Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3 +-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3 + gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3 +-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.


Introduction
Yeast vacuoles are large organelles that function as digestive compartments and also serve as major storage compartments for various amino acids [1][2][3]. In Saccharomyces cerevisiae, the vacuoles contain about 70-90% of the basic amino acids in cells, whereas acidic amino acids are mainly excluded from the organelles, with approximately 90% localized in the cytosol [2]. These differences in the distributions of amino acids suggest the presence of specific transport systems that operate across the vacuolar membrane. It has been reported that 10 amino acids are actively transported into the vacuoles in S. cerevisiae [4]. These active transport systems are probably co-transporters of H + and amino acids, which are driven by a proton electrochemical gradient generated by the vacuolar type H + -ATPase (V-ATPase) [4][5][6].
Several genes for vacuolar amino acid transporters have been identified and characterized in the budding yeast S. cerevisiae based on experiments using isolated vacuolar membrane vesicles [7][8][9][10][11][12][13]. Two gene families, i.e., AVT and VBA, were found to be involved in vacuolar amino acid transport. In the VBA family, which belongs to the major facilitator superfamily, it has been shown that Vba1p, Vba2p, and Vba3p are involved in the uptake of basic amino acids into vacuoles [8]. In the AVT family, which belongs to the amino acid/auxin permease family, Avt1p is involved in the vacuolar uptake of neutral amino acids and histidine [9,10]. Avt3p and Avt4p are involved in the extrusion of neutral and neutral/basic amino acids from vacuoles, respectively [9,12]. Avt6p is involved in the efflux of acidic amino acids [9,13], and Avt7p is involved in the efflux of several neutral amino acids from vacuoles [11]. Furthermore, other genes that belong to the amino acid/polyamine/choline family and the lysosomal cystine transporter family have been identified as vacuolar amino acid transporters [14,15].
Relatively fewer homologs of the vacuolar amino acid transporters have been found in the genome of the fission yeast Schizosaccharomyces pombe compared with S. cerevisiae, thereby implying less redundancy among the functions of its transporters. Therefore, S. pombe may be advantageous to understand the physiological roles of vacuolar amino acid transporters. Previously, based on phylogenetic analysis of S. cerevisiae and S. pombe genomic database, we found that the genes fnx1 + , fnx2 + , avt5 + , vba2 + , and atg22 + are homologs of the vacuolar amino acid transporters identified in S. cerevisiae. However, it is quite difficult to characterize the genes of vacuolar transporters in S. pombe using isolated vacuolar membrane vesicles because the vacuoles are too small in S. pombe and a procedure has not been established for purifying the vacuolar membrane vesicles from S. pombe cells. We also found that V-ATPase-dependent vacuolar compartmentalization had a large effect on amino acid uptake by S. pombe whole cells, so assessing the vacuolar transport activity of amino acids was possible using an indirect assay with whole cells of S. pombe [16]. Using this whole cell assay, we found that Fnx1p and Fnx2p are involved in the uptake of lysine, asparagine, and isoleucine into vacuoles [16]. In addition, Avt5p is involved in the vacuolar uptake of various amino acids [17]. Vba2p is involved in the uptake of basic amino acids into vacuoles [18], and Atg22p is involved in the uptake of several amino acids into vacuoles, as well as in the maintenance of cellular and vacuolar amino acid pools [19]. In any case, establishing an in vitro membrane vesicle system is indispensable for investigating the net transport activities of these transporters.
Under nitrogen starvation, cells utilize the vacuolar amino acid pool as a nitrogen source [20], which is important for maintaining cellular functions [20][21][22]. Thus, exporters of vacuolar amino acids are expected to be important for recycling amino acids for de novo protein synthesis or metabolic pathways [22]. However, the genes of the vacuolar amino acid exporters have not been well characterized in S. pombe. Recently, Mukaiyama et al. indicated that S. pombe Avt3p (SPAC3H1.09c, SpAvt3p), a homologue of S. cerevisiae Avt3p (YKL146w) and Avt4p (YNL101w), is involved in spore formation by S. pombe cells [23].
In this study, the avt3 + gene of S. pombe was heterologously expressed in S. cerevisiae cells, and characterized using isolated vacuolar membrane vesicles. We suggest that SpAvt3p is a vacuolar membrane transporter involved in the extrusion of amino acids from vacuoles.

Plasmid construction and fluorescence microscopy
To tag the SpAvt3p protein with green fluorescence protein (GFP) at its N-terminus, the open reading frame was amplified by PCR and subcloned into pTN54, a derivative of the thiaminerepressible expression vector pREP41 [29], thereby yielding pTN54-avt3 + . S. pombe cells transformed with pTN54-avt3 + were grown in MM medium without leucine and thiamine at 30°C for 20 h, and then labeled with FM4-64, a lipophilic dye for vacuolar membrane staining, as described previously [30].

Transport assays
Cells were cultured in SD+CA medium at 30°C, and then harvested at an OD 660 of 1.0-1.5. Vacuolar membrane vesicles of S. cerevisiae were prepared as described previously [8]. To measure the export activity of amino acids, vesicles were preloaded with L-[ 14 C]-labeled specific amino acids by incubating for 10 min at 25°C in an assay mixture (500 μL), which comprised 25 mM 2-(N-morpholino)ethanosulfonic (MES)-tris(hydroxymethyl) aminomethane (Tris) (pH 7.0), 5 mM MgCl 2 , 25 mM KCl, vesicles (200 μg of protein), and [ 14 C]-labeled amino acid (0.1 mM; 4.8-11.1 GBq/mmol). For the CCA treatment, vesicles were incubated with 1 μM CCA for 10 min at 25°C before preloading with [ 14 C]-labeled amino acid. The reaction was initiated by adding of 2 mM ATP and then stopped by diluting a 100 μL sample with 5 mL of ice-cold wash buffer (25 mM MES-Tris, pH 7.0, 5 mM MgCl 2 , 25 mM KCl) at specific time points. Vesicles were filtered through cellulose acetate membrane filters (0.45 μm; ADVANTEC, Japan) and washed with 5 mL of ice-cold wash buffer. Radioactivity was measured using a liquid scintillation counter with a xylene scintillator. The uptake activity of amino acids was determined as described previously [8].

Analysis of amino acids
Ten OD 660 units of cells were harvested and washed once with 2.5 mM potassium phosphate buffer (pH 6.0) containing 10 mM glucose, and 1.5 M sorbitol (for S. pombe) or 0.6 M sorbitol (for S. cerevisiae). Cupric ion treatment was used to prepare vacuolar fractions of S. pombe and S. cerevisiae [33,34]. The amino acid contents in the fractions were analyzed with an amino acid analyzer (Hitachi L-8800).

Immunoblot analysis
Vacuolar membrane vesicles (20 μg of protein) were applied to an SDS-PAGE gel containing 8% polyacrylamide and analyzed by immunoblotting. Anti-GFP serum was purchased from Invitrogen, and anti-vacuolar alkaline phosphatase (Pho8) antibody was obtained from Molecular Probes (Eugene, OR). Immunoblot signals were detected using the ECL system (GE Healthcare). Protein concentrations were determined by the method proposed by Lowry et al. [35] with bovine serum albumin as the standard.

Intracellular localization of GFP-Avt3p in S. pombe cells
Recently, we showed that Avt3p (YKL146w) and Avt4p (YNL101w) are involved in amino acid export from the vacuoles in S. cerevisiae [12]. Based on phylogenetic relationships with Avt3p and Avt4p, we found that Avt3p in S. pombe (SPAC3H1.09c, SpAvt3p) is the closest relative to these proteins in S. cerevisiae (the shared identities between SpAvt3p with Avt3p and Avt4p were 41% and 34%, respectively). SpAvt3p was predicted to possess 11-transmembrane domains using several programs. Fig 1A shows the topology model of SpAvt3p predicted using the SOSUI program (SOSUI; http://harrier.nagahama-i-bio.ac.jp/sosui/). The alignment of SpAvt3p homologs showed that a glutamate residue at position 469 (E469), which was embedded in the putative sixth transmembrane domain of SpAvt3p, was highly conserved not only in Avt3p and Avt4p in S. cerevisiae, but also in other eukaryotic homologs ( Fig 1A). This conserved glutamate residue was suggested to be essential for the activity of Avt4p in S. cerevisiae [12].
In addition to its homology with Avt3p and Avt4p, Mukaiyama et al. recently indicated that deletion of the avt3 + gene impaired sporulation of S. pombe cells [23], suggesting that SpAvt3p might play an important role in the export of amino acids from vacuoles to the cytosol to maintain cellular functions under starvation conditions. Therefore, we hypothesized that SpAvt3p could be a vacuolar amino acid exporter in S. pombe.
To investigate the subcellular localization of SpAvt3p in S. pombe cells, GFP was tagged at the N-terminus of SpAvt3p and expressed in S. pombe avt3Δ cells. The fluorescence image of GFP-SpAvt3p was merged with the signal of FM4-64, a specific dye for the vacuolar membrane, thereby indicating that SpAvt3p localized exclusively at the vacuolar membrane in S. pombe (Fig 1B).
Effects of avt3 + expression on the amino acid compositions in vacuoles and the vacuolar morphology of S. pombe To assess the involvement of SpAvt3p in the vacuolar extrusion of amino acids, we determined the amino acid contents of the vacuolar pools in avt3Δ or avt3 + -overexpressing S. pombe cells.
The S. pombe wild-type, avt3Δ, and avt3Δ/pTN54-avt3 + cells were cultured in MM medium without thiamine, and vacuolar amino acid pools were prepared using the cupric ion method [33]. As shown in Table 1, the vacuolar levels of threonine, serine, asparagine, glutamine, glycine, alanine, and proline, as well as basic amino acids (histidine, arginine, and lysine), were obviously increased in the avt3Δ cells compared with the wild-type cells. The avt3Δ mutation also had moderate effects on the levels of valine and tyrosine, but little effect on those of other amino acids. The increased levels of these amino acids in the vacuolar pool of avt3Δ cells were significantly reduced by the overexpression of avt3 + . These results suggest that SpAvt3p is involved in the efflux of these amino acids from vacuoles.
Interestingly, we found that the size of vacuoles in avt3Δ cells was strikingly increased compared with that in the wild-type cells, and decreased in avt3 + -overexpressing cells (Fig 2), thereby indicating that the avt3 + gene expression also affected the vacuolar morphology. SpAvt3p-dependent extrusion of amino acids from S. cerevisiae vacuolar membrane vesicles Our analysis of the vacuolar amino acid contents indicated that the overproduction of SpAvt3p decreased the levels of various neutral and basic amino acids in the vacuolar pools in S. pombe cells (Table 1). Further, we examined whether SpAvt3p is involved in the vacuolar export of amino acids by using isolated vacuolar membrane vesicles. Many vacuolar transporters have been characterized using isolated vacuolar membrane vesicles in S. cerevisiae [8][9][10][11][12]. However, the isolation of vacuolar membrane vesicles is difficult in S. pombe because a standard method has not been established for isolating vacuoles due to their small size. Thus, SpAvt3p was heterologously overproduced in S. cerevisiae cells to examine its transport activity. To reduce the background activities of efflux and influx of amino acids by vacuolar membrane vesicles, either the avt3Δavt4Δ double mutant [12] or the avt1Δavt3Δavt4Δ triple mutant [10] of S. cerevisiae was used as the host strain. Both GFP-SpAvt3p and GFP-SpAvt3p E496A localized exclusively at the vacuolar membrane in avt3Δavt4Δ and avt1Δavt3Δavt4Δ cells (Fig 3), and the cellular levels of these proteins in the total cell lysates were almost equal (data not shown).
To examine the export activity of amino acids, the vacuolar membrane vesicles were isolated from avt1Δavt3Δavt4Δ cells that overproduced SpAvt3p. The expression levels of GFP-SpAvt3p and GFP-SpAvt3p E469A in the vacuolar membrane vesicles were almost equal (Fig 4A). [ 14 C]labeled alanine or tyrosine was preloaded into vacuolar membrane vesicles for 10 min, and the  amounts of these [ 14 C]-labeled amino acids remaining in the vesicles after the addition of 2 mM ATP were determined. The efflux of these amino acids from the vesicles was slight in the absence of ATP. The amount of alanine, as well as tyrosine, remaining in the avt1Δavt3Δavt4Δ vesicles was reduced partially by the addition of ATP (Fig 4B, black circles). In agreement with the data of amino acid composition (Table 1), the ATP-dependent export of alanine was enhanced greatly by the overproduction of SpAvt3p and that of tyrosine was enhanced moderately (Fig 4B, black triangles). The overproduction of SpAvt3p E469A did not affect the activity (Fig 4B, black diamonds). As shown in Fig 4C, these export activities were inhibited by the addition of CCA, a specific inhibitor of V-ATPase, suggesting that the activity of SpAvt3p depends on a proton electrochemical gradient generated by the action of V-ATPase.
Effects of avt3 + expression on the uptake activity of basic amino acids by S. cerevisiae vacuolar membrane vesicles We examined whether the overproduction of SpAvt3p affected the transport activity of basic amino acids using isolated vacuolar membrane vesicles. It is difficult to detect the export activity of basic amino acids, which are imported naturally rather than exported from vacuoles, so we examined the effect of SpAvt3p overproduction on the net uptake of basic amino acids using vacuolar membrane vesicles without preloading. The vacuolar membrane vesicles were isolated from the wild-type or the avt3Δavt4Δ cells carrying an empty plasmid, pGPD-GFP-avt3 + , or pGPD-GFP-avt3 E469A . As shown in Fig 5, the ATP-dependent uptake of basic amino acids was higher in the vesicles from the avt3Δavt4Δ cells compared with those from the wildtype cells, indicating that the disruption of AVT3 and AVT4 blocked the efflux of basic amino acids from vacuoles. The overproduction of SpAvt3p significantly decreased the uptake activities (Fig 5, black triangles), whereas the overproduction of SpAvt3p E469A had no effect (Fig 5, black diamonds). These results suggest that SpAvt3p mediates the export of basic amino acids from vacuoles, as well as neutral ones.  The results of in vitro experiments using isolated vacuolar membrane vesicles indicated that SpAvt3p is involved in the export of neutral and basic amino acids from vacuoles (Figs 4 and 5). Further, we examined the effect of avt3 + gene overexpression on vacuolar amino acid compartmentalization in S. cerevisiae cells. The neutral and basic amino acids, but not the acidic ones, were highly accumulated in the vacuolar pool of avt3Δavt4Δ cells compared with that of the wild-type cells (Fig 6) [12]. In agreement with the results of the transport assay into the vacuolar membrane vesicles isolated from avt1Δavt3Δavt4Δ cells carrying an empty plasmid (circles), pGPD-GFP-avt3 + (triangles), or pGPD-GFP-avt3 E469A (diamonds). The export assay was performed in the presence (black symbols) or absence (white symbols) of 2 mM ATP. Preloaded vesicles were removed immediately before (0 min) or at 1, 2, 4, and 8 min after the addition of ATP, and collected on cellulose acetate membrane filters. The amount of preloaded [ 14 C]-labeled amino acids at 0 min was taken as 100%. The relative amounts trapped on the filters are shown. The values represent the mean ± SD based on at least three independent experiments. (C) Effects of CCA on ATP-driven alanine and tyrosine export. The experiments were performed as described above. Vacuolar membrane vesicles were incubated with 1 μM CCA for 10 min before loading with [ 14 C]-labeled amino acids. The amount of preloaded [ 14 C]-labeled amino acids at 0 min was taken as 100%. The relative amounts trapped on the filters at 8 min after the addition of ATP are shown. The values represent the mean ± SD based on at least three independent experiments: avt1Δavt3Δavt4Δ cells carrying an empty plasmid without (white bar) or with (black bar) CCA, and pGPD-GFP-avt3 + without (light gray bar) or with CCA (dark gray bar).
doi:10.1371/journal.pone.0130542.g004 (Figs 4 and 5), the increase in the vacuolar levels of these amino acids were decreased by overexpressing avt3 + in avt3Δavt4Δ cells, which suggests that SpAvt3p was functional in the vacuolar compartmentalization of amino acids in S. cerevisiae cells. By contrast, the overexpression of avt3 E469A had no effect on the vacuolar levels of neutral and basic amino acids in avt3Δavt4Δ cells. The levels of GFP-SpAvt3p and GFP-SpAvt3p E469A levels were almost equal according to western blotting with anti-GFP serum (data not shown), which indicates that the difference in their activities was not caused by unequal amounts of the overproduced proteins. These results suggest that the SpAvt3p transporter is involved in the export of various amino acids from vacuoles in S. pombe cells.

Discussion
In this study, we obtained evidence that SpAvt3p localizes to the vacuolar membrane (Fig 1) where it plays a crucial role in the compartmentalization of amino acids in the vacuoles of S. pombe cells ( Table 1). The overproduction of SpAvt3p in S. cerevisiae cells enhanced the efflux of alanine and tyrosine and reduced the uptake of basic amino acids by vacuolar membrane vesicles (Figs 4 and 5). The contents of amino acids in S. cerevisiae vacuoles were reduced by the overproduction of SpAvt3p (Fig 6). These results suggest that the SpAvt3p mediates the export of various amino acids from the vacuoles of S. pombe. The ATP-dependent export of alanine and tyrosine by vacuolar membrane vesicles was accelerated by the overproduction of SpAvt3p. Alanine was totally extruded from the vesicles isolated from the SpAvt3p-overproducing cells, whereas tyrosine still remained in the vesicles (Fig 4B). The residual amount of tyrosine in vesicles might be due to transport systems related to the ATP-dependent uptake of tyrosine. The ATP-dependent uptake activity of basic amino acids into the vesicles was impaired by the overexpression of avt3 + (Fig 5), which suggests that SpAvt3p is also involved in the export of basic amino acids. However, a method has not been established for detecting the ATP-dependent export activity of basic amino acids. In the transport assay, we used purified vacuolar membrane vesicles from the vba1Δvba2Δvba3Δ strain, in which the genes involved in the uptake of basic amino acids into vacuoles were disrupted [8], but basic amino acids were still highly accumulated in the vesicles after the addition of ATP. We hypothesize that unknown vacuolar transporters are responsible for the Vacuolar Amino Acid Exporter in Schizosaccharomyces pombe uptake of basic amino acids. It should be noted that Ypq1p, a member of the transporteropsin-G protein-coupled receptor superfamily, has been suggested to be involved in the vacuolar uptake of lysine and arginine [36]. Analyses of the export activity using the cells, in which the genes for Ypq1p and its homologs were disrupted, are currently in progress.
We suggest that SpAvt3p is a vacuolar exporter for basic and neutral amino acids; however, the phylogenetic analysis indicated that SpAvt3p shared higher similarity with Avt3p rather than Avt4p in S. cerevisiae. Avt3p and Avt4p are involved in the extrusion of neutral and neutral/basic amino acids from vacuoles, respectively [12]. After comparing the amino acid sequences of SpAvt3p, Avt3p, and Avt4p, we could not find any characteristic residue(s) that might determine the specificity for amino acids. Thus, the binding sites for the substrates of these amino acid exporters should be investigated further.
In normal growth conditions, S. pombe cells contain many small vacuoles. The vacuoles fuse rapidly when cells are suspended in water in response to hypotonic stress. By contrast, vacuole fission can occur when cells are under hypertonic stress [1,37], thereby suggesting that changes in the vacuolar size and compartment number may correspond to the uptake or release of water and ions by vacuoles. Furthermore, the lipid composition, proton homeostasis, and regulatory proteins are also important for vacuolar morphology [38][39][40]. We found that the vacuoles were larger in avt3Δ cells (Fig 2). It has been reported that the deletion of btn1 + , a homolog of the human Batten disease gene CLN3, results in enlarged and more alkaline vacuoles [41]. Interestingly, Btn1p is a vacuolar protein that appears to maintain the balance of cellular basic amino acid levels in yeast [42]. We postulate that the unusual levels of vacuolar amino acids in avt3Δ cells might have affected the vacuolar morphology. However, further experiments are needed to determine how SpAvt3p is involved in the vacuolar morphology.
During nitrogen starvation, the sporulation of yeast cells is impaired by atg mutations, which cause a defect in autophagy, a bulk protein degradation process that generates amino acids in vacuoles under nitrogen starvation [22,23]. Mukaiyama et al. reported that defects in the sporulation of fission yeast was caused by the insufficient expression of proteins due to a lack of amino acids in the cytosol, which suggests that vacuolar amino acid exporters are important during nitrogen starvation [23]. Interestingly, it has been reported that the avt3Δ mutant cells exhibit a defect in sporulation [23]. Our studies showed that amino acids were largely accumulated in the vacuolar pool of avt3Δ mutant in nutrient-rich condition (Table 1). Thus, we propose that a vacuolar amino acid exporter, SpAvt3p, may play an important role in recycling amino acids from vacuoles into the cytosol during nitrogen starvation in S. pombe cells. In addition, the predicted topology of SpAvt3p shows that the protein has a long hydrophilic region at its N-terminus ( Fig 1A). Surprisingly, we found that deleting the N-terminal region of SpAvt3p caused enlargement of the vacuoles (unpublished data), suggesting that the N-terminal region of SpAvt3p might be involved in its activity. Currently, we are studying the effects of the N-terminal region of SpAvt3p on spore formation and vacuolar amino acid compartmentalization under nitrogen starvation.
In this study, we successfully achieved the heterologous expression of S. pombe avt3 + in the vacuolar membrane of S. cerevisiae cells and characterized SpAvt3p as a vacuolar amino acid exporter. Previously, we determined the amino acid uptake activity of S. pombe Vba2p using isolated vacuolar membrane vesicles from vba2 + -overexpressing S. cerevisiae cells [43]. However, GFP-Atg22p and GFP-Avt5p did not localize appropriately to the vacuolar membrane in S. cerevisiae cells (unpublished data). Thus, the sorting signals of vacuolar membrane proteins require further elucidation.