All fungal genomes harbour numerous ABC (ATP-binding cassette) proteins located in various cellular compartments such as the plasma membrane, vacuoles, peroxisomes and mitochondria. Most of them have initially been discovered through their ability to confer resistance to a multitude of drugs, a phenomenon called PDR (pleiotropic drug resistance) or MDR (multidrug resistance). Studying the mechanisms underlying PDR/MDR in yeast is of importance in two ways: first, ABC proteins can confer drug resistance on pathogenic fungi such as Candida spp., Aspergillus spp. or Cryptococcus neoformans; secondly, the well-established genetic, biochemical and cell biological tractability of Saccharomyces cerevisiae makes it an ideal tool to study basic mechanisms of drug transport by ABC proteins. In the past, knowledge from yeast has complemented work on human ABC transporters involved in anticancer drug resistance or genetic diseases. Interestingly, increasing evidence available from yeast and other organisms suggests that ABC proteins play a physiological role in membrane homoeostasis and lipid distribution, although this is being intensely debated in the literature.
As in all other organisms, ABC (ATP-binding cassette) proteins in fungi use ATP hydrolysis to energize transport of a huge variety of compounds across biological membranes. Remarkably, although some yeast ABC proteins have a tremendously broad substrate specificity (e.g. Pdr5 or Snq2), others are known to transport only a limited number of substrates (Ste6), and, in some cases, transport substrates have not even been discovered (e.g. Pdr18). Despite enormous efforts in dissecting the mechanism of ABC transporters, the basis of this variable but restricted substrate specificity, as well as the precise translocation mechanism, remain enigmatic. In this chapter, we discuss the current knowledge about fungal ABC proteins, with special focus on their structure, physiological roles and function in conferring MDR (multidrug resistance) including their transcriptional control. We limit the discussion to the yeast Saccharomyces cerevisiae and, to some extent, to fungal pathogens such as Candida spp., Aspergillus fumigatus and Cryptococcus neoformans.
Inventory of S. cerevisiae ABC proteins
The genome of S. cerevisiae encodes 30 confirmed or predicted ABC proteins . On the basis of phylogenetic relationships, ABC proteins have been grouped into five subfamilies referred to as the PDR (pleiotropic drug resistance), MDR, MRP (multidrug-resistance protein)/CFTR (cystic fibrosis transmembrane conductance regulator), ALDP (adrenoleukodystrophy protein) and YEF3 (yeast elongation factor 3)/RLI (RNase L inhibitor 1) subfamilies. As is evident from Table 1, the classification into subfamilies is also based on the predicted topology of the proteins.
An essential component of every ABC protein is the NBD (nucleotide-binding domain). Most of the proteins also have one or two TMDs (transmembrane domains), usually consisting of six predicted TMSs (transmembrane segments). The domain architecture (Figure 1) includes full-size transporters [(TMS6−NBD)2 or (NBD−TMS6)2], half-size transporters (TMS6−NBD) and members lacking obvious TMDs (NBD or NBD2). Some proteins hold additional N-terminal extensions [NTE−(TMS6−NBD)2] and some proteins carry an atypical TMD (e.g. TMS2–NBD–TMS7). Most yeast ABC proteins are found in cellular membranes, although some are present in the cytosol or associated with polysomes (Figure 2).
The PDR subfamily
The PDR subfamily is the largest ABC subfamily and contains two of the most extensively studied ABC proteins in yeast, namely Pdr5 and Snq2. They localize to the plasma membrane and their overexpression confers resistance to hundreds of chemically unrelated drugs, including agricultural fungicides, azoles, mycotoxins, herbicides, anticancer drugs and many others (reviewed in [2,3]). Interestingly, Pdr10 and Pdr15, two closely related members, share a high overall sequence identity with Pdr5 exceeding 70%. In contrast, limited data are available on these ABC pumps and only very few substrates are known. Pdr15, mainly expressed in cells approaching stationary phase, has been linked to herbicide resistance, as well as to both general stress and membrane stress responses . Pdr10 is involved in modulating the membrane microenvironment, thereby influencing activity and trafficking of other surface proteins .
Sharing approximately 60% identity, Pdr12 is more closely related to Snq2. The Pdr12 transporter effluxes aromatic and branched-chain organic acids, as well as carboxylic acids often used in food preservation [6,7]. Aus1 and Pdr11, Pdr18, Adp1 and the YOL075C gene product are further members of the PDR subfamily. However, except for Aus1 and Pdr11, a PDR phenotype or other biological functions have not been associated with any of these proteins. Interestingly, Aus1 and Pdr11 have been implicated in the import of sterols into the cell . Although ADP1, PDR18 and YOL075C remain functionally uncharacterized, knockouts show impaired fitness on certain drugs [5,9].
The MDR subfamily
The MDR subfamily contains four members: Ste6, Atm1, Mdl1 and Mdl2. Ste6 is localized in the plasma membrane and is required for the export of the yeast mating pheromone, a-factor [10–12]. The half-size transporters Atm1, Mdl1 and Mdl2 are present in the inner mitochondrial membrane. Atm1 is involved in regulation of cellular iron homoeostasis and in cytosolic iron–sulfur cluster biogenesis . Mdl1 has been proposed to be a peptide exporter , whereas the function of Mdl2 remains unknown.
The MRP/CFTR subfamily
Next to Pdr5 and Snq2, the plasma membrane transporter Yor1 is the third major player in PDR in S. cerevisiae [15,16]. A yor1 deletion mutant displays hypersensitivity to many xenobiotics, including oligomycin, reveromycin A and antibiotics, as well as anticancer drugs and different azoles. Yor1 shares some similarity to human CFTR, an ABC transporter acting as a Cl− channel whose defects cause cystic fibrosis. The transporters Ycf1, Ybt1 and Bpt1 contribute to pleiotropic drug resistance by vacuolar sequestration of conjugated and unconjugated xenobiotics or heavy metals [17,18]. Two additional transporters of the MRP/CFTR subfamily are Nft1 and Vmr1. Nft1 might be involved in resistance to cadmium and arsenite , but nothing is known about the function of Vmr1.
The ALDP subfamily
The ALDP subfamily contains only two half-size transporters: Pxa1 and Pxa2. Both localize to the peroxisomal membrane and presumably form heterodimers, which may mediate peroxisomal uptake of very-long-chain fatty acids [20,21].
The YEF3/RLI subfamily
All members of the YEF3/RLI subfamily, except for New1, lack predicted TMSs and consequently localize to the cytosol. Three of them, Yef3, Arb1 and Rli1, are essential for viability under standard growth conditions and have functions connected to protein synthesis or ribosome biogenesis . Hef3 shares 84% overall identity with Yef3, implying a similar or overlapping function. Like Yef3, Gcn20 has a functional role in ribosomal translation by activating Gcn2, a protein kinase that phosphorylates eIF2α (eukaryotic initiation factor 2α) . New1 is the only member of this subfamily that contains three predicted TMSs. However, rather than forming a classical TMD, they have been identified as a prion-forming domain .
YDR061W and Caf16 are represented by only one NBD and do not belong to any of the previous subfamilies. Neither of them has been associated with resistance phenotypes, although YDR061W expression is controlled in a similar manner as some other MDR genes . Interestingly, Caf16 interacts with components of the RNA polymerase II holoenzyme and thus may have a role in transcription .
Inventory of ABC proteins in Candida species and other pathogenic fungi
Although the genomes of Candida spp., Cryptococcus neoformans and Aspergillus spp. encode numerous putative ABC transporters , only a few of them have been investigated in detail so far (Table 2). We refer to comprehensive recent reviews for further details [28,29]. Most ABC transporters studied in various Candida species are orthologues of S. cerevisiae Pdr5 and likewise have been implicated in MDR. The C. albicans genome harbours two genes, namely CaCDR1 and CaCDR2, whose products share high homology and domain organization with Pdr5. Lack of CaCDR1 causes hypersensitivity to azoles, terbinafine, amorolfine and several other metabolic inhibitors. Two additional ABC subfamily members in C. albicans, CaCdr3 and CaCdr4, are close homologues of CaCdr1 and CaCdr2, but neither of them has been associated with an MDR phenotype. Instead, selective expression of CaCdr3 in opaque cells suggests a physiological role in this specific growth phase. CaHst6 is a functional homologue of the S. cerevisiae Ste6 mating pheromone exporter, and so far the only MDR subfamily transporter characterized in C. albicans. Notably, three members of the C. albicans MRP/CFTR subfamily have been associated with drug transport and virulence, namely CaYor1 , CaYcf1  and CaMlt1 .
So far, four ABC proteins have been studied in C. glabrata. CgCdr1, CgCdr2 and CgSnq2 confer MDR phenotypes, whereas CgAus1 mediates sterol uptake, similar to its S. cerevisiae counterpart, Aus1 . Importantly, transcriptional up-regulation of CgCdr1 and CgCdr2 occurs in azole-resistant clinical isolates of C. glabrata, C. dubliniensis, C. krusei and C. tropicalis. In fact, clinical antifungal resistance in C. glabrata is predominantly caused by transcriptional up-regulation of ABC transporters.
ABC transporters in Cryptococcus neoformans are implicated in azole transport, as shown for CnMDR1 by heterologous expression in S. cerevisiae or as in the case of CnAFR1 in C. neoformans directly. Furthermore, interaction studies with a CnAFR1-overexpressing strain and microglia (macrophages residing in the brain) showed reduced acidification and delayed maturation of the phagolysosome . This leads to an interesting connection between azole resistance and virulence, which has also been observed for C. glabrata azole-resistant strains . Finally, at least four ABC proteins (AfuMDR1, AfuMDR2, AfuMDR4 and atrF) mediate itraconazole resistance in A. fumigatus in vitro.
Physiological roles of yeast ABC proteins
Yeast ABC proteins are frequently referred to as drug-efflux pumps associated with PDR or MDR phenomena usually observed when overexpression occurs. Interestingly, members of at least three subfamilies (ALDP, MDR and YEF3/RLI) are not involved in any drug resistance. Within the PDR subfamily, Aus1 and Pdr11 have been linked to uptake processes rather than to excretion of drugs. In this section, we focus on roles of ABC proteins other than drug efflux.
As mentioned above, Pdr12 effluxes toxic weak acid intermediates derived from amino acid catabolism including phenylacetic, indolacetic or fusel acids [6,7]. Thus ABC proteins could function in detoxification of potentially harmful intermediates from diverse metabolic pathways. In fact, Ycf1 detoxifies the red pigment in ade2 mutants by means of vacuolar sequestration . In concert with Ybt1 and Bpt1, Ycf1 also mediates tolerance to various heavy metals [17,18].
Ste6 is a prototypical peptide-transporting ABC protein, extruding the S. cerevisiae mating pheromone a-factor, an isoprenylated and methylated oligopeptide with lipid-like features . Other ABC proteins transporting peptide substrates include Mdl1, releasing proteolytic degradation products generated from mitochondrial membrane proteins . Atm1, a close homologue of Mdl1, is crucial for the export of extra-mitochondrial Fe/S cluster proteins .
Physiological roles have also been identified for cytosolic ABC proteins lacking TMDs: Yef3 is a translational elongation factor, Arb1 has been proposed to stimulate ribosome biogenesis , and Rli1 is involved in translation initiation, termination as well as ribosome recycling . In addition, the peroxisomal ABC proteins Pxa1 and Pxa2 are thought to mediate peroxisomal uptake of very-long-chain fatty acids such as palmitate or oleate for degradation through β-oxidation [20,21].
There is growing evidence linking ABC transporters to membrane lipid transport. Indeed, many transport substrates are lipophilic in nature. For example, Pdr5 and Yor1 negatively affect accumulation of fluorescently labelled NBD–PE (nitrobenzoxadiazole–phosphatidylethanolamine) in vitro, suggesting a role in the movement of membrane lipids [39,40]. Similarly, CaCdr1, CaCdr2 and CaCdr3 behave as general phospholipid translocators . Aus1 and Pdr11 in yeast seem to be involved in uptake of the essential fungal membrane constituent ergosterol. Their absence completely blocks sterol uptake and results in an inviable phenotype under conditions where cells are unable to produce ergosterol, e.g. in anaerobiosis . In C. glabrata, CgAus1 initiates uptake of serum cholesterol, thus protecting this pathogen against azole toxicity in vivo . In addition, CaCdr1 and S. cerevisiae Pdr5 and Snq2 efflux sterol-derived molecules such as β-oestradiol and corticosterone [42,43]. Notably, steroids also induce CaCDR1 expression . These results suggest that steroids and sterols could possibly represent physiological substrates of yeast ABC proteins. Yeast Pdr10 seems to be involved in membrane lipid homoeostasis as well . Even though substrate(s) and the molecular mechanism for Pdr10 remain unclear, it may modulate formation and maintenance of membrane microdomains important for membrane protein trafficking . Furthermore, membrane-damaging agents such as detergents and lysophospholipids, all of which exert massive membrane stress, strongly increase Pdr15 levels . Hence, certain fungal ABC transporters may operate in controlling or modulating membrane lipid homoeostasis, regulation of membrane permeability or even phospholipid bilayer distribution. It is also tantalizing to speculate that ABC pumps could remove oxidized or damaged membrane lipids, which are otherwise detrimental to cells. Unfortunately, available facts are modest at best, as most studies addressing membrane lipid asymmetry employed short-chain fluorescent lipids as transport or ‘flippase’ substrates, which may be recognized as ‘drugs’ rather than natural membrane lipids. Hence natural lipid substrates [46,47] will be necessary to unequivocally demonstrate functions for ABC transporters in lipid homoeostasis.
Transcriptional control of MDR
Members of several subfamilies are involved in rendering cells resistant to xenobiotics and naturally occurring toxic compounds. It is therefore not surprising that many yeast ABC proteins are tightly controlled by various transcriptional regulators. In this section, we want to depict the regulatory network that controls members of the PDR subfamily.
Two master transcription factors, Pdr1 and Pdr3, regulate expression of the PDR network, including proteins such as Pdr5, Snq2, Pdr10 and Pdr15 (reviewed in ). They are typical binuclear Zn2Cys6 transcription factors and bind to the promoters of their target genes through so-called PDREs (Pdr1/Pdr3-response elements). Pdr1 and Pdr3 consist of an N-terminal DNA-binding domain, a central regulatory domain and a C-terminal acidic activation domain. They act as either homo- or hetero-dimers, and their activation requires external stimuli, including membrane-perturbing agents and xenobiotics, as well as loss of the mitochondrial genome (ρ0 cells). This retrograde signalling from mitochondria to the nucleus interestingly also requires Psd1, the mitochondrial phosphatidylserine decarboxylase, thereby linking PDR pumps to lipid metabolism (reviewed in ).
Pdr1 and Pdr3 bind various compounds directly, leading to their activation and subsequent transcriptional induction of their target genes . It is thought that xenobiotic binding triggers structural changes in Pdr1/Pdr3, thereby enhancing recruitment of Med15, a component of the RNA polymerase II mediator complex, to target promoters.
Additionally, other transcription factors such as Yrr1 and Yrm1 for SNQ2 or Msn2 for PDR15 modulate transcription of Pdr1/Pdr3-controlled genes. Interestingly, PDR12 is not regulated via Pdr1/Pdr3, but through its dedicated transcription factor War1 . Weak acid stress leads to conformational changes and enhanced binding of War1 to the PDR12 promoter .
A couple of regulatory elements were found in the promoter region of CaCDR1: a BEE (basal expression element), two SREs (steroid-responsive elements) and an NRE (negative regulatory element). Furthermore, both CaCDR1 and CaCDR2 have a DRE (drug-responsive element) in their promoters mediating induction by oestradiol or fluphenazine. CaFcr1, CaFcr2 and CaFcr3 (fluconazole resistance) were among the first putative regulators of C. albicans PDR genes. When expressed in S. cerevisiae, all of them increase Pdr5-dependent resistance to fluconazole and cycloheximide. Surprisingly, a Cafcr1 deletion in C. albicans leads to hyperresistance to CaCdr1/2 substrates, implying that CaFcr1 rather is a negative regulator of transcription.
Furthermore, CaNdt80 regulates basal CaCDR1 expression and resistance to azoles. It binds to the promoter regions of CaCDR1, CaCDR2, as well as those of ergosterol-biosynthesis genes. In contrast with CaNdt80, CaTac1 (transcriptional activator of CDR genes) does not influence basal expression of CaCDR1/CaCDR2, but up-regulates these genes upon exposure to fluphenazine and other drugs through DREs in their promoter regions. CaTAC1 mutations causing constitutive activation are responsible for CaCdr1 and CaCdr2 overexpression in azole-resistant clinical isolates .
Similar to S. cerevisiae and C. albicans, CgCDR1, CgCDR2 and CgSNQ2 are strongly up-regulated in response to xenobiotics, as well as to the loss of the mitochondrial genome [28,29]. Furthermore, respiratory-deficient mutants are fluconazole-resistant due to CgCDR1 and CgCDR2 overexpression. However, they are rarely found among azole-resistant clinical isolates, probably because of their strong growth defect and reduced virulence. CgPdr1, the orthologue of S. cerevisiae Pdr1 regulates expression of CgCDR1, CgCDR2 and CgSNQ2. Cgpdr1 gain-of-function mutations cause a constitutive overexpression of these transporters in clinical isolates, as well as in in vitro generated resistant strains. Like Pdr1 in yeast, the C. glabrata regulator binds directly to different CgCdr1/CgCdr2 substrates and to the transcriptional mediator complex .
Structure and mechanism of fungal ABC transporters
Detailed information on structural features of ABC transporters and our current understanding of the catalytic cycle are discussed in Chapters 3 and 4 of this book. As for ABC transporters in general, limited structural information is available for yeast ABC proteins. However, there has been progress in understanding underlying molecular principles of action through random as well as site-directed mutagenesis approaches. Most of this work has been carried out on Pdr5, the best characterized ABC transporter in S. cerevisiae, as well as on CaCdr1. Recent work highlights some of the unique features of yeast drug ABC transporters. One of the key questions is how ABC proteins recognize their substrates, since knowledge about drug-binding sites would facilitate drug discovery aimed at generating specific inhibitors that can reverse MDR in pathogenic fungi . TMDs vary considerably in amino acid sequence even within subfamilies, thus making the identification of crucial residues more difficult. The second key question is how drug binding is communicated between TMDs and NBDs to allow for efficient coupling of ATP hydrolysis and drug translocation.
Different strategies were used to answer both questions. Random mutagenesis identified many residues important for Pdr5 function, including folding, trafficking and stability, substrate specificity and inhibitor susceptibility of the transporter . Interestingly, a mutation in TMS11 (S1360F) changes substrate specificity and abolishes Pdr5 inhibition by the immunosuppressant macrolide FK506. This indicates that substrate recognition and transporter inhibition might be dependent on common structural features. Notably, S558Y in predicted helix 2 of Pdr5 leads to cycloheximide sensitivity without affecting ATPase activity , suggesting that ATP hydrolysis and drug transport are uncoupled. A second mutation (N242K) located in the N-terminal NBD was able to restore function. Hence the short intracellular loop between TMS2 and TMS3 may be involved in interdomain communication, similar to the function of the so-called coupling helices found in the prokaryotic ABC transporter Sav1866.
A different way of elucidating substrate recognition is by systematically manipulating the chemistry of potential drug substrates of Pdr5 (reviewed in ). Essentially, several distinct drug-binding sites exist and all of them seem to use different chemical properties for substrate selection. A common feature, however, is the size-dependency of substrate–transporter interaction.
Alanine-scanning mutagenesis of CaCdr1 TMS5 and TMS11 yields insights into the role of these helices regarding substrate binding and coupling of ATPase activity to drug transport. Out of 21 residues in TMS11, only seven mutant forms show increased sensitivity to drug substrates. Interestingly, five of them cluster to the more hydrophilic side of the helix . In contrast, all residues in TMS5 influenced drug resistance . Mutated CaCdr1 variants fell into two groups, one with diminished resistance to all tested substrates and one with impaired resistance to a subset of drugs used. As ATPase activity is normal in most of the mutants, TMS5 may couple ATP hydrolysis to transport of substrates.
Although nucleotide hydrolysis by ABC proteins seems to be a highly conserved mechanism, some distinct features of fungal transporters have been identified. In contrast with human P-glycoprotein, whose ATPase activity is stimulated in the presence of substrate, fungal ABC proteins only show basal ATPase activities [59,60]. Rather than being a waste of energy, this might represent a way of always keeping the protein in a transport-competent state to enable yeasts to rapidly react to toxic substances entering the cell.
One of the most remarkable features of yeast NBDs is their functional non-equivalence. Both NBDs contain all hallmark features of ABC transporters, namely Walker A (consensus sequence GXXGXGKS/T, with X representing any amino acid), Walker B (ΦΦΦΦD, with Φ being any aliphatic residue) and the so-called signature motif (LSGGQ). These motifs are important for ATP hydrolysis and Mg2+ co-ordination in all ABC transporters from bacteria to humans. However, the N-terminal NBD (NBD1) of Pdr5 and other PDR transporters contains a replacement in the Walker A motif: instead of the lysine residue known to be crucial for ATPase activity , one finds a cysteine residue. Furthermore, the histidine residue in the N-terminal H-loop is missing and the signature motif in the C-terminal domain (NBD2) is degenerate. The available crystal structures of bacterial and mammalian NBDs indicate a head-to-tail arrangement of both NBDs with the Walker A motif from NBD1 and the ABC signature motif of NBD2 building one ATP-binding site, and vice versa. Consequently, Pdr5 would contain one intact and one altered ATP-binding site. Indeed, replacing Cys199 of the Walker A motif in NBD1 does not influence drug resistance, R6G (Rhodamine 6G) transport and ATPase activity of Pdr5. In contrast, replacing Lys911 in the Walker A motif of NBD2 renders cells sensitive to known Pdr5 substrates and abolishes R6G transport as well as ATPase activity . Similar indications for distinct roles for the N- and C-terminal NBDs exist for CaCdr1.
Moreover, the function of the putative ‘catalytic carboxylate’ (Glu1036) and the ‘catalytic dyad histidine’ (His1068) has been analysed. As predicted, the E1036A mutant is unable to hydrolyse ATP. Consequently, cells are drug-susceptible and no R6G transport is possible. Surprisingly, the substitution of alanine for His1068 does not affect steady-state ATPase activity, but selectively abolishes R6G transport, whereas cells remain resistant to azoles. These findings led to the suggestion that overall kinetics of drug–transporter interaction and ATP hydrolysis influence substrate selection .
In this chapter, we have briefly summarized ABC proteins and their functions in the model organism S. cerevisiae, as well as in pathogenic fungi such as Candida spp. Whereas yeast ABC research mainly focuses on drug efflux, there is growing evidence implicating yeast ABC transporters in the modulation of membrane lipid homoeostasis. The purification and functional reconstitution of ABC transporters into proteoliposomes and the use of appropriate drug and/or lipid substrates will be necessary to better understand molecular mechanisms underlying ATP hydrolysis and the cross-talk of NBDs and TMDs, and might even prove a possible function in lipid transport. In addition, attempts to crystallize purified proteins must continue, despite difficulties encountered during purification. High-resolution structures of ABC proteins may uncover the basis of substrate recognition, and, along with molecular structure–function studies, facilitate the development of novel drugs specifically designed to inhibit actions of ABC proteins. Since S. cerevisiae offers a wide range of well-established genetic and biochemical tools, it will remain a valuable model organism to study the function of ABC proteins.
• ABC proteins are involved in various physiological and stress-related processes in yeasts, including drug detoxification, transport of metabolic intermediates, pheromones and lipids across cellular membranes, ribosomal biogenesis and translation, and mitochondrial iron homoeostasis.
• Overexpression of ABC transporters in pathogenic fungi can lead to MDR strains, thus counteracting therapy of fungal infections.
• Random, as well as site-directed, mutagenesis studies can help to identify residues influencing substrate selection and specificity of ABC transporters.
• Studies on Pdr5 have provided interesting insights into how ATPase kinetics might influence substrate selection, a principle so far unique for yeast ABC transporters.
We apologize to all colleagues in the field whose work could not be properly cited due to space limitations. Research in our laboratory on ABC proteins is supported by the Austrian Science Foundation [grant number FWF-SFB35-04], and the Framework Programme 6 Marie Curie Training Networks Flippases [grant number MRTN-CT-2004-5330], as well as in part by the Christian Doppler Research Society, the EraNet Pathogenomics project FunPath [grant number FWF-I-0125), the Framework Programme 6 EURESFUN project [grant number STREP-2004-CT-PL518199], and the Austrian GenAU research programme (SysMo-MOSES).
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