to fluconazole [84]. This showed that, although the regulators in these two yeast might be orthologues, they have evolved to perform different functions. Another gene responsible for regulating the expression of CaCdr1p is called CaNDT80 (Ca non-dityrosine) [85]. CaNdt80p in S. cerevisiae functions as a regulator of meiosis genes. Interestingly, in C. albicans, it was shown to up-regulate CaCdr1p expression. Furthermore, a Candt80-null mutant was susceptible to antifungals, which was consistent with the abolishment of CaCdr1p expression in this strain. Recently, another novel zinc-finger-containing regulator, Tac1p (transcription activator of Cdr genes 1), was identified as the main positive regulator of CaCdr1p and CaCdr2p [86]. In terms of the drug-response elements that it binds to in the promoters of CaCdr1p and CaCdr2p, it appears to be the closest functional homologue of Pdr1p/Pdr3p [86]. Hyperactive alleles of Tac1p have been isolated from azole-resistant clinical isolates, indicating that, indeed, a mutation in this TF in clinical isolates enables overexpression of genes of Tac1p targets therein [86]. This probably is one of the mechanisms by which C. albicans cells develop azole resistance during prolonged exposure to these antifungals.

In S. cerevisae Ecm22 (extracellular mutant 22) and Upc2p (uptake control 2) are known to be involved in the regulation of genes involved in the ergosterol biosynthesis pathway. CaUpc2p, a homologue of S. cerevisiae Upc2p, in C. albicans has also been shown to regulate ergosterol biosynthetic pathway genes. Interestingly, C. albicans cells with CaUPC2 deleted are susceptible to antifungal therapy [87,88]. This study provides additional evidence of a link between membrane sterols and their effects on function of efflux pumps, leading to sensitivity to antifungals.

MEMBRANE RAFT CONSTITUENTS AND GENES ENCODING DRUG-EFFLUX PUMPS ARE CO-ORDINATELY REGULATED

Microarray analysis has provided an insight into the genes that are co-regulated with the drug-resistance genes [89,90]. This would indicate that these genes, which are co-regulated with MDR genes, either share a common function or common regulatory sequences. The most interesting genes co-ordinately regulated with the MDR genes are those involved in sphingolipid biosynthesis and in lipid metabolism, which contribute to the formation of the membrane rafts [89,90]. As drugs are not a part of the normal cell mileu, this raises the question as to the normal physiological substrates for these efflux pumps. The fact that drug-efflux pump proteins, such as Pdr5p and Yor1p of S. cerevisiae and CaCdr1p and CaCdr2p of C. albicans, are also phospholipid translocators suggests that these pumps might co-ordinate the synthesis and transport of the important lipid constituents of the plasma membrane [92]. Superimposed with these facts, the requirement of lipid rafts for the proper membrane localization of the drug-efflux pump proteins further strengthens the possibility of a connection between the raft constituents and MDR genes, particularly those encoding drug-extrusion pumps. We have demonstrated recently [93] that CaCdr1p is exclusively localized within membrane rafts, whereas CaMdr1p does not show such selectivity (Figure 4). Additionally, any imbalance in lipid metabolism, particularly with sphingolipid or ergosterol synthesis, leads to selective mislocalization of the ABC transporter CaCdr1p [93].

Localization of Cdr1p and CaMdr1p within the plasma membrane

Figure 4
Localization of Cdr1p and CaMdr1p within the plasma membrane

An OptiPrep® gradient showing distribution of the detergent-resistant and detergent-soluble membrane fractions. Membrane lipid rafts are localized in the top two fractions. (B) CaCDR1–GFP (green fluorescent protein) and CaMDR1–GFP were overexpressed in a wild-type S. cerevisiae strain. Immunoblot analysis with anti-GFP antibody of the lipid-raft fractions clearly show the presence of CaCdr1p in the top two floating raft fractions (upper panel). The rest of the panels show the mislocalization of CaCdr1p–GFP in lipid metabolism mutants. (C) The distribution of CaMdr1p in OptiPrep® gradient shows that CaMdr1p is a non-raft protein, the localization of which is not affected in various lipid mutants. fen1, sur4 and ipt1 are enzymes of the sphingolipid biosynthesis pathway, whereas erg24, erg6 and erg4 are enzymes of the ergosterol biosynthesis pathway. WT, wild-type.

Figure 4
Localization of Cdr1p and CaMdr1p within the plasma membrane

An OptiPrep® gradient showing distribution of the detergent-resistant and detergent-soluble membrane fractions. Membrane lipid rafts are localized in the top two fractions. (B) CaCDR1–GFP (green fluorescent protein) and CaMDR1–GFP were overexpressed in a wild-type S. cerevisiae strain. Immunoblot analysis with anti-GFP antibody of the lipid-raft fractions clearly show the presence of CaCdr1p in the top two floating raft fractions (upper panel). The rest of the panels show the mislocalization of CaCdr1p–GFP in lipid metabolism mutants. (C) The distribution of CaMdr1p in OptiPrep® gradient shows that CaMdr1p is a non-raft protein, the localization of which is not affected in various lipid mutants. fen1, sur4 and ipt1 are enzymes of the sphingolipid biosynthesis pathway, whereas erg24, erg6 and erg4 are enzymes of the ergosterol biosynthesis pathway. WT, wild-type.

The first evidence for the existence of such a co-regulation came from the studies performed with cancer cells. In cancer cells, the development of drug resistance involves the up-regulation of human Pgp/MDR1, with a simultaneous overexpression of lipid and protein constituents that are required for the formation of lipid rafts and caveolar membranes [16]. Typical caveolae are defined as 50–100 nm non-clathrin-coated invaginations of the plasma membrane, which are characterized by the presence of an integral membrane protein called caveolin. Drug-resistant cell lines have high levels of caveolin, as compared with drug-sensitive cell lines [16]. Pgp/MDR1 is shown to be associated with caveolin-rich membrane domains, which may be required to ensure its proper functioning.

Although the link between membrane-raft lipids and their impact on drug transporters has been partly evaluated in mammalian systems, such a correlation in yeast is only beginning to be realized. Interestingly, microarray analysis performed with the hyperactive alleles of TFs such as Pdr1p (Pdr1-3) and Pdr3p (Pdr3-7) of S. cerevisiae confirms this assumption [89]. One study showed that a number of genes involved in sphingolipid metabolism and in the homoeostasis of its precursors are up-regulated along with the ABC transporter genes PDR5, SNQ2 and YOR1 [89]. Additionally, PDR16 and IPT1 which have a known role in sphingolipid metabolism and genes such as RTA1 (resistance to 7-aminocholesterol 1) and RSB1 (resistance to sphingoid base 1), which are integral membrane proteins, were also up-regulated along with the ABC transporter genes [9497].

The first experimental evidence demonstrating a link between the PDR pathway and sphingolipid biosynthesis came from studies involving IPT1 in S. cerevisiae [56]. The gene IPT1 catalyses the last step in the sphingolipid biosynthetic pathway in yeast. The transcriptional induction of IPT1 in the presence of hyperactive alleles of Pdr1p and Pdr3p occurs via a single PDRE in its promoter region. An IPT1 null mutant displayed increased resistance to cycloheximide and decreased resistance to oligomycin [56]. This study showed that alterations in the sphingolipid levels in the plasma membrane selectively affect the drug resistance and membrane drug transporters. Interestingly, all the membrane drug-transporter proteins are not necessarily susceptible to membrane lipid fluctuations. For example, it has been observed that the MFS transporter of C. albicans, CaMdr1p, does not respond to lipid changes and its functions remain unaffected, whereas the functions of the ABC transporter CaCdr1p are abrogated under similar conditions [98].

Analysis of other genes of the sphingolipid pathway revealed that LAC (longevity-assurance gene cognate 1) is also responsive to the changes in the activity of the PDR pathway [99]. In the sphingolipid biosynthetic pathway, enzymes such as Dpl1p, ceramidases and transporters, which are responsible for the efflux of LCBs (long-chain bases) out of the cell, comprise of a regulatory circuit that maintains normal concentrations of the LCBs. LCBs are the precursors of LCBPs (LCB phosphates) and ceramide, which promote cell proliferation and trigger apoptosis in the cell. AS LCBPs and ceramide control antagonistic processes, an appropriate balance of LCBP/ceramide is required by the cell [50,101].

RSB1, a recently identified membrane transporter, has been shown to be responsible for the efflux of LCBs in S. cerevisiae (Figure 5). RSB1 was identified on the basis of its ability, when expressed via a high-copy number plasmid, to suppress sensitivity to PHS (phytosphingosine; one of the LCBs) of dpl1Δ cells [97]. The expression of Rsb1p was up-regulated in pdr5 null cells in a Pdr1p-dependent manner [102]. These strains therefore were highly resistant to exogenously added PHS, with a concomitant increase in LCB efflux. Two independent microarray analyses demonstrated that this gene is overexpressed with Pdr5p and other ABC drug transporters in the presence of a hyperactive allele of Pdr1p and in ρ0 cells [89,90]. The role of Pdr5p as a phospholipid translocator and its connection with Rsb1p shows that the PDR pathway probably regulates the transport of phospholipids and LCBs to ensure proper membrane-raft mileu [103]. Kihara and Igarashi [102] showed that there was a reciprocal relationship between Pdr5p and Rsb1p, where Rsb1p is overexpressed in pdr5 null cells. Contradictory to this, another study showed that, although Δpdr5 cells are highly resistant to PHS, it is not necessarily by activation of Rsb1p [104]. This would suggest an independent mechanism is activated in the absence of pdr5 and renders the cells resistant to PHS. The latter study [104] instead points to the fact that both Pdr5p and Rsb1p are up-regulated in ρ0 cells, which places them both in the retrograde regulon in S. cerevisiae. Together, these studies support the idea that the PDR pathway has a major role to play in controlling the lipid mileu in the plasma membrane.

Co-ordinate regulation of the sphingolipid biosynthetic pathway and drug-efflux pumps in S. cerevisiae

Figure 5
Co-ordinate regulation of the sphingolipid biosynthetic pathway and drug-efflux pumps in S. cerevisiae

Genes regulated by Pdr1p/Pdr3p are shown in the sphingolipid biosynthetic pathway. Inhibitors used to block this pathway are shown in grey circles. Rsb1p, a membrane protein that effluxes the LCBs (dihydrosphingosine/DHS and PHS), is also a part of the PDR regulon. The inset shows targets for Pdr1p/Pdr3p, some of which are up-regulated in ρ0 cells. Pdr5p and Yor1p function as phospholipid translocators in S. cerevisiae. PM, plasma membrane.

Figure 5
Co-ordinate regulation of the sphingolipid biosynthetic pathway and drug-efflux pumps in S. cerevisiae

Genes regulated by Pdr1p/Pdr3p are shown in the sphingolipid biosynthetic pathway. Inhibitors used to block this pathway are shown in grey circles. Rsb1p, a membrane protein that effluxes the LCBs (dihydrosphingosine/DHS and PHS), is also a part of the PDR regulon. The inset shows targets for Pdr1p/Pdr3p, some of which are up-regulated in ρ0 cells. Pdr5p and Yor1p function as phospholipid translocators in S. cerevisiae. PM, plasma membrane.

In C. albicans, comparison of gene expression profiles in the azole-resistant clinical isolates and drug (fluphenazine)-induced laboratory strains show that, in addition to the multidrug transporters CaCdr1p and CaCdr2p, genes such as RTA3 (homologue of Rsb1p), IFU5 and HSP12 (heat-shock protein 12) and IPF4065 (putatively involved in stress response) are the most highly expressed genes. A total of 42 genes were commonly regulated when the fluphenazine-exposed cells were compared with the azole-resistant isolates. All the above genes, except for HSP12 and IPF4065, have a drug-response element in their promoter regions and hence are targets for Tac1p [86,105].

The studies conducted so far establish that MDR in yeast is a result of the simultaneous activation of many factors in the cells in response to a drug. While it involves the overexpression of genes encoding drug-efflux pumps, it is also accompanied by additional biochemical changes, which include up-regulation of the lipid-raft constituents. The results discussed above imply that yeast possess a co-ordinated programme for up-regulation of both lipid-raft constituents and drug-efflux pumps.

Abbreviations

     
  • ABC

    ATP-binding cassette

  •  
  • Ca

    Candida albicans

  •  
  • FLR/FLU

    fluconazole resistance

  •  
  • HSP12

    heat-shock protein 12

  •  
  • IPC

    inositol phosphoceramide

  •  
  • IPT

    inositol phosphotransferase

  •  
  • LCB

    long-chain base

  •  
  • LCBP

    LCB phosphate

  •  
  • M(IP)2C

    mannosyl bi-inositol diphosphoceramide

  •  
  • MDR

    multidrug resistance

  •  
  • MFS

    major facilitator superfamily

  •  
  • MIPC

    mannosyl IPC

  •  
  • NBD

    nucleotide-binding domain

  •  
  • NDT

    non-dityrosine

  •  
  • PDR

    pleiotropic drug resistance

  •  
  • PDRE

    pleiotropic drug-response element

  •  
  • Pgp

    P-glycoprotein

  •  
  • RSB

    resistance to sphingoid base

  •  
  • RTA

    resistance to 7-aminocholesterol

  •  
  • SNQ

    sensitivity to 4-nitroquinoline-N-oxide

  •  
  • Tac

    transcription activator of Cdr genes

  •  
  • TF

    transcription factor

  •  
  • TMD

    transmembrane domain

  •  
  • TMS

    transmembrane-spanning segment

  •  
  • Upc

    uptake control

  •  
  • YAP1

    yeast activator protein 1

  •  
  • YOR

    yeast oligomycin resistance

  •  
  • YRR

    yeast reveromycin resistance

References

References
Prasad
 
R.
Gaur
 
N. A.
Gaur
 
M.
Komath
 
S. S.
 
Efflux pumps in drug resistance of Candida
Infect. Disord. Drug Targets
2006
, vol. 
6
 (pg. 
69
-
83
)
Ernst
 
R.
Klemm
 
R.
Schmitt
 
L.
Kuchler
 
K.
 
Yeast ATP-binding cassette transporters: cellular cleaning pumps
Methods Enzymol.
2005
, vol. 
400
 (pg. 
460
-
484
)
Lupetti
 
A.
Danesi
 
R.
Campa
 
M.
Tacca
 
M. D.
Kelly
 
S.
 
Molecular basis of resistance to azole antifungals
Trends Mol. Med.
2002
, vol. 
8
 (pg. 
76
-
81
)
Ambudkar
 
S. V.
Kimchi-Sarfaty
 
C.
Sauna
 
Z. E.
Gottesman
 
M. M.
 
P-glycoprotein: from genomics to mechanism
Oncogene
2003
, vol. 
22
 (pg. 
7468
-
7485
)
Pumbwe
 
L.
Glass
 
D.
Wexler
 
H. M.
 
Efflux pump overexpression in multiple-antibiotic-resistant mutants of Bacteroides fragilis
Antimicrob. Agents Chemother.
2006
, vol. 
50
 (pg. 
3150
-
3153
)
Zgurskaya
 
H. I.
 
Molecular analysis of efflux pump-based antibiotic resistance
Int. J. Med. Microbiol.
2002
, vol. 
292
 (pg. 
95
-
105
)
Gaur
 
M.
Choudhury
 
D.
Prasad
 
R.
 
Complete inventory of ABC proteins in human pathogenic yeast, Candida albicans
J. Mol. Microbiol. Biotechnol.
2005
, vol. 
9
 (pg. 
3
-
15
)
Decottignies
 
A.
Goffeau
 
A.
 
Complete inventory of the yeast ABC proteins
Nat. Genet.
1997
, vol. 
15
 (pg. 
137
-
145
)
Demeule
 
M.
Jodoin
 
J.
Gingras
 
D.
Beliveau
 
R.
 
P-glycoprotein is localized in caveolae in resistant cells and in brain capillaries
FEBS Lett.
2000
, vol. 
466
 (pg. 
219
-
224
)
Garrigues
 
A.
Escargueil
 
A. E.
Orlowski
 
S.
 
The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane
Proc. Natl. Acad. Sci. U.S.A.
2002
, vol. 
99
 (pg. 
10347
-
10352
)
Mukhopadhyay
 
K.
Prasad
 
T.
Saini
 
P.
Pucadyil
 
T. J.
Chattopadhyay
 
A.
Prasad
 
R.
 
Membrane sphingolipidergosterol interactions are important determinants of multidrug resistance in Candida albicans
Antimicrob. Agents Chemother
2004
, vol. 
48
 (pg. 
1778
-
1787
)
Prasad
 
T.
Saini
 
P.
Gaur
 
N. A.
Vishwakarma
 
R. A.
Khan
 
L. A.
Haq
 
Q. M.
Prasad
 
R.
 
Functional analysis of CaIPT1, a sphingolipid biosynthetic gene involved in multidrug resistance and morphogenesis of Candida albicans
Antimicrob. Agents Chemother
2005
, vol. 
49
 (pg. 
3442
-
3452
)
Pasrija
 
R.
Krishnamurthy
 
S.
Prasad
 
T.
Ernst
 
J. F.
Prasad
 
R.
 
Squalene epoxidase encoded by ERG1 affects morphogenesis and drug susceptibilities of Candida albicans
J. Antimicrob. Chemother.
2005
, vol. 
55
 (pg. 
905
-
913
)
Martin
 
S. W.
Konopka
 
J. B.
 
Lipid rafts polarization contributes to hyphal growth in Candida albicans
Eukaryot. Cell
2004
, vol. 
3
 (pg. 
675
-
684
)
Wachtler
 
V.
Rajagopalan
 
S.
Balasubramanian
 
M. K.
 
Sterol-rich plasma membrane domains in the fission yeast Schizosaccharomyces pombe
J. Cell Sci.
2003
, vol. 
116
 (pg. 
867
-
874
)
Lavie
 
Y.
Liscovitch
 
M.
 
Changes in lipid and protein constituents of rafts and caveolae in multidrug resistance cancer cells and their functional consequences
Glycoconj. J.
2001
, vol. 
17
 (pg. 
253
-
259
)
Ravaud
 
S.
Do Cao
 
M. A.
Jidenko
 
M.
Ebel
 
C.
Le
 
M. M.
Jault
 
J. M.
Di
 
P. A.
Haser
 
R.
Aghajari
 
N.
 
The ABC transporter BmrA from Bacillus subtilis is a functional dimer when in a detergent-solubilized state
Biochem. J.
2006
, vol. 
395
 (pg. 
345
-
353
)
Poelarends
 
G. J.
Mazurkiewicz
 
P.
Konings
 
W. N.
 
Multidrug transporters and antibiotic resistance in Lactococcus lactis
Biochim. Biophys. Acta
2002
, vol. 
1555
 (pg. 
1
-
7
)
Balzi
 
E.
Wang
 
M.
Leterme
 
S.
Van Dyck
 
L.
Goffeau
 
A.
 
PDR5, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDR1
J. Biol. Chem.
1994
, vol. 
269
 (pg. 
2206
-
2214
)
Sanglard
 
D.
Ischer
 
F.
Monod
 
M.
Bille
 
J.
 
Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterisation of CDR2, a new multidrug ABC transporter gene
Microbiology
1997
, vol. 
143
 (pg. 
405
-
416
)
20a
Bissinger
 
P. H.
Kuchler
 
K.
 
Molecular cloning and expression of the Saccharomyces cerevisiae STS1 gene product. A yeast ABC transporter conferring mycotoxin resistance
J. Biol. Chem.
1994
, vol. 
269
 (pg. 
4180
-
4186
)
20b
Mahé
 
Y.
Parle-McDermott
 
A.
Nourani
 
A.
Delahodde
 
A.
Lamprecht
 
A.
Kuchler
 
K.
 
The ATP-binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3
Mol. Microbiol.
1996
, vol. 
20
 (pg. 
109
-
117
)
20c
Katzmann
 
D. J.
Epping
 
E. A.
Moye-Rowley
 
W. S.
 
Mutational disruption of plasma membrane trafficking of Saccharomyces cerevisiae Yor1p, a homologue of mammalian multidrug resistance protein
Mol. Cell. Biol.
1999
, vol. 
19
 (pg. 
2998
-
3009
)
20d
Prasad
 
R.
De Wergifosse
 
P.
Goffeau
 
A.
Balzi
 
E.
 
Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance of drugs and antifungals
Curr. Genet.
1995
, vol. 
27
 (pg. 
320
-
329
)
Jha
 
S.
Dabas
 
N.
Karnani
 
N.
Saini
 
P.
Prasad
 
R.
 
ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry
FEMS Yeast Res.
2004
, vol. 
5
 (pg. 
63
-
72
)
Jha
 
S.
Karnani
 
N.
Dhar
 
S. K.
Mukhopadhayay
 
K.
Shukla
 
S.
Saini
 
P.
Mukhopadhayay
 
G.
Prasad
 
R.
 
Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis
Biochemistry
2003
, vol. 
42
 (pg. 
10822
-
10832
)
Altenberg
 
G. A.
 
Structure of multidrug-resistance proteins of the ATP-binding cassette (ABC) superfamily
Curr. Med. Chem. Anticancer Agents
2004
, vol. 
4
 (pg. 
53
-
62
)
Saini
 
P.
Prasad
 
T.
Gaur
 
N. A.
Shukla
 
S.
Jha
 
S.
Komath
 
S. S.
Khan
 
L. A.
Haq
 
Q. M.
Prasad
 
R.
 
Alanine scanning of transmembrane helix 11 of Cdr1p ABC antifungal efflux pump of Candida albicans: identification of amino acid residues critical for drug efflux
J. Antimicrob. Chemother.
2005
, vol. 
56
 (pg. 
77
-
86
)
Ginn
 
S. L.
Brown
 
M. H.
Skurray
 
R. A.
 
The TetAK tetracycline/H+ antiporter from Staphylococcus aureus: mutagenesis and functional analysis of motif C
J. Bacteriol.
2000
, vol. 
182
 (pg. 
1492
-
1498
)
Sharoni
 
M.
Steiner-Mordoch
 
S.
Schuldiner
 
S.
 
Exploring the binding domain of EmrE, the smallest multidrug transporter
J. Biol. Chem.
2005
, vol. 
280
 (pg. 
32849
-
32855
)
Sigal
 
N.
Vardy
 
E.
Molshanski-Mor
 
S.
Eitan
 
A.
Pilpel
 
Y.
Schuldiner
 
S.
Bibi
 
E.
 
3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membraneembedded positive charge
Biochemistry
2005
, vol. 
44
 (pg. 
14870
-
14880
)
De
 
R. E.
Arrigo
 
P.
Bellinzoni
 
M.
Silva
 
P. A.
Martin
 
C.
Ainsa
 
J. A.
Guglierame
 
P.
Riccardi
 
G.
 
The multidrug transporters belonging to major facilitator superfamily in Mycobacterium tuberculosis
Mol. Med.
2002
, vol. 
8
 (pg. 
714
-
724
)
Varela
 
M. F.
Sansom
 
C. E.
Griffith
 
J. K.
 
Mutational analysis and molecular modelling of an amino acid sequence motif conserved in antiporters but not symporters in a transporter superfamily
Mol. Membr. Biol.
1995
, vol. 
12
 (pg. 
313
-
319
)
Kimura-Someya
 
T.
Iwaki
 
S.
Konishi
 
S.
Tamura
 
N.
Kubo
 
Y.
Yamaguchi
 
A.
 
Cysteine-scanning mutagenesis around transmembrane segments 1 and 11 and their flanking loop regions of Tn10-encoded metal-tetracycline/H+ antiporter
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
18692
-
18697
)
Kimura
 
T.
Nakatani
 
M.
Kawabe
 
T.
Yamaguchi
 
A.
 
Roles of conserved arginine residues in the metal-tetracycline/H+ antiporter of Escherichia coli
Biochemistry
1998
, vol. 
37
 (pg. 
5475
-
5480
)
Yamaguchi
 
A.
Someya
 
Y.
Sawai
 
T.
 
Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3
J. Biol. Chem.
1992
, vol. 
267
 (pg. 
19155
-
19162
)
Someya
 
Y.
Yamaguchi
 
A.
 
Mercaptide formed between the residue Cys70 and Hg2+ or Co2+ behaves as a functional positively charged side chain operative in the Arg70→Cys mutant of the metal-tetracycline/H+ antiporter of Escherichia coli
Biochemistry
1996
, vol. 
35
 (pg. 
9385
-
9391
)
Paulsen
 
I. T.
Brown
 
M. H.
Skurray
 
R. A.
 
Proton-dependent multidrug efflux systems
Microbiol. Rev.
1996
, vol. 
60
 (pg. 
575
-
608
)
Broco
 
N.
Tenreiro
 
S.
Viegas
 
C. A.
Sa-Correja
 
I.
 
FLR1 gene (ORFYBR008c) is required for benomyl and methotrexate resistance in Saccharomyces cerevisiae and its benomyl induced expression is dependent on Pdr3 transcriptional regulator
Yeast
1999
, vol. 
15
 (pg. 
1595
-
1608
)
Fling
 
M. E.
Kopf
 
J.
Tamarkin
 
A.
Gorman
 
J. A.
Smith
 
H. A.
Koltin
 
Y.
 
Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate
Mol. Gen. Genet.
1991
, vol. 
227
 (pg. 
318
-
329
)
Braun
 
B. R.
van het Hoog
 
M.
d'Enfert
 
C.
Martchenko
 
M.
Dungan
 
J.
Kuo
 
A.
Inglis
 
D. O.
Uhl
 
M. A.
Hogues
 
H.
Berriman
 
M.
, et al 
A human-curated annotation of the Candida albicans genome
PLoS Genet.
2005
, vol. 
1
 (pg. 
36
-
57
)
Pasrija
 
R.
Banerjee
 
D.
Prasad
 
R.
 
Structure and function analysis of CaMdr1p, a MFS antifungal efflux transporter protein of Candida albicans: identification of amino acid residues critical for drug/H+ transport
Eukaryot. Cell.
2007
, vol. 
6
 (pg. 
443
-
453
)
Hiller
 
D.
Sanglard
 
D.
Morschhauser
 
J.
 
Overexpression of the MDR1 gene is sufficient to confer increased resistance to toxic compounds in Candida albicans
Antimicrob. Agents Chemother.
2006
, vol. 
50
 (pg. 
1365
-
1371
)
Calabrese
 
D.
Bille
 
J.
Sanglard
 
D.
 
A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole
Microbiology
2000
(pg. 
146
(pg. 
2743
-
2754
)
Simmons
 
C. R.
Fridlender
 
M.
Navarro
 
P. A.
Yalpani
 
N.
 
A maize defense-inducible gene is a major facilitator superfamily member related to bacterial multidrug resistance efflux antiporters
Plant Mol. Biol.
2003
, vol. 
52
 (pg. 
433
-
446
)
Kaur
 
R.
Bachhawat
 
A. K.
 
The yeast multidrug resistance pump, Pdr5p, confers reduced drug resistance in erg mutants of Saccharomyces cerevisiae
Microbiology
1999
, vol. 
145
 (pg. 
809
-
818
)
Decottignies
 
A.
Grant
 
A. M.
Nichols
 
J. W.
De Wet
 
H.
McIntosh
 
D. B.
Goffeau
 
A.
 
ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p
J. Biol. Chem.
1998
, vol. 
273
 (pg. 
12612
-
12622
)
Debry
 
P.
Nash
 
E. A.
Nekalson
 
D. W.
Metherall
 
J. E.
 
Role of multidrug resistance P-glycoproteins in cholesterol esterification
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
1026
-
1031
)
Hitchcock
 
C. A.
Barrett-Bee
 
K.
Russel
 
N. J.
 
The lipid composition of azole-sensitive and azole-resistant strains of Candida albicans
J. Gen. Microbiol.
1986
, vol. 
132
 (pg. 
2421
-
2431
)
Reference deleted
Gaber
 
R. F.
Copple
 
D. M.
Kennedy
 
B. K.
Vidal
 
M.
Bard
 
M.
 
The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol
Mol. Cell. Biol.
1989
, vol. 
9
 (pg. 
3447
-
3456
)
Reference deleted
Dickson
 
R. C
Lester
 
R. L.
 
Sphingolipid functions in Saccharomyces cerevisiae
Biochim. Biophys. Acta
2002
, vol. 
1583
 (pg. 
13
-
25
)
Hannun
 
Y. A.
Obeid
 
L. M.
 
The ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
25847
-
25850
)
Mandala
 
S. M.
Thornton
 
R. A.
Rosenbach
 
M.
Milligan
 
J.
Garcia-Calvo
 
M.
Bull
 
H. G.
Kurtz
 
M. B.
 
Khafrefungin, a novel inhibitor of sphingolipid synthesis
J. Biol. Chem.
2002
, vol. 
272
 (pg. 
32709
-
32714
)
Nagiec
 
M. M.
Nagiec
 
E. E.
Baltisberger
 
J. A.
Wells
 
G. B.
Lester
 
R. L.
Dickson
 
R. C.
 
Sphingolipid synthesis as a target for antifungal drugs
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
9809
-
9817
)
Dickson
 
R. C
Lester
 
R. L.
 
Metabolism and selected functions of sphingolipids in the yeast Saccharomyces cerevisiae
Biochim. Biophys. Acta
1999
, vol. 
1438
 (pg. 
305
-
321
)
Dickson
 
R. C
Lester
 
R. L.
 
Yeast sphingolipids
Biochim. Biophys. Acta
1999
, vol. 
1426
 (pg. 
347
-
357
)
Dickson
 
R. C.
Nagiec
 
E. E.
Wells
 
G. B.
Nagiec
 
M. M.
Lester
 
R. L.
 
Synthesis of mannose-(inositol-P)2-ceramide, the major sphingolipid in Saccharomyces cerevisiae, requires the IPT1 (YDR072c) gene
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
29620
-
29625
)
Hallstrom
 
T. C.
Lambert
 
L.
Schorling
 
S.
Balzi
 
E.
Goffeau
 
A.
Moye-Rowley
 
W. S.
 
Coordinate control of sphingolipid biosynthesis and multidrug resistance in Saccharomyces cerevisiae
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
23674
-
23680
)
Wachtler
 
V.
Balasubramanian
 
M. K.
 
Yeast lipid rafts? – an emerging view
Trends Cell Biol.
2006
, vol. 
1
 (pg. 
1
-
4
)
Dupre
 
S.
Tsapis
 
R. H.
 
Raft partioning of the yeast uracil permease during trafficking along the endocytic pathway
Traffic
2003
, vol. 
4
 (pg. 
83
-
96
)
Reference deleted
Pike
 
L. J.
Han
 
X.
Gross
 
R. W.
 
Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids
J. Biol. Chem.
2005
, vol. 
280
 (pg. 
26796
-
26804
)
Becher
 
A.
McIlhinney
 
R. A.
 
Consequences of lipid raft association on G-protein-coupled receptor function
Biochem. Soc. Symp.
2005
, vol. 
72
 (pg. 
151
-
164
)
Bacso
 
Z.
Nagy
 
H.
Goda
 
K.
Bene
 
L.
Fenyvesi
 
F.
Matko
 
J.
Szabo
 
G.
 
Raft and cytoskeleton associations of an ABC transporter: P-glycoprotein
Cytometry A.
2004
, vol. 
61
 (pg. 
105
-
116
)
Dustin
 
M. L.
 
Shmoos, rafts, and uropods – the many facets of cell polarity
Cell
2002
, vol. 
110
 (pg. 
13
-
18
)
Bagnat
 
M.
Simons
 
K.
 
Cell surface polarization during yeast mating
Proc. Natl. Acad. Sci. U.S.A.
2002
, vol. 
99
 (pg. 
14183
-
14188
)
Sanglard
 
D.
Kuchler
 
K.
Ischer
 
F.
Pagani
 
J.-L.
Monod
 
M.
Bille
 
J.
 
Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters
Antimicrob. Agents Chemother.
1995
, vol. 
39
 (pg. 
2378
-
2386
)
Krishnamurthy
 
S.
Gupta
 
V.
Prasad
 
R.
Panwar
 
S. L.
Prasad
 
R.
 
Expression of CDR1, a multidrug resistance gene of Candida albicans: In vitro transcriptional activation by heat shock, drugs and human steroid hormones
FEMS Microbiol. Lett.
1998
, vol. 
160
 (pg. 
191
-
197
)
MacPherson
 
S.
Larochelle
 
M.
Turcotte
 
B.
 
A fungal family of transcriptional regulators: the zinc cluster proteins
Microbiol. Mol. Biol. Rev.
2006
, vol. 
70
 (pg. 
583
-
604
)
Moye-Rowley
 
W. S.
 
Transcriptional control of multidrug resistance in the yeast Saccharomyces
Prog. Nucleic Acid Res.
2003
, vol. 
73
 (pg. 
251
-
279
)
Alarco
 
A. M.
Balan
 
I.
Talibi
 
D.
Mainville
 
N.
Raymond
 
M.
 
AP1-mediated multidrug resistance in Saccharomyces cerevisiae requires FLR1 encoding a transporter of the major facilitator superfamily
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
19304
-
19313
)
Coleman
 
S. T.
Epping
 
E. A.
Steggerda
 
S. M.
Moye-Rowley
 
W. S.
 
Yap1p activates gene transcription in an oxidant specific fashion
Mol. Cell. Biol.
1999
, vol. 
19
 (pg. 
8302
-
8313
)
Miyahara
 
K.
Hirata
 
D.
Miyakawa
 
T.
 
yAP-1- and yAP-2-mediated, heat shock-induced transcriptional activation of the multidrug resistance ABC transporter genes in Saccharomyces cerevisiae
Curr. Genet.
1996
, vol. 
29
 (pg. 
103
-
105
)
Wendler
 
F.
Bergler
 
H.
Prutej
 
K.
Jungwirth
 
H.
Zisser
 
G.
Kuchler
 
K.
Hogenauer
 
G.
 
Diazoborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleoitropic drug resistance genes PDR1 and PDR3
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
27091
-
27098
)
Le Crom
 
S.
Devaux
 
P. F.
Marc
 
P.
Zhang
 
X.
Moye-Rowley
 
W. S.
Jacq
 
C.
 
New insights into the pleiotropic drug resistance network from genome-wide characterization of the YRR1 transcription factor regulation system
Mol. Cell. Biol.
2002
, vol. 
22
 (pg. 
2642
-
2649
)
Moye-Rowley
 
W. S.
 
Regulation of the transcriptional response to oxidative stress in fungi: similarities and differences
Eukaryot. Cell
2003
, vol. 
2
 (pg. 
381
-
389
)
Hellauer
 
K.
Akache
 
B.
MacPherson
 
S.
Sirard
 
E.
Turcotte
 
B.
 
Zinc cluster protein Rdr1p is a transcriptional repressor of the PDR5 gene encoding a multidrug transporter
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
17671
-
17676
)
Akache
 
B.
Turcotte
 
B.
 
New regulators of drug sensitivity in the family of yeast zinc cluster proteins
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
21254
-
21260
)
Akache
 
B.
MacPherson
 
S.
Sylvain
 
M. A.
Turcotte
 
B.
 
Complex interplay among regulators of drug resistance genes in Saccharomyces cerevisiae
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
27855
-
27860
)
Lopez-Ribot
 
J. L.
McAtee
 
R. K.
Lee
 
L. N.
Kirkpatrick
 
W. R.
White
 
T. C.
Sanglard
 
D.
Patterson
 
T. F.
 
Distinct patterns of gene expression associated with development of fluconazole resistance in serial Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal Candidiasis
Antimicrob. Agents Chemother.
1998
, vol. 
42
 (pg. 
2932
-
2937
)
Perea
 
S.
Lopez-Ribot
 
J. L.
Kirkpatrick
 
W. R.
McAtee
 
R. K.
Santillan
 
R. A.
Martinez
 
M.
Calabrese
 
D.
Sanglard
 
D.
Patterson
 
T. F.
 
Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients
Antimicrob. Agents Chemother.
2001
, vol. 
45
 (pg. 
2676
-
2684
)
De Deken
 
X.
Raymond
 
M.
 
Constitutive activation of the PDR16 promoter in a Candida albicans azole-resistant clinical isolate overexpressing CDR1 and CDR2
Antimicrob. Agents Chemother.
2004
, vol. 
48
 (pg. 
2700
-
2703
)
Wirsching
 
S.
Michel
 
S.
Kohler
 
G.
Morschhauser
 
J.
 
Activation of the multidrug resistance gene MDR1 in fluconazole resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor
J. Bacteriol.
2000
, vol. 
182
 (pg. 
400
-
404
)
Goto
 
M.
Masuda
 
S.
Saito
 
H.
Inui
 
K.
 
Decreased expression of P-glycoprotein during differentiation in the human intestinal cell line Caco-2
Biochem. Pharmacol.
2003
, vol. 
66
 (pg. 
163
-
170
)
Laurencot
 
C. M.
Scheffer
 
G. L.
Scheper
 
R. J.
Shoemaker
 
R. H.
 
Increased LRP mRNA expression is associated with the MDR phenotype in intrinsically resistant human cancer cell lines
Int. J. Cancer
1997
, vol. 
72
 (pg. 
1021
-
1026
)
Talibi
 
D.
Raymond
 
M.
 
Isolation of a putative Candida albicans transcriptional regulator involved in pleiotropic drug resistance by functional complementation of a pdr1 pdr3 mutation in Saccharomyces cerevisiae
J. Bacteriol.
1999
, vol. 
181
 (pg. 
231
-
240
)
Wang
 
J. S.
Yang
 
Y. L.
Wu
 
C. J.
Ouyang
 
K. J.
Tseng
 
K. Y.
Chen
 
C. G.
Wang
 
H.
Lo
 
H. J.
 
The DNA-binding domain of CaNdt80p is required to activate CDR1 involved in drug resistance in Candida albicans
J. Med. Microbiol.
2006
, vol. 
55
 (pg. 
1403
-
1411
)
Coste
 
A. T.
Turner
 
V.
Ischer
 
F.
Morschhauser
 
J.
Forche
 
A.
Selmecki
 
A.
Berman
 
J.
Bille
 
J.
Sanglard
 
D.
 
A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans
Genetics
2006
, vol. 
172
 (pg. 
2139
-
2156
)
MacPherson
 
S.
Akache
 
B.
Weber
 
S.
De Deken
 
X.
Raymond
 
M.
Turcotte
 
B.
 
Candida albicans zinc cluster protein Upc2p confers resistance to antifungal drugs and is an activator of ergosterol biosynthetic genes
Antimicrob. Agents Chemother.
2005
, vol. 
49
 (pg. 
1745
-
1752
)
White
 
T. C.
Silver
 
P. M.
 
Regulation of sterol metabolism in Candida albicans by the UPC2 gene
Biochem. Soc. Trans.
2005
, vol. 
33
 (pg. 
1215
-
1218
)
DeRisi
 
J.
van den Hazel
 
B.
Marc
 
P.
Balzi
 
E.
Brown
 
P.
Jacq
 
C.
Goffeau
 
A.
 
Genome microarray analysis of transcriptional activation in multidrug resistance yeast mutants
FEBS Lett.
2000
, vol. 
470
 (pg. 
156
-
160
)
Devaux
 
F.
Carvajal
 
E.
Moye-Rowley
 
S.
Jacq
 
C.
 
Genome-wide studies on the nuclear PDR3-controlled response to mitochondrial dysfunction in yeast
FEBS Lett.
2002
, vol. 
515
 (pg. 
25
-
28
)
Reference deleted
Smriti Krishnamurthy
 
S.
Dixit
 
B. L.
Gupta
 
C. M.
Milewski
 
S.
Prasad
 
R.
 
ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators
Yeast
2002
, vol. 
19
 (pg. 
303
-
318
)
Pasrija
 
R.
Panwar
 
S. L.
Prasad
 
R.
 
Multidrug transporters CaCdr1p and CaMdr1p of Candida albicans display different lipid specificities: both Ergosterol and Sphingolipids are essential for targeting of CaCdr1p to membrane rafts
Antimicrob. Agents Chemother.
2008
, vol. 
52
 (pg. 
694
-
704
)
Saidane
 
S.
Weber
 
S.
De Deken
 
X.
St-Germain
 
G.
Raymond
 
M.
 
PDR16-mediated azole resistance in Candida albicans
Mol. Microbiol.
2006
, vol. 
60
 (pg. 
1546
-
1562
)
Reference deleted
Soustre
 
I.
Letourneux
 
Y.
Karst
 
F.
 
Characterization of the Saccharomyces cerevisiae RTA1 gene involved in 7-aminocholesterol resistance
Curr. Genet.
1996
, vol. 
30
 (pg. 
121
-
125
)
Kihara
 
A.
Igarashi
 
Y.
 
Identification and characterization of a Saccharomyces cerevisiae gene, RSB1, involved in sphingoid long-chain base release
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
30048
-
30054
)
Mukhopadhyay
 
K.
Kohli
 
A. K.
Prasad
 
R.
 
Drug susceptibilities of yeast cells are affected by membrane lipid composition
Antimicrob. Agents Chemother.
2002
, vol. 
46
 (pg. 
3695
-
3705
)
Kolaczkowski
 
M.
Kolaczkowska
 
A.
Gaigg
 
B.
Schneiter
 
R.
Moye-Rowley
 
W. S.
 
Differential regulation of ceramide synthase components LAC1 and LAG1 in Saccharomyces cerevisiae
Eukaryot. Cell
2004
, vol. 
3
 (pg. 
880
-
892
)
Reference deleted
Maceyka
 
M.
Payne
 
S. G.
Milstien
 
S.
Spiegel
 
S.
 
Sphingosine kinase, sphingosine-1-phosphate, and apoptosis
Biochim. Biophys. Acta
2002
, vol. 
1585
 (pg. 
193
-
201
)
Kihara
 
A.
Igarashi
 
Y.
 
Cross talk between sphingolipids and glycerophospholipids in the establishment of plasma membrane asymmetry
Mol. Biol. Cell
2004
, vol. 
15
 (pg. 
4949
-
4959
)
Kean
 
L. S.
Grant
 
A. M.
Angeletti
 
C.
Mahe
 
Y.
Kuchler
 
K.
Fuller
 
R. S.
Nichols
 
J. W.
 
Plasma membrane translocation of fluorescent-labeled phosphatidylethanolamine is controlled by transcription regulators, PDR1 and PDR3
J. Cell Biol.
1997
, vol. 
138
 (pg. 
255
-
270
)
Panwar
 
S. L.
Moye-Rowley
 
W. S.
 
Long chain base tolerance in Saccharomyces cerevisiae is induced by retrograde signals from the mitochondria
J. Biol. Chem.
2006
, vol. 
281
 (pg. 
6376
-
6384
)
Karababa
 
M.
Coste
 
A. T.
Rognon
 
B.
Bille
 
J.
Sanglard
 
D.
 
Comparison of gene expression profiles of Candida albicans azole-resistant clinical isolates and laboratory strains exposed to drugs inducing multidrug transporters
Antimicrob. Agents Chemother.
2004
, vol. 
48
 (pg. 
3064
-
3079
)

Author notes

1

Present address: Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar, India.