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Keywords:

  • antifungal drug resistance;
  • genome evolution;
  • Hemiascomycetes;
  • yeast;
  • ABC transporter

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information

The available genomic sequences of five closely related hemiascomycetous yeast species (Kluyveromyces lactis, Kluyveromyces waltii, Candida glabrata, Ashbya (Eremothecium) gossypii with Saccharomyces cerevisiae as a reference) were analysed to identify multidrug resistance (MDR) transport proteins belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies (MFS), respectively. The phylogenetic trees clearly demonstrate that a similar set of gene (sub)families already existed in the common ancestor of all five fungal species studied. However, striking differences exist between the two superfamilies with respect to the evolution of the various subfamilies. Within the ABC superfamily all six half-size transporters with six transmembrane-spanning domains (TMs) and most full-size transporters with 12 TMs have one and only one gene per genome. An exception is the PDR family, in which gene duplications and deletions have occurred independently in individual genomes. Among the MFS transporters, the DHA2 family (TC 2.A.1.3) is more variable between species than the DHA1 family (TC 2.A.1.2). Conserved gene order relationships allow to trace the evolution of most (sub)families, for which the Kluyveromyces lactis genome can serve as an optimal scaffold. Cross-species sequence alignment of orthologous upstream gene sequences led to the identification of conserved sequence motifs (“phylogenetic footprints”). Almost half of them match known sequence motifs for the MDR regulators described in S. cerevisiae. The biological significance of those and of the novel predicted motifs awaits to be confirmed experimentally.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information

Multidrug resistance (MDR) is a ubiquitous biological phenomenon causing serious problems in the treatment of human cancers and infections of bacterial and fungal origin. MDR is usually associated with specific transport systems that catalyse efflux of structurally and functionally unrelated compounds out of the cell. The most important MDR transporters belong either to the ATP-binding cassette (ABC) family or to the major facilitator superfamily (MFS), which differ by the way they are energized. Whereas the ABC transporters bind ATP and require ATP hydrolysis for transport activity, the MFS-mediated transport is driven by the proton-motive force. Members of both classes are found in all three kingdoms of life and are apparently involved in transport of solutes across the plasma membrane or across intracellular membranes. Subfamilies have been defined on the basis of structural and functional criteria (Pao et al., 1998; Saier, 2000; De Hertog et al., 2002), but only for a few transporters the physiological substrates are known. Both ABC and MFS transporters are encoded by large gene families that have been characterized extensively, we refer to recent reviews for more comprehensive comparisons (Decottignies & Goffeau, 1997; Goffeau et al., 1997; Nelissen et al., 1997; Bauer et al., 1999; Wolfger et al., 2001; Sá-Correia & Tenreiro, 2002). This review focuses on the evolution of the gene families of MDR-related membrane transporters of both ABC and MFS type, by comparing the genomes of five related Hemiascomycetes. Such comparisons should help to understand to which extent functional overlap in transporter activity contributes to MDR and whether the evolution of the transporter subfamilies arose before or after the divergence of these species. Particular emphasis was therefore put on the comparison between the Kluyveromyces lactis and S. cerevisiae genomes, which diverged before the genome duplication event that had occurred in the Saccharomyces cerevisiae branch (Wolfe & Shields, 1997; Wong et al., 2002). The genomes of Candida glabrata, closely related to S. cerevisiae, of Kluyveromyces waltii, and of Ashbya (Eremothecium) gossypii (A. gossypii) were included in the analysis of the complete complement of ABC- and MDR-related MFS membrane transporters (Dietrich, 2004; Dujon, 2004; Kellis et al., 2004). A. gossypii contains the smallest genome among completely sequenced yeast genomes so far (Dietrich, 2004). The phylogenetic trees of ABC and MFS transporters reveal striking differences in the evolution of the gene families and subfamilies. However, the results clearly demonstrate that the individual gene families already existed in the common ancestor of all these fungal species and were remarkably conserved in evolution.

ABC transporters

  1. Top of page
  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information

In the S. cerevisiae genome the products of 22 genes are classified as primary active transporters of the ABC family (Transport Classification TC 3.A.1) (Table S1). Among those, 16 proteins are about twice as long as the other six. The larger proteins have 12 transmembrane-spanning domains (TMS) and probably arose by duplication of the half-size ABC transporters with six TMS. This latter class is exceptionally conserved with respect to gene number per genome: in each of the five hemiascomycete genomes one and only one closely related open reading frame is found, which is considered a true orthologue. These orthologues of different species share a higher degree of similarity than the paralogues of one species, giving a phylogenetic tree with six branches splitting up into five ends for the five species included in the analysis (Fig. 1). One and only one orthologue for each subfamily is also found in more distantly related yeasts like Candida albicans (Table S1). Obviously, a strong selection pressure against gene duplication and gene loss exists for these ABC half-size transporters. The structural similarity is likely to reflect functional equivalence such that each branch defines a functional category.

image

Figure 1.  Phylogenetic relationship of ATP-binding cassette (ABC) half-size transporters determined by primary sequence comparison (ClustalW) of translated ORFs from Saccharomyces cerevisiae (Y), Candida glabrata (CAGL), Kluyveromyces lactis (KLLA), Kluyveromyces waltii (numbers only), and Ashbya gossypii (A). ORF names are indicated and K. lactis ORFs are printed bold face. Results of cluster analyses are shown as unrooted neighbour-joining trees. The standard names of the S. cerevisiae representatives of each branch are given.

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The two Peroxisomal Fatty Acyl CoA Transporter (P-FAT) Subfamily (TC 3.A.1.203) members Pxa1 and Pxa2 branch off from a common stem, as do the two MHC Peptide Transporter (TAP) Subfamily (TC 3.A.1.209) members Mdl1 and Mdl2. Pxa1 and Pxa2 are subunits of a heterodimeric transporter necessary for transport of long-chain fatty acids into peroxisomes (Shani et al., 1996). Mdl1 and Mdl2 are associated with the mitochondrial inner membrane and Mdl1 was shown to mediate peptide export from mitochondria (Young et al., 2001). Atm1 belongs to the Heavy Metal Transporter (HMT) Subfamily (TC 3.A.1.210) (Kispal et al., 1999) and is also located in the inner mitochondrial membrane, whereas Adp1p, Eye Pigment Precursor (EPP) Transporter Subfamily (TC 3.A.1.204), is located in the endoplasmic reticulum (Kumar et al., 2002). Thus, all six classes of half-size ABC transporters in Ascomycetes are associated with intracellular compartments. Probably their physiological role is not primarily devoted to drug efflux.

Among the full-size ABC transporters, the uncharacterized ORF YOL075C, genes related to the Sex Pheromone Exporter (STE family, ABCB, TC3.A.1.206), and among the Conjugate Transporters (CT Family, ABCC, TC 3.A.1.208) the branches of YOR1 (TC 3.A.1.208.3), YCF1 (TC 3.A.1.208.11), and BPT1 form subfamilies with one member in each of the five species like the half-size transporters (with the exception of BPT1 and YOL 075C orthologues in A. gossypii) (Fig. 2). In none of these branches gene duplications that had occurred in the ancestor of C. glabrata and S. cerevisiae (Wolfe & Shields, 1997; Wong et al., 2002; Dietrich, 2004; Kellis et al., 2004) have been retained in the recent genomes (see also below). In contrast, the YBT1/VMR1 branch of Conjugate Transporters (TC 3.A.1.208.12) has three S. cerevisiae and two C. glabrata members and only one of each of the other three species.

image

Figure 2.  Phylogenetic relationship of multidrug resistance-related ATP-binding cassette transporters (TC 3.A.1 families). The trees were constructed as described in the legend to Fig. 1. Half-size transporter families shown in Fig. 1 are included. ORF names for the different species are colour-coded: Saccharomyces cerevisiae black, Candida glabrata blue, Kluyveromyces lactis red, Kluyveromyces waltii brown, and Ashbya gossypii green. The highly variable pleiotropic drug resistance gene families are marked in yellow.

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The Pleiotropic Drug Resistance (PDR) Transporters (ABCG, TC 3.A.1.205), which play an important role in detoxification of endogenously and exogenously derived toxic compounds, represent a gene family that indicates a different evolution and does not allow to assign true orthologues. Altogether the S. cerevisiae genome has nine PDR genes whereas the small genome of A. gossypii contains four. The PDR5 branch (TC 3.A.1.205.1) is most expanded and has at least two representatives in each species. The well-characterized C. albicans drug resistance determinants CDR1, CDR2 and CDR4 belong to this branch (Table S1). Remarkably no close relatives of PDR12 (TC 3.A.1.205.3) exist in A. gossypii and K. waltii and PDR11/AUS1 (TC 3.A.1.205.8) orthologues are lacking in A. gossypii, K. lactis and K. waltiiPDR11 and AUS1 mediate sterol movement from the plasma membrane to the endoplasmic reticulum and can contribute to the uptake of exogenous sterol (Li & Prinz, 2004).

In summary, we conclude that expansion of the ABC superfamily occurred early in evolution, since the same set of subfamilies is found in all five Hemiascomycetes genomes examined. For most subfamilies a strong selection pressure against further duplications and gene loss has retained a 1 : 1 correspondence for each gene. Exceptions are some conjugate transporters and especially the PDR subfamilies, which have evolved and expanded during divergence of these species. These transporters are most directly involved in MDR.

MFS Drug:H+ Antiporters

  1. Top of page
  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information

The MFS-MDR transporters are classified into two families according to the number of predicted transmembrane spans: The Drug:H+ Antiporter-1 (12-Spanner; DHA1) Family, TC 2.A.1.2, and the Drug:H+ Antiporter-2 (14-Spanner; DHA2) Family, TC 2.A.1.3 (Table S2).

On average the DHA2 family is more variable between genomes than the DHA1 family and both are more variable than the ABC transporters, although again marked differences exist between the various branches.

The DHA1 family

In the DHA1 family (TC 2.A.1.2) 51 members were found in the five genomes. Comparison of primary sequences for all 51 genes gives two large clusters as described previously (Nelissen et al., 1997; Sá-Correia & Tenreiro, 2002) (Fig. 3). Cluster 1 is represented by the S. cerevisiae genes AQR1, QDR1/2, DTR1, QDR3, and Cluster 2 by TPO1, TPO2/3, TPO4, FLR1 (and HOL1). The function of the 12 S. cerevisiae family members as MDR determinants has been confirmed following their systematic individual disruption. As for the ABC transporters, each of these genes has orthologues in the other fungal genomes that are more closely related than the paralogues, giving nine distinct subclusters. It can be anticipated that the proteins within each subcluster will exhibit similar functional characteristics in spite of the fact that clustering reflects structural rather than substrate specificity constraints.

image

Figure 3.  Phylogenetic relationship of the DHA1 (TC 2.A.1.2) family of Drug:H+ antiporters (major facilitator superfamily). Gene coding as described in the legend to Fig. 2.

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The subclusters represented by the DTR1 and the AQR1 genes have the five-ended branches typical for the ABC transporters. As argued above, this strict conservation of gene number indicating selection pressure against gene duplication suggests that the drug resistance phenotypes associated with mutations in these genes may be only indirectly related to the physiological function of the encoded transporters. Indeed, the AQR1 gene product was recently shown to be involved in excretion of excess amino acids (Velasco et al., 2004) whereas Dtr1, which is localized in the prospore membrane, plays an important role in spore wall synthesis (Felder et al., 2002). It facilitates the translocation of bisformyl dityrosine, the major building block of the spore surface, through the prospore membrane. The expression of DTR1 in vegetative cells renders the cells slightly more resistant against antimalarial drugs and food-grade organic acid preservatives.

The products of the genes QDR1, QDR2 and QDR3 were characterized as drug efflux pumps. They are located in the plasma membrane and confer resistance to quinidine and barban and to the anticancer agents cisplatin and bleomycin (Nunes et al., 2001; Vargas et al., 2004; Tenreiro et al., 2005). Despite the fact that Qdr1 and Qdr2 are encoded by tandemly duplicated genes and are highly similar, Qdr2 is more effective in active export of quinidine than Qdr1. Orthologues of QDR1/2 are lacking in A. gossypii. K. lactis has two linked QDR1/2-like genes but this gene duplication apparently arose independently from the one in S. cerevisiae and is not found in K. waltii and C. glabrata. QDR3 is more distantly related and an orthologue is lacking in C. glabrata.

In cluster 2 of the DHA1 family the TPO genes dominate. TPO1 to TPO4 influence polyamine toxicity by detoxifying excess spermidine and putrescine (Albertsen et al., 2003). These transporters were originally proposed to be localized in the yeast vacuolar membrane, but recent experimental results have localized them in the plasma membrane (Sickmann et al., 2003). In S. cerevisiae two closely related genes, TPO2 and TPO3, are present whereas each of the other four species only has a single orthologue. Conversely, the TPO1 orthologue is duplicated in C. glabrata but unique in S. cerevisiae. The same is true for the FLR1 gene. Mutations in FLR1 confer resistance to fluconazole, 4-nitroquinoline-N-oxide (4-NQO), cycloheximide, the fungicide benomyl and the antitumor agent methotrexate (Alarco et al., 1997). Remarkably, members of cluster-2 genes are underrepresented in the more ancient genomes (FLR1 and TPO1 orthologues are lacking in the A. gossypii genome, FLR1 and TPO4 in K. lactis) but (some) are duplicated in S. cerevisiae or C. glabrata indicating that these subfamilies expanded more recently.

In contrast, extensive gene loss after divergence of the yeast species apparently occurred in the HOL1 branch. Whereas A. gossypii has three paralogues, K. lactis and K. waltii have two genes each, there is no HOL1-like gene in C. glabrata and only a single one in S. cerevisiae. The HOL1 genes form a separate cluster that is only distantly related to the cluster 1 and 2 of the DHA1 family. S. cerevisiae Hol1p was shown to be involved in the uptake of histidinol and was detected in the S. cerevisiae mitochondrial proteome (Sickmann et al., 2003; Wright et al., 1996), but otherwise HOL1 is poorly characterized.

The DHA2 family

The DHA2 family of drug: H+ antiporters with 14 predicted membrane-spanning segments (TC 2.A.1.3) consists of 35 members including 10 S. cerevisiae but only two A. gossypii genes (Fig. 4). Clustering by sequence similarity gives four distinct branches and several additional singletons or twin genes. Two of the branches, ATR1 and VBA3, have no A. gossypii but multiple Kluyveromyces members. The S. cerevisiae ATR1/SNQ1 gene product was one of the first yeast drug efflux pumps characterized. It is involved in resistance to aminotriazole and 4-NQO (Kanazawa et al., 1988; Gompel-Klein & Brendel, 1990). ATR1 is closely related to YMR279C and more distantly to YOR378W. The latter forms a separate branch with two ORFs from K. lactis and K. waltii.

image

Figure 4.  Phylogenetic relationship of the DHA2 (TC 2.A.1.3) Family of Drug:H+ antiporters (major facilitator superfamily). Gene coding as described in the legend to Fig. 2.

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The VBA3 branch contains two K. lactis ORFs and four S. cerevisiae genes (VBA3,YKR105C, AZR1 and SGE1). SGE1 is responsible for resistance to crystal violet, ethidium bromide, 10-N-nonyl acridine orange, malachite green and methyl methanesulphonate, AZR1 confers resistance to weak organic acids, ketoconazole and crystal violet (Ehrenhofer-Murray et al., 1998; Tenreiro et al., 2000). The finding that the proteins encoded by AZR1 and SGE1 are localized in the plasma membrane whereas VBA3 encodes a vacuolar protein suggests that there is functional divergence in the cluster of VBA3-related genes, at least in S. cerevisiae.

VBA1, VBA2 like VBA3 encode vacuolar membrane proteins that are apparently involved in transport of basic amino acids (Shimazu et al., 2005). The related VBA1 and VBA2 genes differ in that close homologues to VBA1 but not VBA2 exist in each of the genomes. Instead, five related genes, including the A. gossypii ORF AGR076C and two K. lactis genes, have no close relative in the S. cerevisiae genome, although synteny exists between AGR076C and the uncharacterized, distantly related ORF YDR119W (Dietrich, 2004). The functional link of the VBA branches to MDR remains to be established. Since only VBA1/2-type genes are present in all five genomes this may represent the minimum equipment for vacuolar transport in yeast.

The ORFs that do not cluster to VBA- or ATR1-like genes include K. lactis KNQ1, which was recently shown to confer resistance to 4-NQO, aminotriazole, bifonazole and ketoconazole (Takáčováet al., 2004). KNQ1 has a close relative in K. waltii only.

In summary, most gene families of MDR-related MFS transporters have also diverged early in evolution and orthologues of the S. cerevisiae genes are found in other species. The K. lactis genome has at least one ORF in each of the subfamilies with the exception of TPO4 and FLR1. However, the strict 1 : 1 correspondence for all five genomes typical of ABC transporters is found in a few branches only. There are many branches lacking an A. gossypii orthologue and the K. lactis KNQ1 branch is unique to Kluyveromyces. The DHA2 family shows the highest degree of variation and in several cases true orthologues cannot be assigned simply based on sequence comparison. Nevertheless, the phylogenetic trees are a good indication for potential functional equivalence.

Evolution of MDR gene families

  1. Top of page
  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information

To get further insight into the evolution of MDR-related gene families, synteny of individual membrane transporter orthologues was analysed. As an example, the environment of the DTR1 orthologues is shown (Fig. 5). In the genomes of K. lactis and K. waltii these ORFs (red arrows) are flanked by orthologues of S. cerevisiae genes found on chromosome XVI (YPL094C, YPL093W, YPL092W, YPL091W, bottom line), whereas DTR1/YBR180W itself is located on chromosome II (top line). In both Kluyveromyces species, the DTR1 orthologue is sandwiched between YPL092W and YPL093W and flanked by YBR179C orthologues, indicating that this represents the gene order before genome duplication. In line with this, S. cerevisiae genes to the right of DTR1 (YBR181/RPS6B etc.) have syntenic twin genes on chromosome XVI (YPL090C/RPS6A etc) reminiscent of the genome duplication. In the evolution of S. cerevisiae and C. glabrata (identical to S. cerevisiae, not shown) the hypothetical DTR1 twin gene must have been deleted from chromosome XVI. Conversely, YPL094C to YPL091W twin genes are lacking on chromosome II where DTR1 has been retained. Interestingly, the YPL092W/SSU1 orthologue flanking the DTR1 orthologue in Kluyveromyces encodes a sulfite transporter of the MFS family (Park & Bakalinsky, 2000) that exhibits weak sequence similarity to DTR1. An orthologue of SSU1 is lacking in A. gossypii suggesting an origin by DTR1 gene duplication or gene conversion in the Kluyveromyces lineage. Sequence similarity may thus have facilitated deletion of SSU1 or DTR1 in S. cerevisiae (and C. glabrata) on chromosome II and XVI, respectively.

image

Figure 5.  Evolution of the DTR1 locus. ORFs with significant sequence similarity are shown in the same colour. Chromosomes II and XVI of Saccharomyces cerevisiae are shown at the top and bottom, respectively, representing twin genes retained after genome duplication. The twin region of Candida glabrata located on chromosome H (not shown) has the same gene order as chromosome XVI of S. cerevisiae.

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Deletion of one or the other duplicated gene in a block of synteny is a common phenomenon in the S. cerevisiae and C. glabrata genomes. An easy access to such data is provided by the yeast gene order browser (http://wolfe.gen.tcd.ie/ygob/) (Byrne & Wolfe, 2005). This tool shows that most ABC transporter genes reside in blocks of synteny in which one of the twin genes has been lost. An exception is the PDR5/PDR15/PDR10 branch (Fig. 2). Whereas PDR10 is unique in S. cerevisiae in an otherwise conserved block of genes on chromosome XV, PDR5 and PDR15 are twin genes in syntenic blocks on chromosomes XV and IV, respectively. Already in the A. gossypii genome two members of the PDR5/PDR15/PDR10 branch are present. AGL42C is flanked by the rDNA repeat and ABR126W is flanked by the SNQ2/YPL058C orthologue ABR125C. This situation may have favoured the rapid evolution of the PDR subfamily. In K. lactis, there are two PDR5-related genes. KLLA0D03476 g is located adjacent to the SNQ2 orthologue KLLA0D03432 g, like in A. gossypii, whereas KLLA0F21692g/KlPDR5 is in a unique position in K. lactis, surrounded by genes syntenic with the A. gossypii and K. waltii genomes, but completely rearranged in S. cerevisiae. With genomic information from additional species becoming available it may be possible to trace back the evolution of the PDR family.

Putative regulatory sequences

  1. Top of page
  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information

The publicly available fungal genomic sequences also provide a rich source of information for the identification of conserved, functionally important noncoding sequences (Kellis et al., 2003). We have looked for sequence conservation in the upstream sequences of MDR membrane transporter genes in the five yeast species using the MEME motif discovery program (Bailey & Gribskov, 1998). Several conserved sequence motifs were found that may serve as novel protein binding sites (Table 1). Moreover, well-characterized regulatory elements were detected by the MEME and also by the YEASTRACT program (YEASTRACT, 2005). The PDR responsive elements (PDREs) and YRREs (Yrr1 responsive elements), which bind the partially redundant transcription factors Pdr1 and/or Pdr3 and Yrr1, respectively, play an important role in controlling expression of ABC transporters mostly of the pleiotropic drug resistance (PDR) family in S. cerevisiae and other yeasts. Indeed, putative PDREs and YRREs were found in the promoters of many PDR subfamily members (Table 1). Remarkably, the absence of such sites in A. gossypii suggests that regulation of PDR in this organism is independent of the PDR regulatory network. The program also identified the well-known stress response element STRE (AGGGG or CCCCT) in promoters of many ABC transporter genes. The biological targets of the response to stress/drugs are highly conserved in yeasts (Martinez-Pastor et al., 1996; Chiang et al., 2003). Interestingly, STREs were found in many K. lactis but not in S. cerevisiae ABC half size transporter promoters. Moreover, the presence of a putative Cbf1p-binding motif in ABC half-size promoters of each of the yeast species except S. cerevisiae is interesting and could be of significance. A motif binding War1 (WARE motif, CGCN6CCG), which is responsible for transcriptional induction of the S. cerevisiae PDR12 in response to weak-acid stress (Kren et al., 2003) is also present in the K. lactis PDR12 gene orthologue. This promoter shares several motifs, including a new motif GCCAAACGACC with the promoter of the SNQ2 orthologue KLLA0D03423, suggesting that these two K. lactis genes are similarly regulated.

Table 1.   Putative regulatory sites in promoters of ATP-binding cassette transporters
MotifSequence logo identifiedS. c. geneS. c.C. g.K. w.K. l.A. g.
  1. S. c., Saccharomyces cerevisiae; C. g., Candida glabrata; K. w., Kluyveromyces waltii; K. l., Kluyveromyces lactis; A. g., Ashbya gossypii.

Novel conserved motifsTaCCCCa (Kellis No.56)PXA1+++
PXA2++
MDL2++
ADP1++
CACgGagAcAcGGAAYOR1+++
YBT1++
TACCAGCCGgCPDR12++Gene absent+Gene absent
PDR5++
BPT1+Gene absent
PDRETCCgcGgaPDR5+++
PDR10+Gene absentGene absentGene absentGene absent
PDR15++++
YRREtCCGcggaaYOR1++++
NFT1/YBT1+
SNQ2+++
STRECCCCTPDR12+Gene absent+Gene absent
PDR15+++
STE6+
YBT1+
PXA1++
PXA2++
MDL1+
MDL2++
ADP1++
ATM1+
Cbf1paTCACgTGSTE6++
YOR1+
SNQ2++
YCF1++
PXA1+++
PXA2++
MDL1++
MDL2+
ATM1++++
ADP1+++

We expected to find more motifs conserved between species among the six classes of unique half-size ABC transporters. However, high scoring sequences were not found. Only in pair-wise comparisons significant similarities were detected, mostly in the PXA1, PXA2, ADP1 cluster. We speculate that physiological divergence and niche specialization, going along with adaptive processes, have changed the regulation of orthologous genes. Alternatively but less likely, the binding sites for some of these transcription factors may have diverged too far to be detected by our approach.

In general, the conservation of known binding sites across these species is roughly related to their phylogenetic distance, S. cerevisiae and C. glabrata performing far better than the rest. As suggested previously, the detection of common regulatory pattern by sequence comparison is most promising with very closely related species (Kellis et al., 2003). We conclude that the evolutionary distance between the five selected genomes is optimal to trace the evolution of coding sequences by sequence comparison. However, it is highly speculative to propose regulatory mechanisms solely on the basis of comparison of noncoding regions.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information

Comparison of gene families related to MDR between five Hemiascomycete genomes provided insight into their evolution as well as their possible functional role. Both superfamilies encoding ABC and MFS transporters have multiple members even in the small genome of A. gossypii, indicating that they arose before the divergence of these yeast species. The relative distance between those genes has been maintained throughout yeast evolution such that the recent representatives form subclusters of orthologues distinct from the paralogues. There are striking differences between the subfamilies with respect to the variability of gene number per species. For example, the genes of the six ABC half-size transporters are strictly limited to one copy per genome; the DHA2 family, on the other hand, shows great variations. We propose that these differences are primarily related to the function of the gene products and only partially to the location of the coding sequences in the genome. Among the numerous transporter genes originally characterized in genetic screens for drug resistance many have a role in transport of physiological substances across cellular membranes. These ones are typically found in the distinct five-ended branches, i.e. they are unique genes in each genome. Most likely, functional equivalence exists for the gene products of this class. Their influence on resistance towards toxic compounds may often follow only indirectly from their physiological function.

On the other hand, functional divergence is likely among those gene products where expansion of the subfamily had occurred. An example is the VBA3 cluster encoding vacuolar permeases as well as plasma membrane-localized transporters. Thus, the high variability among MFS transporters, in particular of those shown in Fig. 4, makes it difficult to predict function from sequence similarity.

For studies on the evolution of true drug efflux pumps the PDR family is most instructive. As opposed to many duplicated genes in other subfamilies, the expansion of the PDR family from four genes in A. gossypii to eight in S. cerevisiae does not reflect genome duplication. The phylogenetic trees rather indicate that gene amplification has occurred more recently and independently in each of the genomes.

The Kluyveromyces lactis genome can serve as an excellent scaffold to trace genome evolution. It contains a number of genes not found in the small genome of A. gossypii and contains many neighbouring genes of the ancient genome that are separated on different chromosomes in S. cerevisiae. We would like to point out that the genome environment in the ancient genomes may have functional relevance such that closer inspection of the K. lactis genome sometimes provides information about a possible functional context of an uncharacterized gene product.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information

Determination of orthologous ORFs

The sequences corresponding to the ABC and MFS membrane transporters are hypothetical translations of the indicated open reading frames. The Saccharomyces cerevisiae sequences (strain S288C) served as starting point and were obtained from the SGD website http://www.yeastgenome.org/. The genomic sequences of Candida glabrata strain CBS138 (http://cbi.labri.fr/Genolevures/elt/CAGL), Kluyveromyces lactis strain NRRLY-1140 (CBS2359) (http://cbi.labri.fr/Genolevures/elt/KLLA), Kluyveromyces waltii strain NCYC2644 and Ashbya (Eremothecium) gossypii (http://agd.unibas.ch/) (Dujon, 2004; Dietrich, 2004; Kellis et al., 2004) obtained from NCBI website (http://www.ncbi.nlm.nih.gov/) were scanned for orthologous and paralogous ORFs using TBLASTN (Altschul et al., 1990). Reciprocal best matches were considered as orthologous ORFs.

Multiple sequence alignment and evolutionary tree construction

Within each superfamily of membrane transporters analysed (ABC and MFS superfamily, respectively), the protein sequences obtained for five yeast species were aligned with the multiple sequence alignment program ClustalW at GenomeNet using default parameters (http://www.genome.jp). The program was used for phylogenetic tree construction and for the identification of gene clusters.

Analysis of gene order conservation

For each yeast species 15 kb of DNA sequence centred around the gene encoding the analysed membrane transporter was taken into consideration. The order and orientation of genes (annotated according to the Genbank entry), on each DNA fragment were then recorded. In addition, the yeast gene order browser (http://wolfe.gen.tcd.ie/ygob/) (Byrne & Wolfe, 2005) was consulted for comparison.

Motif searching and comparison

The putative regulatory regions of orthologous genes (approximately 1200 basepairs upstream of the translation start, basepairs in other coding regions were excluded) were taken for the search of significant motifs (the identification of putative regulatory sequences) using the Motif Alignment and Search Tool (Bailey & Gribskov, 1998). MEME program, version 3.0 (http://meme.sdsc.edu) was used on the sets of promoter sequences belonging to the orthologous genes in five yeast species, as well as on the sets of promoter sequences belonging to the same transporter subfamily for each yeast species separately or altogether, with the following parameters: motif width was allowed to range between 5-25 and 6-50, respectively, and both strands of the promoter were searched. For computationally predicted binding sites, occurrences were taken to be those listed in the MEME outputs for both motif widths.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information

We gratefully acknowledge financial support by DAAD travelling grant as well VEGA 1/2338/05 and DFG Br 921/6-1 grants.

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  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. ABC transporters
  5. MFS Drug:H+ Antiporters
  6. Evolution of MDR gene families
  7. Putative regulatory sequences
  8. Conclusions
  9. Methods
  10. Acknowledgement
  11. References
  12. Supporting Information

Table S1. List of all ABC transporter genes in six yeast species (including Candida albicans). Genes present in one copy in each genome analysed are marked in blue. Putative orthologs are listed in the same line. Table S2. List of genes in the major facilitator superfamily related to multidrug resistance. Comparison of five yeast species. Putative orthologs are listed in the same line.

FilenameFormatSizeDescription
FEMSYR+058+Table+S1.xls23KSupporting info item
FEMSYR+058+Table+S2.xls24KSupporting info item

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