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1 Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan
2 Research Center, Asahi Glass Co. Ltd, Yokohama, Kanagawa 221-8755, Japan
Correspondence
Kaoru Takegawa
takegawa{at}ag.kagawa-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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abc4 exhibited drug sensitivity, and a decreased accumulation of monochlorobimane, suggesting that both of the proteins encoded by these genes are involved in detoxification of xenobiotics, and vacuolar sequestration of glutathione S-conjugates.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Most, but not all family members, are membrane proteins (ABC transporters) active in the transport of a broad range of compounds, including xenobiotics, organic anions and cations, and conjugates of glutathione and glucuronic acid (Bauer et al., 1999). All ABC transporters share a similar molecular architecture that includes at least one nucleotide-binding domain (NBD), and several predicted integral membrane domains (transmembrane domains, TMDs). While the TMDs form the transmembrane channel, and are thought to contain the substrate-binding sites, the NBDs are the molecular motors that transform the chemical energy of ATP into protein conformational changes (Jones & George, 2004). While the NBDs and TMDs are normally arranged in duplicated forward (TMD-NBD)2 or reverse (NBD-TMD)2 configurations, half-size transporters have various topologies. The complete core structures for half-size transporters can be homo- or heterodimers.
Thirty-one ABC transporter genes are known in humans, and 55 in Caenorhabditis elegans (Dean et al., 2001a, b; Sheps et al., 2004). Animal ABC proteins are classified into eight subfamilies: ABCA–ABCH (Dean et al., 2001a, b). ABCA has no homologues in yeast, and ABCE and ABCF have no TMDs. ABCH proteins are not found in humans, but are found in Drosophila and budding yeast. The ABCH protein YDR061w in yeast possesses no TMDs (Dean et al., 2001b). Arabidopsis has over 100 ABC transporter genes (Sánchez-Fernández et al., 2001). Fewer ABC transporters are found in unicellular eukaryotes. Saccharomyces cerevisiae has 29 ABC proteins, and 23 of these have TMDs (Bauer et al., 1999; Decottignies & Goffeau, 1997). Candida albicans has 21 ABC transporters (Gaur et al., 2005).
In Sac. cerevisiae, ABC proteins are classified into six families corresponding to clusters of amino acid sequence similarity (Decottignies & Goffeau, 1997). Cluster I is equivalent to the mammalian ABCG family. Most Cluster I proteins have (NBD-TMD)2 configurations, and are divided into three subfamilies. Cluster I. 1 has eight members, including Pdr5p and Snq2p, which confer multidrug resistance (Decottignies & Goffeau, 1997; Bauer et al., 1999), while Cluster I. 2 has one member, YOL075C. Members of Cluster I. 1 and 2 are full-size molecules. Cluster I. 3 has one member, Adp1p, with an unknown function, but with a unique TMD-NBD-TMD topology.
Cluster II members comprise three subfamilies consisting of full-size molecules with a forward orientation, (TMD-NBD)2. Cluster II. 1 is equivalent to the mammalian ABCC family, while the ABCB family includes Cluster II. 2 and 3. Cluster II. 1 contains Ycf1p, Bpt1p and Ybt1p/Bat1p, which have been shown to transport glutathione conjugates, bile acid and the characteristic red pigment (ade-pigment) of mutants affected in the adenine biosynthetic pathway (Chaudhuri et al., 1996; Li et al., 1996; Sharma et al., 2002, 2003; Klein et al., 2002; Ortiz et al., 1997). Another Cluster II. 1 member, Yor1p, is known to provide resistance to oligomycin and other compounds (Katzmann et al., 1995; Cui et al., 1996; Decottignies et al., 1998). Cluster II. 2 has one member, Ste6p, which exports the a-factor pheromone (Kuchler et al., 1989). Cluster II. 3 proteins are half-size ABC transporters, and three members have been found in Sac. cerevisiae: Atm1p, Mdl1p and Mdl2p. Atm1p functions in the export of Fe–S clusters from mitochondria (Leighton & Schatz, 1995; Kispal et al., 1997). Mdl1p localizes to mitochondria, mediates peptide transport from mitochondria, and has a known homologue, Mdl2p (Young et al., 2001; Dean et al., 1994).
Cluster III proteins are also half-size ABC transporters equivalent to mammalian ABCD family members. Two members of this family, Pxa1 and Pxa2, play roles in the transport of long-chain fatty acids into peroxisomes (Shani et al., 1995; Shani & Valle, 1996). Cluster IV and VI members have no TMDs. Cluster V consists of an RNase L inhibitor homologue Rli1p, which was predicted to have TMDs (Decottignies & Goffeau, 1997), but has since been found to localize to the cytoplasm and nucleus, and to function in translation initiation and ribosome biogenesis (Dong et al., 2004).
Prior to the present study, six ABC transporters of Schizosaccharomyces pombe had been previously identified. Bfr1p confers brefeldin A (BfA) resistance (Nagao et al., 1995; Turi & Rose, 1995). Mam1p is known to function as a pheromone M-factor transporter (Christensen et al., 1997a), and Pmd1p is a multidrug efflux transporter that recognizes leptomycin B (Nishi et al., 1992). The sequence and expression pattern of abc1+ has been analysed, but its precise role remains to be defined (Christensen et al., 1997b). A half-size molecule, Atm1p, has a role in mitochondrial function (Chen & Cowan, 2003; Iwaki et al., 2005). Hmt1p is the best-characterized ABC transporter in fission yeast to date, and is required for cadmium tolerance, and sequestration of phytochelatin–Cd2+ complexes in vacuoles (Ortiz et al., 1995, 1992). Although phytochelatin belongs to the family of glutathione-related peptides having the structure (
-Glu-Cys)n-Gly (where n is 
2–11) (Cobbett, 2000), it has been shown that Hmt1p does not catalyse glutathione (GS) transport (Ortiz et al., 1995).
Identification of ABC transporter genes in fission yeast has been facilitated by the recent completion of the Sch. pombe genome sequencing project (Wood et al., 2002). A BLAST search of protein databases indicated that Sch. pombe contains 11 ABC transporters, which were assigned to subfamilies according to the Sac. cerevisiae classification system (Fig. 1a; Decottignies & Goffeau, 1997). Gene disruption was used to characterize the ABC tranporters. Localization of the ABC transporters was performed by GFP tagging. The results of the present study demonstrate that two ABC transporters are major vacuolar glutathione S-conjugate (GS-X) pumps in fission yeast.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Strains, media and materials.
Escherichia coli strain XL-1 Blue (Stratagene) was used for all cloning procedures. The Sch. pombe gene disruption mutants were constructed using wild-type strain YF016 (h– leu1-32 ura4-C190T ade7 : : ura4), which is a derivative of ARC039 (h– leu1-32 ura4-C190T). Standard rich medium (YES), synthetic minimal medium (MM) and YE for Sch. pombe cells were used as described (Moreno et al., 1991). FM4-64, MitoTracker Red CMXRos and monochlorobimane (MClB) were from Molecular Probes. All other chemicals were from Sigma or Wako Pure Chemicals.
Cloning and disruption of genes encoding ABC transporters.
Genes encoding ABC transporter proteins were cloned into pGEM-T EASY or pGEM-T vectors (Promega) following PCR amplification using the primers listed in Table 1. The resulting plasmids were digested with the restriction enzymes listed in Table 1, followed by insertion of a ura4+ gene cassette to generate gene disruption constructs (Fig. 1b
). The fission yeast ade7 strain YF016 was transformed by electroporation (Suga & Hatakeyama, 2001), with the PCR products amplified from these constructs. Gene disruptions were confirmed by colony-PCR, using appropriate primers.
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). SDS, at a concentration of 0.006 % (w/v), was added to medium containing BfA to enhance the toxicity of this inhibitor (Nagao et al., 1995). Growth was scored after 3 days.
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. The corresponding PCR products were digested with the appropriate restriction enzymes, and cloned into the corresponding sites of pTN197 derived from pREP41 (Nakamura et al., 2001), which is a thiamine-repressible expression vector.
Organelle staining and fluorescence microscopy.
Nuclei were stained by using the DNA-specific dye Hoechst 33342 (Dojindo Laboratories). Cells were collected by centrifugation, and suspended in 1 µg ml–1 Hoechst 33342 solution. After a 10 min incubation at room temperature, cells were washed twice with distilled water, and visualized by fluorescence microscopy.
Mitochondria were stained using the mitochondrion-specific dye MitoTracker Red CMXRos. Cells were suspended in 10 mM HEPES, pH 7.4, containing 3 % glucose, and MitoTracker Red CMXRos was added to yield a final concentration of 100 nM. After a 15 min incubation at room temperature, cells were visualized by fluorescence microscopy.
To visualize the fission yeast vacuole, cells were labelled with the lipophilic dye FM4-64, as described by Vida & Emr (1995). Briefly, 1 ml of exponentially growing cells in YES was harvested by centrifugation, and suspended in 0.5 ml YES medium containing 16 µM FM4-64, followed by incubation at 30 °C for 30 min, with shaking for pulse labelling. The labelled cells were then washed once with fresh medium, resuspended in 1 ml YES medium without dye, and incubated at 30 °C for 90 min. The cells were then pelleted by centrifugation, resuspended in distilled water to induce vacuolar fusion under hypotonic stress, and examined by fluorescence microscopy.
Uptake of MClB was visualized after harvesting exponential-phase cells, resuspending them in 1 ml YES medium containing 100 µm MClB, and incubating at 30 °C for 5 h. The cells were then pelleted by centrifugation, and washed twice with YES medium. The cells were viewed by fluorescence microscopy, and photographed using exposure times of 10 ms.
Stained cells were observed under a fluorescence microscope (model BX-60; Olympus) using a U-MWIG filter set (Olympus) for FM 4-64 and MitoTracker Red CMXRos, a U-MGFPHQ filter set (Olympus) for GFP, and a U-MWU filter set (Olympus) for Hoechst 33342 and MClB. Images were captured with a Sensys Cooled CCD camera using MetaMorph (Roper Scientific).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, Fig. 1a). Sch. pombe has no homologues of Cluster I. 3 or Cluster III members. In addition to the six ABC transporters identified in fission yeast prior to this study, five new ABC transporters were revealed by this analysis. These uncharacterized genes are named in Table 1.
Phenotypic characterization of all ABC transporters
All 11 Sch. pombe ORFs were cloned, and disruption mutants were constructed (Fig. 1b). All were found to be viable. The atm1 mutants grew slowly, and exhibited sensitivity to diamide (Fig. 2), H2O2 (0.01 %), DTT (5 mM), CuCl2 (1 mM), NaCl (0.3 M), LiCl (5 mM), G418 (10 µg ml–1), hygromycin B (15 µg ml–1), nalidixic acid (180 µg ml–1), caffeine (6 mM) and high temperature (37 °C) (data not shown). Growth of the other deletants was not inhibited by these compounds, or by high temperature. The growth of bfr1 cells was severely inhibited by BfA, cerulenin and clotrimazole, and slightly inhibited by cycloheximide (CHX; Fig. 2). The growth of pmd1 cells was also slightly inhibited by CHX, but less so by BfA than the wild type (Fig. 2). The hmt1 strain was found to be more sensitive to cadmium than any other deletants, and its growth was inhibited by clotrimazole (Fig. 2). Most of these mutants exhibited partial sensitivity to cerulenin, while atm1, pmd1 and abc2 were found to be tolerant. Other ions, including Ca2+ (0.1 M), Mn2+ (1 mM), Zn2+ (2mM), Mg2+ (10 mM), Hg2+ (10 µM) and Fe3+ (2 mM), chelators including EGTA (10 mM) and o-phenanthroline (30 µg ml–1), an alkaloid (caffeine, 2 mM), antibiotics, including amphotericin B (0.1 µg ml–1), nystatin (2 µg ml–1), staurosporine (1 µg ml–1) and tunicamycin (0.1 µg ml–1), a weak acid (acetic acid, 80 mM), and 4-nitroquinoline oxide (4-NQO) (Fig. 2), had no observed effect on the growth of these mutants (data not shown).
Mutations in ade7 of Sch. pombe (equivalent to ADE1 in S. cerevisiae) led to accumulation of characteristic red pigments (ade pigments) in vacuoles. The precursor of the ade pigments is toxic, and is conjugated by glutathione prior to transport to the vacuole mediated by a GS-X-transporting ATPase (GS-X pump) (Chaudhuri et al., 1996; Smirnov et al., 1967). These intermediates accumulate only when the mutants are grown in adenine-limiting medium. When cells are grown in adenine-limiting YE, atm1 mutants have been observed to form pale-pink colonies because of a mild petite phenotype (Iwaki et al., 2005). Other deletants were indistinguishable from the wild type, suggesting that multiple proteins may contribute to Sch. pombe GS-X pump activity.
Localization of ABC transporters in fission yeast
To confirm localization of fission yeast ABC transporters, GFP was fused to their C termini, and the tagged constructs were expressed under control of the attenuated nmt1 promoter (Fig. 3).
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; Huyer et al., 2004). Atm1-GFP and Mdl1-GFP co-localized with the mitochondrion, as visualized by MitoTracker Red CMXRos. Hmt1-GFP has previously been shown to localize to the vacuolar membrane (Iwaki et al., 2003). The patterns of fluorescence of three Cluster II. 1 proteins, Abc2-GFP, Abc3-GFP and Abc4-GFP, were similar to that of Hmt1-GFP, and co-localized with FM4-64, a vacuolar membrane-specific dye. Abc1-GFP and Pdr1-GFP were found in the cell perimeter and around the nucleus, as visualized by Hoechst 33342, in a typical endoplasmic reticulum (ER) pattern.
Search for ABC transporters required for red pigment accumulation
A defect in the ADE1 gene of Sac. cerevisiae leads to accumulation of the purine intermediate phosphoribosylaminoimidazole carboxylate (Fisher, 1969). Glutathione conjugates of the ade pigments are transported to the vacuole mediated by the Cluster II. 1 proteins Ycf1p, Bpt1p and Ybt1p/Bat1p (Chaudhuri et al., 1996; Sharma et al., 2002, 2003). Studies in Sac. cerevisiae have shown that colony colour of ade1 and ade2 mutants is a good indicator of GS-X pump activity.
Mutations in gcs1+, encoding the first enzyme in glutathione biosynthesis, -glutamylcysteine synthetase, exhibit a defect in pigment formation, indicating that the ade pigment is probably a glutathione conjugate in fission yeast (Chaudhuri et al., 1996). However, the ade-pigment pump of Sch. pombe remains to be characterized. To date, the only finding is that it is distinct from the vacuolar pump Hmt1p (Chaudhuri et al., 1996).
Phenotypes of single-gene deletants, localization of GFP-tagged proteins, and similarity to budding yeast Ycf1p, strongly suggest that ade-pigment transport is mediated by two or more Cluster II. 1 proteins. Therefore, we constructed a quadruple deletion mutant, abc1 abc2 abc3 abc4. This mutant formed paler colonies than the wild type (Fig. 4a). While the abc2 mutant formed a somewhat pale colony, its tolerance for xenobiotics was indistinguishable from that of the wild type. When two Cluster II. 1 proteins, abc2 and abc4, were deleted, the double disruption mutant formed pale colonies similar to the quadruple mutant. This double deletion caused sensitivity to CHX and 4-NQO (Fig. 4b). Unlike Sac. cerevisiae ycf1 cells, Cd2+ had no effect on growth of the double disruptant. Although all Cluster II. 1 proteins share a high degree of similarity to one another, disruption of abc3 or abc1 had no additional effect on colony colour, or tolerance for xenobiotics. From these results, we conclude that two Cluster II. 1 proteins, Abc2p and Abc4p, are responsible for ade-pigment transport into vacuoles.
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abc4 hmt1, was constructed. While this mutant was found to be more sensitive to 0.1 mM CdCl2 than hmt1, the double mutant abc2 abc4 was able to grow as described above (Fig. 4c). This observation suggests that transport of glutathione–Cd2+ plays a significant role in Cd2+ tolerance, but a minor role relative to phytochelatin–Cd2+ transport by Hmt1p.
Vacuolar accumulation of MClB-GS in vivo
MClB, a membrane-permeable nonfluorescent compound, is specifically conjugated with glutathione by cytosolic glutathione S-transferases to generate the intensely fluorescent membrane-impermeable S-conjugate MClB–glutathione (Shrieve et al., 1988; Oude Elferink et al., 1993; Ishikawa et al., 1994). GS-X pumps exhibit activity toward a broad range of S-conjugates, including MClB–glutathione (Ishikawa et al., 1994; Li et al., 1996). Thus, MClB is commonly used as a sensitive probe for monitoring the intracellular transport and localization of its S-conjugate. In Sac. cerevisiae, vacuolar transport of MClB–glutathione is mediated by Ycf1p (Li et al., 1996).
To determine if Cluster II. 1 proteins play a role in vacuolar accumulation of MClB–glutathione in intact cells, wild-type and mutant cells were observed by fluorescence microscopy after incubation in medium containing MClB (Fig. 5). Wild-type cells exhibited an intense punctate fluorescence, corresponding to vacuoles, as determined by Nomarski optics. A similar pattern was observed in the abc4 mutant, although fluorescence was slightly lower in the abc2 mutant. Accumulation of MClB decreased remarkably when both abc2 and abc4 were deleted. Deletion of the two Cluster II. 1 genes abc3 and abc1 had little effect on accumulation of MClB under these conditions. These results indicate that activity of the GS-X pump was affected significantly by loss of Abc2p and Abc4p.
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). Thus, it is believed that a factor depending on the H+ gradient across the vacuolar membrane is partly responsible for GS-X transport into vacuoles, although the ABC transporters are thought to play the major role (Sharma et al., 2003; Penninckx, 2002). To test the contribution of V-ATPase to pigmentation, we constructed a vma3 disruptant in an ade7 background. As shown in Fig. 6(a), the vma3 mutant formed white colonies. This was partly a consequence of a petite (respiration-defective) phenotype (data not shown), although less pigmentation was observed in the vma3 mutant than in the mild petite atm1 mutant.
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). While intense fluorescence was observed in the vacuoles of vma3, MClB–glutathione was also detected in the cytosol. This cytosolic fluorescence was not observed in other strains examined (compare with Fig. 5). These observations suggest that an unknown transporter may be involved in GS-X transport to vacuoles, and that its activity may depend on the H+-gradient generated by V-ATPases. Sorting to the vacuoles may also be affected because of the severe defect reported in vacuolar protein sorting (Iwaki et al., 2004). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Decottignies & Goffeau, 1997).
In Sch. pombe, a phenotypic survey of all ABC transporter gene disruptants revealed that only the atm1 cells showed a decrease in vacuolar accumulation of the ade pigment. Further characterization demonstrated that atm1 exhibited a mild petite phenotype, and was defective in vacuolar function, thereby affecting colony colour (Iwaki et al., 2005). This observation strongly suggested that multiple transporters are involved in vacuolar accumulation of ade pigment and GS-X, raising the question of which is the actual pump that transports ade pigment directly into vacuoles. We speculated that it might be one of the Cluster II. 1 proteins, similar to Ycf1p, Bpt1p and Ybt1p/Bat1p of Sac. cerevisiae. While four Cluster II. 1 proteins were found in fission yeast, notable differences in colour were not observed in these single-gene disruptants. Because Cluster II. 1 proteins have redundant functions, significant reduction in pigmentation may not be detectable in the single deletants. To determine if Cluster II. 1 proteins transport ade pigment into vacuoles, all Cluster II. 1 genes were disrupted. Quadruple disruption of Cluster II. 1 proteins revealed that transport of GS-X is mediated by Cluster II. 1 proteins. Moreover, a major role for Abc2p and Abc4p in fission yeast was discovered, because there was almost no difference in pigmentation and uptake of MClB between the abc2 abc4 mutant and the quadruple mutant. These observations indicate that the contributions of Abc1p and Abc3p are minor, and that the ade pigment is not a preferred substrate of these Cluster II. 1 proteins.
While loss of the ABC transporters caused a reduction in colony colour, pigmentation was not completely lost in these mutants. Pigmentation may derive from the contribution of a transporter dependent on the vacuolar H+-ATPase-generated pH gradient. In Sac. cerevisiae, the contributions of this transporter are thought to be minor compared with the contributions of the Cluster II. 1 proteins (Sharma et al., 2003; Penninckx, 2002). However, these contributions may not be negligible in Sch. pombe, because loss of V-ATPase activity gave rise to white colonies lacking pigmentation. As previously reported, deletion of vma3 encoding V-ATPase subunit c causes a severe defect in vacuolar protein sorting of carboxypeptidase Y, although vacuolar ABC transporter Hmt1p was correctly sorted to vacuoles (Iwaki et al., 2004). These results suggest that loss of V-ATPase activity results in loss of colony colour due to a combination of effects. In V-ATPase mutants, unknown H+-gradient-dependent mechanisms will be impaired. Elevated vacuolar pH may also affect ABC transporter activity, thereby reducing vacuolar accumulation of GS-X. Potential sorting defects of these transporters must also be considered.
The Cd2+ sensitivity of the abc2 abc4 hmt1 mutant indicates that GS-X transport also plays a role in heavy-metal tolerance. It has been shown that GS-X transport by Ycf1p is required for detoxification of heavy metals in budding yeast (Li et al., 1996). However, GS-metal transport in fission yeast is not yet well understood because Sch. pombe has been observed to synthesize phytochelatin to chelate heavy metals with subsequent transport of the phytochelatin–metal complex into vacuoles by Hmt1p (Cobbett, 2000; Ortiz et al., 1992, 1995). hmt1 was found to be Cd2+ sensitive, and to form faint pink colonies on rich medium containing 20 µM Cd2+ (data not shown). This suggests that GS-X transport by Abc2p and Abc4p is activated in hmt1 cells. A double disruption mutant, abc2 abc4, and a quadruple deletion mutant, abc1 abc2 abc3 abc4, were able to grow in the presence of 0.1 mM Cd2+, strongly suggesting that the contribution of glutathione–metal transport to heavy-metal tolerance is much less significant than phytochelatin–metal transport by Hmt1p.
The number of ABC transporters found in fission yeast is about half that found in Sac. cerevisiae, which has 23 ABC transporters, although this number contains the duplicated ORFs YKR103w/YKR104w. The Cluster III proteins, including Pxa1 and Pxa2, necessary for transport of long-chain fatty acids into peroxisomes in budding yeast (Shani et al., 1995; Shani & Valle, 1996), and the Cluster I. 3 proteins, were not found in the fission yeast genome. Recently, it was reported that budding yeast takes up exogenous sterol, and that two Cluster I proteins, Aus1p and Pdr11p, facilitate sterol cycling between the plasma membrane and ER (Li & Printz, 2004). In contrast, exogenous sterol cannot be incorporated into fission yeast cells (Hughes et al., 2005), presumably due to lack of the corresponding Cluster I proteins. The budding yeast contains eight Cluster I. 1 proteins, and one Cluster I. 2 protein, involved in multidrug resistance (Bauer et al., 1999; Decottignies & Goffeau, 1997), while only two Cluster I proteins were found in the Sch. pombe genome. However, fission yeast has one additional Cluster II. 2 protein, Pmd1p, which is involved in leptomycin B resistance (Christensen et al., 1997a). Pmd1p may functionally substitute for Cluster I proteins, some of which have apparently been lost during evolution. These findings indicate that most fission yeast ABC transporters may have multiple specificities or functions, except for the phytochelatin transporter Hmt1p (Ortiz et al., 1995, 1992).
In this report, all ABC transporters were characterized and localized by GFP tagging and fluorescence microscopy. ER-localized ABC transporters within the Cluster I. 1, I. 2 and II. 1 families have not been reported in budding yeast to date, but two fission yeast proteins exhibited an ER pattern of fluorescence. ER localization of Adp1p, a Cluster I. 3 protein, has been inferred from direct assay (Kumar et al., 2002), and human Cluster III (ABCD) proteins are known to localize in the ER (Bresnahan et al., 1997). In fission yeast, overexpression on a multicopy plasmid might cause aberrant mislocalization of Abc1p and Pdr1p to the ER. Abc1-GFP and Pdr1-GFP function could not be confirmed by complementation of the disruption mutants because these mutants exhibited no apparent phenotypes relative to the tested inhibitors. Further analysis is needed to confirm correct localization of the tagged proteins, and expression levels of the ABC transporter genes.
The present study also detected an additional and intriguing phenotype of a fission yeast strain lacking Pmd1p. While Pmd1p was originally isolated as a homologue of human P-glycoprotein, which catalyses efflux of leptomycin B (Nishi et al., 1992), we found that the pmd1 mutant had an increased tolerance for BfA, indicating that Pmd1p is directly or indirectly involved in uptake of BfA. Lactococcus lactis LmrA, which is a homologue of human P-glycoprotein, can take up and efflux ethidium bromide (Balakrishnan et al., 2004). Ethidium uptake by LmrA has been found to be mediated by proton-ethidium symport, without a direct requirement for ATP (Venter et al., 2003). Similar mechanisms may be conserved in Pmd1p. The present results provide an important starting point for future detailed analysis of the functions of fission yeast ABC transporter proteins.
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ACKNOWLEDGEMENTS |
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Received 28 February 2006;
revised 15 April 2006;
accepted 19 April 2006.
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