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Avt5p is required for vacuolar uptake of amino acids in the fission yeast Schizosaccharomyces pombe Soracom Chardwiriyapreecha a , Hiroyuki Mukaiyama b , Takayuki Sekito a , Tomoko Iwaki a , Kaoru Takegawa b , Yoshimi Kakinuma a, * a Department of Applied Bioresource Science, Faculty of Agriculture, Ehime University, Ehime 790-8566, Japan b Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan article info Article history: Received 11 March 2010 Accepted 6 April 2010 Available online 11 April 2010 Edited by Francesc Posas Keywords: Vacuolar H + -ATPase Vacuolar amino acid transporter Nitrogen starvation Concanamycin A Schizosaccharomyces pombe abstract We identified SPBC1685.07c of Schizosaccharomyces pombe as a novel vacuolar protein, Avt5p, with similarity to vacuolar amino acid transporters Avt5p from Saccharomyces cerevisiae. Avt5p localizes to the vacuolar membrane and upon disruption of avt5, uptake of histidine, glutamate, tyrosine, arginine, lysine or serine was impaired. During nitrogen starvation, the transient increase of vacu- olar lysine transport observed for wild-type cells still occurred in the mutant cells, however, uptake of glutamate did not significantly increase in response to nitrogen starvation. Our results show that under diverse growth conditions Avt5p is involved in vacuolar transport of a selective set of amino acids. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction In all living organisms, maintenance of the cytosolic amino acid concentration at a constant level is crucial for effective protein syn- thesis. Up to now, the regulatory mechanism for the amino acid homeostasis including uptake from extracellular environment and biosynthesis has been extensively investigated in the budding yeast Saccharomyces cerevisiae [1,2]. The yeast vacuole is the large- size organelle that functions as a digestive compartment and also serves as a major storage compartment for amino acids [3]. In S. cerevisiae, several amino acids, especially basic amino acids, are highly concentrated in vacuoles, whereas acidic amino acids are particularly localized in the cytosol [4–6]. It has been reported that 10 amino acids were taken up by active transport into vacuoles of S. cerevisiae [7,8]. Active transport of these amino acids was driven by a proton electrochemical gradient across the vacuolar mem- brane, generated by a vacuolar H + -ATPase (V-ATPase) and likely mediated by an H + /amino acid antiport system [9]. The vacuole is thus the selective compartment for regulating the cytosolic level of amino acids [8]. So far, several genes for vacuolar amino acid transporters are identified in S. cerevisiae [8,10,11]. Two gene families, i.e. the vacu- olar basic amino acids transporter (VBA) family, which belongs to the major facilitator superfamily (MFS) [12], and the amino acid vacuolar transport (AVT) family, were found to be involved in vac- uolar amino acid transport. Three members of the VBA family, Vba1p, Vba2p and Vba3p, are involved in uptake of basic amino acids into vacuoles [10], whereas the function of Vba4p and Vba5p is still unknown. In the AVT family, which belongs to the amino acid/auxin permease family (AAAP) [13], Avt1p is involved in up- take of neutral amino acids, whereas Avt3p and Avt4p participate in extrusion of these amino acids and Avt6p in efflux of acidic ami- no acids from vacuoles [11]. However, the function of Avt2p, Avt5p and Avt7p has not been characterized. In addition, other vacuolar amino acid transporters, i.e. that belong to the amino acid–poly- amine–choline family (APC) [13] and the lysosomal cystine trans- porter family (LCT) [14], have been reported in S. cerevisiae. Little is known about vacuolar amino acid transport in the fission yeast Schizosaccharomyces pombe. Recently, however, we found that the MFS family proteins Fnx1p and Fnx2p of S. pombe were phyloge- netically related to Vba2p of S. cerevisiae and involved in the up- take of lysine, asparagine or isoleucine into vacuoles [15]. Furthermore, determination of total and vacuolar pools of amino 0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.04.012 Abbreviations: V-ATPase, vacuolar type H + -ATPase; DMSO, dimethyl sulfoxide; CCA, concanamycin A; GFP, green fluorescent protein; 2-DG, 2-deoxy-D-glucose; ABC, ATP-binding cassette * Corresponding author. Address: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan. Fax: +81 89 946 9994. E-mail address: [email protected] (Y. Kakinuma). FEBS Letters 584 (2010) 2339–2345 journal homepage: www.FEBSLetters.org

Avt5p is required for vacuolar uptake of amino acids in the fission yeast Schizosaccharomyces pombe

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FEBS Letters 584 (2010) 2339–2345

journal homepage: www.FEBSLetters .org

Avt5p is required for vacuolar uptake of amino acids in the fission yeastSchizosaccharomyces pombe

Soracom Chardwiriyapreecha a, Hiroyuki Mukaiyama b, Takayuki Sekito a, Tomoko Iwaki a,Kaoru Takegawa b, Yoshimi Kakinuma a,*

a Department of Applied Bioresource Science, Faculty of Agriculture, Ehime University, Ehime 790-8566, Japanb Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

a r t i c l e i n f o

Article history:Received 11 March 2010Accepted 6 April 2010Available online 11 April 2010

Edited by Francesc Posas

Keywords:Vacuolar H+-ATPaseVacuolar amino acid transporterNitrogen starvationConcanamycin ASchizosaccharomyces pombe

0014-5793/$36.00 � 2010 Federation of European Biodoi:10.1016/j.febslet.2010.04.012

Abbreviations: V-ATPase, vacuolar type H+-ATPaseCCA, concanamycin A; GFP, green fluorescent proteiABC, ATP-binding cassette

* Corresponding author. Address: Faculty of AgriculTarumi, Matsuyama 790-8566, Japan. Fax: +81 89 94

E-mail address: [email protected] (Y. Kakin

a b s t r a c t

We identified SPBC1685.07c of Schizosaccharomyces pombe as a novel vacuolar protein, Avt5p, withsimilarity to vacuolar amino acid transporters Avt5p from Saccharomyces cerevisiae. Avt5p localizesto the vacuolar membrane and upon disruption of avt5, uptake of histidine, glutamate, tyrosine,arginine, lysine or serine was impaired. During nitrogen starvation, the transient increase of vacu-olar lysine transport observed for wild-type cells still occurred in the mutant cells, however, uptakeof glutamate did not significantly increase in response to nitrogen starvation. Our results show thatunder diverse growth conditions Avt5p is involved in vacuolar transport of a selective set of aminoacids.� 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

In all living organisms, maintenance of the cytosolic amino acidconcentration at a constant level is crucial for effective protein syn-thesis. Up to now, the regulatory mechanism for the amino acidhomeostasis including uptake from extracellular environmentand biosynthesis has been extensively investigated in the buddingyeast Saccharomyces cerevisiae [1,2]. The yeast vacuole is the large-size organelle that functions as a digestive compartment and alsoserves as a major storage compartment for amino acids [3]. In S.cerevisiae, several amino acids, especially basic amino acids, arehighly concentrated in vacuoles, whereas acidic amino acids areparticularly localized in the cytosol [4–6]. It has been reported that10 amino acids were taken up by active transport into vacuoles ofS. cerevisiae [7,8]. Active transport of these amino acids was drivenby a proton electrochemical gradient across the vacuolar mem-brane, generated by a vacuolar H+-ATPase (V-ATPase) and likelymediated by an H+/amino acid antiport system [9]. The vacuole is

chemical Societies. Published by E

; DMSO, dimethyl sulfoxide;n; 2-DG, 2-deoxy-D-glucose;

ture, Ehime University, 3-5-76 9994.uma).

thus the selective compartment for regulating the cytosolic levelof amino acids [8].

So far, several genes for vacuolar amino acid transporters areidentified in S. cerevisiae [8,10,11]. Two gene families, i.e. the vacu-olar basic amino acids transporter (VBA) family, which belongs tothe major facilitator superfamily (MFS) [12], and the amino acidvacuolar transport (AVT) family, were found to be involved in vac-uolar amino acid transport. Three members of the VBA family,Vba1p, Vba2p and Vba3p, are involved in uptake of basic aminoacids into vacuoles [10], whereas the function of Vba4p and Vba5pis still unknown. In the AVT family, which belongs to the aminoacid/auxin permease family (AAAP) [13], Avt1p is involved in up-take of neutral amino acids, whereas Avt3p and Avt4p participatein extrusion of these amino acids and Avt6p in efflux of acidic ami-no acids from vacuoles [11]. However, the function of Avt2p, Avt5pand Avt7p has not been characterized. In addition, other vacuolaramino acid transporters, i.e. that belong to the amino acid–poly-amine–choline family (APC) [13] and the lysosomal cystine trans-porter family (LCT) [14], have been reported in S. cerevisiae. Littleis known about vacuolar amino acid transport in the fission yeastSchizosaccharomyces pombe. Recently, however, we found that theMFS family proteins Fnx1p and Fnx2p of S. pombe were phyloge-netically related to Vba2p of S. cerevisiae and involved in the up-take of lysine, asparagine or isoleucine into vacuoles [15].Furthermore, determination of total and vacuolar pools of amino

lsevier B.V. All rights reserved.

2340 S. Chardwiriyapreecha et al. / FEBS Letters 584 (2010) 2339–2345

acids by fractionation after cupric ion-treatment indicated vacuo-lar compartmentalization of various amino acids, such as threo-nine, serine, glutamine, glycine, isoleucine, tyrosine,phenylalanine and tryptophan, as well as basic amino acids [6]. Itis noteworthy that most of aspartate and more than 15% of gluta-mate were retained in the vacuole of S. pombe [6]. These resultssuggested that S. pombe and S. cerevisiae have slightly different vac-uolar compartmentalization systems.

Here, we focus on S. pombe SPBC1685.07c, designated as avt5+,which is phylogenetically related to the vacuolar amino acid trans-porters Avt5p and Avt6p of S. cerevisiae [11]. We report that Avt5pis a vacuolar membrane protein involved in acidic, neutral, and ba-sic amino acid uptake into vacuoles.

2. Materials and methods

2.1. Strains, media, and materials

S. pombe strains used in this study were the wild-type strainARC039 (h� leu1-32 ura4-C190T), the Dfnx1 mutant (h� leu1-32ura4-C190T fnx1::ura4) [15] and the Davt5 mutant (h� leu1-32ura4-C190T avt5::ura4) [16] constructed as previously described.These S. pombe cells were cultured as described elsewhere [17]by standard rich medium (YES) and synthetic minimal medium(MM). For nitrogen starvation, MM containing no nitrogen source(MM-N) was used [18]. Concanamycin A (CCA) and FM4-64 werepurchased from Wako Pure Chemicals Co. (Osaka, Japan) and Invit-rogen Corp. (Carlsbad, CA, USA), respectively, and dissolved in di-methyl sulfoxide (DMSO). L-[14C] labeled materials werepurchased from American Radiolabeled Chemical (St. Louis, MO,USA; L-arginine, L-lysine, L-tyrosine, L-asparagine and 2-deoxy-D-glucose), GE healthcare (Buckinghamshire, England; L-histidine, L-isoleucine, L-glutamine, L-valine, L-glutamic acid), and Perkin ElmerInc. (Waltham, MA, USA; L-serine).

2.2. Plasmid construction and fluorescence microscopy

To tag the Avt5p protein with green fluorescent protein (GFP),the ORF was amplified by PCR and subcloned into pTN197, a deriv-ative of the thiamine-repressible expression vector pREP41 [19],resulting in pTN197/avt5. Wild-type cells transformed with thisplasmid were grown in MM medium without leucine at 30 �C for20 h and examined with an Olympus BX-60 fluorescence micro-scope using a U-MGFPHQ filter. Vacuoles were labeled with FM4-64 as described elsewhere [15]. After exposure to lipophilic dyeFM4-64, cells were incubated with sterilized distilled water for3 h to induce vacuolar fusion. Images were captured with a SenSyscooled charge-coupled device (CCD) camera (Roper Scientific)using MetaMorph software (Molecular Devices).

2.3. Transport assay

Amino acid uptake was measured as described previously [15]and values were averaged from three independent experiments. Inthe case of CAA treatment, cells were preincubated for 30 min at30 �C with 10 lM CCA (in DMSO). For nitrogen starvation conditions,cells cultured in YES medium were harvested during logarithmicphase, washed twice with sterilized water, and then incubated inMM-N medium for the indicated durations. Reactions were initiatedby adding specific labeled substrates, (L-[U-14C]histidine (final con-centration 0.04 mM; 11.6 GBq/mmol), L-[U-14C]lysine (0.04 mM;11.8 GBq/mmol), L-[U-14C]arginine (0.04 mM; 12.9 GBq/mmol), L-[U-14C]glutamic acid (0.04 mM; 9.36 GBq/mmol), L-[U-14C]tyrosine(0.04 mM; 16.65 GBq/mmol), L-[U-14C]glutamine (0.04 mM;9.47 GBq/mmol) L-[U-14C]asparagine (0.04 mM; 10.4 GBq/mmol),

L-[U-14C]isoleucine (0.04 mM; 11.1 GBq/mmol), L-[U-14C]serine(0.04 mM; 4.78 GBq/mmol), L-[U-14C]valine (0.04 mM; 9.58 GBq/mmol) or L-[U-14C]2-DG (5 mM; 11.1 GBq/mmol). As a negative con-trol, samples were incubated with DMSO.

2.4. Sequence analysis

ClustalW software (http://www.ebi.ac.uk/clustalw/) was usedto align multiple sequences and transmembrane domains werepredicted by means of the SOSUI algorithm (http://bp.nuap.na-goya-u.ac.jp/sosui/).

3. Results

3.1. Avt5p, the S. pombe homologue of S. cerevisiae Avt5p and Avt6p,localizes to the vacuolar membrane

Homology search by Russnak et al. [11] revealed two S. pombeproteins, SPBC1685.07c and SPAC3H1.09c, as members of theAVT family. Based on phylogenetic comparison with S. cerevisiaeAVT family proteins, which showed that ScAvt5p (YBL089W) andScAvt6p (YER119C) were the closest relatives of SPBC1685.07c[11], we designated the SPBC1685.07c gene as avt5+. The percent-ages of amino acid identity of Avt5p with ScAvt5p, and ScAvt6p are38% and 37%, respectively, with the two S. cerevisiae proteins being53% identical. All three proteins are predicted to have 11 trans-membrane domains. Compared to ScAvt5p and ScAvt6p, the regionlinking the eighth and ninth transmembrane domains is relativelyshort in Avt5p (Fig. 1A). Although the function of ScAvt5p is stillunknown, ScAvt6p has been found to export aspartic acid and glu-tamic acid from vacuoles [11].

The phylogenetic relationships between Avt5p and ScAvt6psuggested that Avt5p could be a vacuolar transporter. To determinethe subcellular localization of Avt5p, GFP was attached at the car-boxyl terminus of Avt5p and the localization of the Avt5p-GFP fu-sion protein was detected by fluorescence microscopy. Wild-typecells expressing Avt5p-GFP exhibited ring-like fluorescence whichlargely co-localized with the vacuolar membrane stained withFM4-64 (Fig. 1B), indicating that Avt5p was a vacuolar membraneprotein.

3.2. Amino acid uptake in S. pombe involves vacuolarcompartmentalization

In S. cerevisiae, many vacuolar transporters have been charac-terized directly with isolated vacuolar membrane vesicles [10,20]and indirectly with whole cells [10]. Since it is difficult to isolatevacuoles from S. pombe, examination of vacuolar transport wasperformed by indirect observation using whole cells, as describedpreviously [6,15]. We determined the uptake of several aminoacids and that of 2-deoxy-D-glucose (2-DG), which is a substratefor the S. pombe glucose/H+ symporter Ght1p on the plasma mem-brane [22]. Furthermore, the effect of CCA, a specific potent inhib-itor of V-ATPase [21], on these activities was studied. We recentlyreported that treatment with 10 lM CCA significantly decreasedthe vacuolar uptake of the basic amino acid histidine and the neu-tral amino acid isoleucine [15]. In this study, similar effects in thecase of the basic amino acid arginine, the aromatic amino acidtyrosine, the acidic amino acid glutamate (Fig. 2) and the neutralamino acid glutamine (Fig. 3) were observed. Compared to aminoacid uptake by untreated cells, treatment with CCA resulted in amore than two-fold decrease of the uptake of arginine (Fig. 2B),tyrosine (Fig. 2C) and glutamine (Fig. 3B), whereas glutamate up-take was inhibited by 40% (Fig. 2D), reflecting V-ATPase-dependentaccumulation of these amino acids [6]. In contrast, uptake of 2-DGwas, as expected, little affected by 10 lM CCA (Fig. 2A). These re-

Fig. 1. Summary of Avt5p. (A) Alignment of the amino acid sequences of Avt5p and the related AVT proteins of S. cerevisiae. Black boxes indicate identical residues and grayboxes indicate similar residues. Black bars indicate transmembrane domains of Avt5p. (B) Fluorescence microscopy of wild-type cells expressing the Avt5p-GFP fusionprotein. Vacuoles are visualized with lipophilic dye FM4-64.

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sults indicate that amino acid uptake by S. pombe involves V-ATP-ase-dependent vacuolar compartmentalization.

3.3. Avt5p is involved in vacuolar uptake of a specific set of aminoacids

In order to establish a role of Avt5p in vacuolar amino acidtransport, the uptake of a variety of amino acids and 2-DG byDavt5 mutant cells was examined (Fig. 3). As shown in Fig. 3A, up-take of histidine, tyrosine and glutamate was significantly im-paired and after 90 min in Davt5 cells only 50% of radio-labeledamino acid accumulated that was taken up by parental cells. Up-take of basic amino acids arginine and lysine was also affected

(Fig. 3A) and serine uptake decreased by about 30% in Davt5 cellsafter 90 min (data not shown). In contrast, cellular accumulationof glutamine, asparagine, isoleucine and valine (data not shown),or of 2-DG (Fig. 3A) was as wild-type in the absence of Avt5p. Thus,the disruption of avt5 had a specific effect and only affected thetransport of a particular set of amino acids. We conclude thatAvt5p significantly contributes to the uptake of histidine, gluta-mate or tyrosine and, to a lesser extent, to that of arginine, lysineor serine.

To clarify whether reduced uptake of these amino acids in theabsence of Avt5p is caused by a deficiency of sequestration intovacuoles, we compared the effect of CCA on the uptake of gluta-mine and glutamate (Fig. 3B). While the uptake of 2-DG was little

Fig. 2. Effect of CCA on the uptake of 2-DG (A) and amino acids arginine (B),tyrosine (C) and glutamate (D) by whole cells. Exponentially growing wild-typecells in YES medium were subjected to transport assay. Normal uptake (open bars)is compared to transport in the presence of DMSO (hatched bars), or 10 lM CCA inDMSO (solid bars). The activity without addition (normal uptake) was taken as100%. The results shown are the means ± standard deviations of three independentexperiments.

2342 S. Chardwiriyapreecha et al. / FEBS Letters 584 (2010) 2339–2345

affected by 10 lM CCA in the case of either mutant or parentalcells, glutamine uptake was equally affected, suggesting vacuolaraccumulation of this amino acid and confirming that this happensvia an Avt5p-independent process. In contrast, glutamate uptakeby parental cells was inhibited by CCA to the same extent as whenAvt5p was absent in untreated cells, by about 40%. Treatment ofDavt5 cells with CCA did not cause a significantly further decreasein glutamate uptake, suggesting that Avt5p mediates or controls V-ATPase-dependent uptake of glutamate into vacuoles. These re-sults indicate that Avt5p is involved in vacuolar uptake of a specificset of amino acids.

3.4. Vacuolar uptake of amino acids during nitrogen starvation

Under nitrogen starvation, cells utilize vacuolar amino acid poolas nitrogen sources [23]. In S. cerevisiae, several vacuolar aminoacid transporters respond to nitrogen starvation and have beeninvestigated for understanding the physiological role of vacuolarcompartmentalization [11,24].

To define the role of Avt5p in the S. pombe response to nitrogenstarvation, we examined the uptake activities of glutamate and ly-sine by parental cells, Davt5 cells and Dfnx1 cells in nitrogen-poor(MM-N) medium. As described above, Avt5p is involved in vacuo-lar uptake of glutamate and lysine (Fig. 3A), and, as reported in ourprevious paper, Fnx1p is a vacuolar transporter for lysine, isoleu-cine and asparagine, but not glutamate [15]. The glutamate and ly-sine uptake varied when wild-type cells were subjected to nitrogenstarvation: after one hour incubation in MN-N medium the uptakeof these amino acids peaked and increased almost three-fold, butupon further incubation this increase reversed and after threehours the uptake was close to normal, i.e. the level observed duringgrowth on rich medium (Fig. 4A). In the absence of Avt5p, however,nitrogen starvation induced a very limited transient increase in theglutamate uptake by about 20%. In contrast, lysine uptake peakedafter one hour on MM-N media as this was still two-fold higherthan in the case of Davt5 cells grown on rich medium. Comparedto the parental cells the overall uptake activities for glutamateand lysine were reduced to 40% and 50%, in line with the expectedtransport defect caused by lack of Avt5p (Fig. 3A). Despite its rolein the uptake of lysine, Avt5p seems not essential for the occur-rence of a transient increase in lysine uptake during nitrogen star-vation. In Davt5 cells this response could be mediated by Fnx1pand Fnx2p [15], which was confirmed by the finding that upon dis-ruption of fnx1, the transient increase in lysine uptake on MM-Nwas limited to about 20% when compared to lysine uptake duringgrowth on rich medium. After 1 h nitrogen starvation the lysineuptake of Dfnx1 cells was only about 25% of that of parent cellsand about 50% of that of Davt5 cells, suggesting that the fnx1/2gene products are major determinants of lysine transport. Gluta-mate uptake, however, seems more under the control of Avt5pand independent of Fnx1p as in Dfnx1 cells this transport was com-parable to that observed for the parental strain during growth onrich media as well as under conditions of nitrogen starvation(Fig. 4A, [15]). These results suggest that nitrogen starvation in-duces Avt5p-dependent glutamate uptake as well as Fnx1p-depen-dent lysine uptake.

The transient increase of amino acid uptake in response tonitrogen starvation also involves vacuolar compartmentalizationas it relies on V-ATPase activity. Exposure to CCA reduced the up-take of glutamate or lysine, as measured after one hour incubationin MM-N medium, to a basal level which was about 25% of theuptake observed for untreated wild-type cells (Fig. 4B). Further-more, all the remaining uptake activities of Davt5 and Dfnx1 cellswere inhibited by CCA to the same basal level. In contrast, uptakeof 2-DG by parental or mutant strains under nitrogen starvationwas constant (Fig. 4A) and not affected by exposure to CAA(Fig. 4B). We conclude that both Avt5p- and Fnx1p-dependent ami-no acid uptake should be attributed to vacuolar transport occurringduring growth on rich medium or under conditions of nitrogenstarvation.

4. Discussion

Although phenotypic analysis revealed a role for S. pombe Avt5pin spore formation during nitrogen starvation [16], this protein isphylogenetically related to vacuolar proteins ScAvt5p and ScAvt6of S. cerevisiae (Fig. 1A). In this study, we have presented evidencethat Avt5p localizes to the vacuolar membrane (Fig. 1B) and thatplays a critical role in amino acid uptake by S. pombe cells. In theabsence of Avt5p the uptake of a variety of amino acids was re-duced to that in wild-type cells was sensitive to CCA, indicatingthat Avt5p is involved in V-ATPase-dependent import of histidine,tyrosine, glutamate, to a lesser extent, lysine, arginine and serineinto vacuoles of S. pombe (Figs. 2–4).

Fig. 3. The effect of avt5 disruption on the uptake of amino acids and 2-DG. (A) Uptake as measured in parental cells (open circles) and the Davt5 mutant (solid circles) inthree independent experiments. (B) Effect of CCA. See also the legend for Fig. 2.

S. Chardwiriyapreecha et al. / FEBS Letters 584 (2010) 2339–2345 2343

In S. cerevisiae, basic amino acids are enriched in vacuoles,whereas the acidic amino acids glutamate and aspartic acid areexclusively localized to the cytosol. The vacuoles of S. pombe re-tains 85–100% of the cellular content of basic amino acids, andmost of aspartic acid and more than 15% of glutamate are accumu-lated in the vacuole, which suggested the presence of vacuolar im-porter(s) for these acidic amino acids [6]. Interestingly,accumulation of acidic amino acids by vacuolar membrane vesiclesof wild-type cells of S. cerevisiae was low in the presence of ATP,and ScAvt6p was found to contribute to the export of acidic aminoacids from vacuoles [11]. Thus one S. cerevisiae homologue of Avt5pappears to be an effluxer of glutamate, but in S. pombe this proteinis required for vacuolar glutamate accumulation, which occurs in aV-ATPase-dependent manner (Figs. 3 and 4). Vacuolar compart-mentalization of amino acids in S. pombe seems therefore funda-mentally similar to that of S. cerevisiae except for the localizationof acidic amino acids [6].

Avt5p was also involved in vacuolar transport of amino acidsunder nitrogen starvation (Fig. 4). Nitrogen starvation has beenknown to induce a set of cellular responses aimed at maintainingsurvivability. Many amino acid transporters and permeases oper-ate at the plasma membrane [25,26] and the vacuolar membrane[15,18] and nitrogen starvation induces the expression of thesegenes [18,27,28]. Our results indicate that nitrogen starvation re-sults in the up-regulation of Avt5p-dependent amino acid uptakeby whole cells, and that this stimulation is V-ATPase dependent(Fig. 4). A similar increase has been observed in the case ofFnx1p-dependent lysine uptake (Fig. 4), suggesting that vacuolaramino acid transporters become temporarily more active undernitrogen starvation of S. pombe. It is unlikely that this up-regula-tion is mediated by an increase in transcription of the genes encod-ing the subunits of V-ATPase [28]. It cannot be excluded, however,that V-ATPase activity may change as a result of an altered interac-tion of the integral V0 domain with the peripheral V1 domain, as

Fig. 4. Relative uptake of glutamate, lysine and 2-DG during nitrogen starvation. (A) The uptake by the Davt5 mutant (hatched bars) and the Dfnx1 mutant (solid bars) ascompared to that by the parental strain (open bars), which was set as 1. The results shown are the means ± standard deviations of three independent experiments. (B) Effect ofCCA during nitrogen starvation. See also the legend for Fig. 2.

2344 S. Chardwiriyapreecha et al. / FEBS Letters 584 (2010) 2339–2345

occurs in cells under glucose starvation [29]. Therefore, it needs tobe determined whether, and if so how, V-ATPase is activated undernitrogen starvation.

It is possible that Avt5p functions as a vacuolar importer with asubstrate specificity that is more wide-ranging than that of Fnx1p[15]. It has been reported that transcription of fnx1+ is inducedupon nitrogen starvation [18], which parallels activation of theFnx1p-dependent uptake of lysine (Fig. 4). Northern blot analysisrevealed, however, that transcription of avt5+ remained as low dur-ing nitrogen starvation as during growth in rich medium (data notshown). In support of this finding, microarray analysis has shownthat in Tor2 depleted cells, which have a phenotype very similarto that of wild-type cells starved for nitrogen, expression of manymembrane transporters is induced, but putative vacuolar aminoacid transporters, except fnx1+, are not up-regulated [27]. There-fore, it remains unclear whether Avt5p is directly or indirectly in-

volved in vacuolar import of amino acids. If, on one hand, Avt5pacts as a vacuolar importer, it could be post-translationally regu-lated by phosphorylation, or the association or dissociation of aregulatory molecule in response to altered growth conditions. Onthe other hand, Avt5p may control as a regulatory protein the dis-tribution of other membrane proteins that are directly involved invacuolar amino acid import. For instance, Avt5p could be impor-tant for the organization of a membrane microdomain, analogousto the role of ATP-binding cassette (ABC) transporter Pdr10p,which was recently found to be required for the incorporation ofanother ABC transporter, Pdr12p, into a detergent-resistant mem-brane microdomain within the plasma membrane of S. cerevisiae[30]. Ergosterol, a constituent of membrane microdomains, is in-volved in homotypic vacuolar fusion [31], which suggests thatsuch, functionally important, microdomains also occur in vacuolarmembranes.

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Our results demonstrate that Avt5p contributes to amino acidimport into vacuoles of S. pombe, in contrast to its S. cerevisiaehomologue ScAvt6p, which mediates vacuolar export. It would beinteresting to determine the directionality of the other S. cerevisiaehomologue and putative amino acid transporter, ScAvt5p. Furtherwork on purification and reconstitution of Avt5p is scheduled toobtain direct evidence for the function of Avt5p in vacuolar aminoacid transport.

Acknowledgements

We thank Shuichi Katoh and Shiro Kajiwara for technical assis-tance and Dr. Taro Nakamura for providing plasmid pTN197. Thiswork was supported in part by a Grant-in-Aid for Scientific Re-search (to Y.K.) from the Ministry of Education, Culture, Sports, Sci-ence, and Technology of Japan.

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