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Avt5p is required for vacuolar uptake of amino acids in the fission yeast Schizosaccharomyces pombe
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 Soracom Chardwiriyapreecha a, Hiroyuki Mukaiyama b, Takayuki Sekito a, Tomoko Iwaki a, Kaoru Takegawa b, Yoshimi Kakinuma a,* a b
Department of Applied Bioresource Science, Faculty of Agriculture, Ehime University, Ehime 790-8566, Japan Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
a r t i c l e
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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
a b s t r a c t 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 vacuolar 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 synthesis. 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 largesize 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 membrane, generated by a vacuolar H+-ATPase (V-ATPase) and likely mediated by an H+/amino acid antiport system [9]. The vacuole is
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).
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 vacuolar 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 vacuolar 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 uptake of neutral amino acids, whereas Avt3p and Avt4p participate in extrusion of these amino acids and Avt6p in efflux of acidic amino 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–polyamine–choline family (APC) [13] and the lysosomal cystine transporter 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 phylogenetically related to Vba2p of S. cerevisiae and involved in the uptake 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
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acids by fractionation after cupric ion-treatment indicated vacuolar compartmentalization of various amino acids, such as threonine, serine, glutamine, glycine, isoleucine, tyrosine, phenylalanine and tryptophan, as well as basic amino acids [6]. It is noteworthy that most of aspartate and more than 15% of glutamate were retained in the vacuole of S. pombe [6]. These results suggested that S. pombe and S. cerevisiae have slightly different vacuolar compartmentalization systems. Here, we focus on S. pombe SPBC1685.07c, designated as avt5+, which is phylogenetically related to the vacuolar amino acid transporters Avt5p and Avt6p of S. cerevisiae [11]. We report that Avt5p is a vacuolar membrane protein involved in acidic, neutral, and basic 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 strain ARC039 (h leu1-32 ura4-C190T), the Dfnx1 mutant (h leu1-32 ura4-C190T fnx1::ura4) [15] and the Davt5 mutant (h leu1-32 ura4-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 were purchased from Wako Pure Chemicals Co. (Osaka, Japan) and Invitrogen Corp. (Carlsbad, CA, USA), respectively, and dissolved in dimethyl sulfoxide (DMSO). L-[14C] labeled materials were purchased from American Radiolabeled Chemical (St. Louis, MO, USA; L-arginine, L-lysine, L-tyrosine, L-asparagine and 2-deoxy-Dglucose), GE healthcare (Buckinghamshire, England; L-histidine, Lisoleucine, L-glutamine, L-valine, L-glutamic acid), and Perkin Elmer Inc. (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 derivative of the thiamine-repressible expression vector pREP41 [19], resulting in pTN197/avt5. Wild-type cells transformed with this plasmid were grown in MM medium without leucine at 30 °C for 20 h and examined with an Olympus BX-60 fluorescence microscope using a U-MGFPHQ filter. Vacuoles were labeled with FM464 as described elsewhere [15]. After exposure to lipophilic dye FM4-64, cells were incubated with sterilized distilled water for 3 h to induce vacuolar fusion. Images were captured with a SenSys cooled 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. In the case of CAA treatment, cells were preincubated for 30 min at 30 °C with 10 lM CCA (in DMSO). For nitrogen starvation conditions, cells cultured in YES medium were harvested during logarithmic phase, washed twice with sterilized water, and then incubated in MM-N medium for the indicated durations. Reactions were initiated by adding specific labeled substrates, (L-[U-14C]histidine (final concentration 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),
14 L-[U- C]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 control, samples were incubated with DMSO. 2.4. Sequence analysis
ClustalW software (http://www.ebi.ac.uk/clustalw/) was used to align multiple sequences and transmembrane domains were predicted by means of the SOSUI algorithm (http://bp.nuap.nagoya-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. pombe proteins, SPBC1685.07c and SPAC3H1.09c, as members of the AVT family. Based on phylogenetic comparison with S. cerevisiae AVT family proteins, which showed that ScAvt5p (YBL089W) and ScAvt6p (YER119C) were the closest relatives of SPBC1685.07c [11], we designated the SPBC1685.07c gene as avt5+. The percentages of amino acid identity of Avt5p with ScAvt5p, and ScAvt6p are 38% and 37%, respectively, with the two S. cerevisiae proteins being 53% identical. All three proteins are predicted to have 11 transmembrane domains. Compared to ScAvt5p and ScAvt6p, the region linking the eighth and ninth transmembrane domains is relatively short in Avt5p (Fig. 1A). Although the function of ScAvt5p is still unknown, ScAvt6p has been found to export aspartic acid and glutamic acid from vacuoles [11]. The phylogenetic relationships between Avt5p and ScAvt6p suggested that Avt5p could be a vacuolar transporter. To determine the subcellular localization of Avt5p, GFP was attached at the carboxyl terminus of Avt5p and the localization of the Avt5p-GFP fusion protein was detected by fluorescence microscopy. Wild-type cells expressing Avt5p-GFP exhibited ring-like fluorescence which largely co-localized with the vacuolar membrane stained with FM4-64 (Fig. 1B), indicating that Avt5p was a vacuolar membrane protein. 3.2. Amino acid uptake in S. pombe involves vacuolar compartmentalization In S. cerevisiae, many vacuolar transporters have been characterized directly with isolated vacuolar membrane vesicles [10,20] and indirectly with whole cells [10]. Since it is difficult to isolate vacuoles from S. pombe, examination of vacuolar transport was performed by indirect observation using whole cells, as described previously [6,15]. We determined the uptake of several amino acids and that of 2-deoxy-D-glucose (2-DG), which is a substrate for the S. pombe glucose/H+ symporter Ght1p on the plasma membrane [22]. Furthermore, the effect of CCA, a specific potent inhibitor of V-ATPase [21], on these activities was studied. We recently reported that treatment with 10 lM CCA significantly decreased the vacuolar uptake of the basic amino acid histidine and the neutral amino acid isoleucine [15]. In this study, similar effects in the case of the basic amino acid arginine, the aromatic amino acid tyrosine, the acidic amino acid glutamate (Fig. 2) and the neutral amino acid glutamine (Fig. 3) were observed. Compared to amino acid uptake by untreated cells, treatment with CCA resulted in a more than two-fold decrease of the uptake of arginine (Fig. 2B), tyrosine (Fig. 2C) and glutamine (Fig. 3B), whereas glutamate uptake was inhibited by 40% (Fig. 2D), reflecting V-ATPase-dependent accumulation of these amino acids [6]. In contrast, uptake of 2-DG was, as expected, little affected by 10 lM CCA (Fig. 2A). These re-
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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 gray boxes indicate similar residues. Black bars indicate transmembrane domains of Avt5p. (B) Fluorescence microscopy of wild-type cells expressing the Avt5p-GFP fusion protein. Vacuoles are visualized with lipophilic dye FM4-64.
sults indicate that amino acid uptake by S. pombe involves V-ATPase-dependent vacuolar compartmentalization. 3.3. Avt5p is involved in vacuolar uptake of a specific set of amino acids In order to establish a role of Avt5p in vacuolar amino acid transport, the uptake of a variety of amino acids and 2-DG by Davt5 mutant cells was examined (Fig. 3). As shown in Fig. 3A, uptake of histidine, tyrosine and glutamate was significantly impaired and after 90 min in Davt5 cells only 50% of radio-labeled amino acid accumulated that was taken up by parental cells. Uptake of basic amino acids arginine and lysine was also affected
(Fig. 3A) and serine uptake decreased by about 30% in Davt5 cells after 90 min (data not shown). In contrast, cellular accumulation of 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 the transport of a particular set of amino acids. We conclude that Avt5p significantly contributes to the uptake of histidine, glutamate or tyrosine and, to a lesser extent, to that of arginine, lysine or serine. To clarify whether reduced uptake of these amino acids in the absence of Avt5p is caused by a deficiency of sequestration into vacuoles, we compared the effect of CCA on the uptake of glutamine and glutamate (Fig. 3B). While the uptake of 2-DG was little
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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-type cells 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 in DMSO (solid bars). The activity without addition (normal uptake) was taken as 100%. The results shown are the means ± standard deviations of three independent experiments.
affected by 10 lM CCA in the case of either mutant or parental cells, glutamine uptake was equally affected, suggesting vacuolar accumulation of this amino acid and confirming that this happens via an Avt5p-independent process. In contrast, glutamate uptake by parental cells was inhibited by CCA to the same extent as when Avt5p was absent in untreated cells, by about 40%. Treatment of Davt5 cells with CCA did not cause a significantly further decrease in glutamate uptake, suggesting that Avt5p mediates or controls VATPase-dependent uptake of glutamate into vacuoles. These results indicate that Avt5p is involved in vacuolar uptake of a specific set of amino acids. 3.4. Vacuolar uptake of amino acids during nitrogen starvation Under nitrogen starvation, cells utilize vacuolar amino acid pool as nitrogen sources [23]. In S. cerevisiae, several vacuolar amino acid transporters respond to nitrogen starvation and have been investigated for understanding the physiological role of vacuolar compartmentalization [11,24].
To define the role of Avt5p in the S. pombe response to nitrogen starvation, we examined the uptake activities of glutamate and lysine by parental cells, Davt5 cells and Dfnx1 cells in nitrogen-poor (MM-N) medium. As described above, Avt5p is involved in vacuolar uptake of glutamate and lysine (Fig. 3A), and, as reported in our previous paper, Fnx1p is a vacuolar transporter for lysine, isoleucine and asparagine, but not glutamate [15]. The glutamate and lysine uptake varied when wild-type cells were subjected to nitrogen starvation: after one hour incubation in MN-N medium the uptake of these amino acids peaked and increased almost three-fold, but upon further incubation this increase reversed and after three hours the uptake was close to normal, i.e. the level observed during growth on rich medium (Fig. 4A). In the absence of Avt5p, however, nitrogen starvation induced a very limited transient increase in the glutamate uptake by about 20%. In contrast, lysine uptake peaked after one hour on MM-N media as this was still two-fold higher than in the case of Davt5 cells grown on rich medium. Compared to the parental cells the overall uptake activities for glutamate and lysine were reduced to 40% and 50%, in line with the expected transport defect caused by lack of Avt5p (Fig. 3A). Despite its role in the uptake of lysine, Avt5p seems not essential for the occurrence of a transient increase in lysine uptake during nitrogen starvation. In Davt5 cells this response could be mediated by Fnx1p and Fnx2p [15], which was confirmed by the finding that upon disruption of fnx1, the transient increase in lysine uptake on MM-N was limited to about 20% when compared to lysine uptake during growth on rich medium. After 1 h nitrogen starvation the lysine uptake of Dfnx1 cells was only about 25% of that of parent cells and about 50% of that of Davt5 cells, suggesting that the fnx1/2 gene products are major determinants of lysine transport. Glutamate uptake, however, seems more under the control of Avt5p and independent of Fnx1p as in Dfnx1 cells this transport was comparable to that observed for the parental strain during growth on rich media as well as under conditions of nitrogen starvation (Fig. 4A, [15]). These results suggest that nitrogen starvation induces Avt5p-dependent glutamate uptake as well as Fnx1p-dependent lysine uptake. The transient increase of amino acid uptake in response to nitrogen starvation also involves vacuolar compartmentalization as it relies on V-ATPase activity. Exposure to CCA reduced the uptake of glutamate or lysine, as measured after one hour incubation in MM-N medium, to a basal level which was about 25% of the uptake observed for untreated wild-type cells (Fig. 4B). Furthermore, all the remaining uptake activities of Davt5 and Dfnx1 cells were inhibited by CCA to the same basal level. In contrast, uptake of 2-DG by parental or mutant strains under nitrogen starvation was constant (Fig. 4A) and not affected by exposure to CAA (Fig. 4B). We conclude that both Avt5p- and Fnx1p-dependent amino acid uptake should be attributed to vacuolar transport occurring during growth on rich medium or under conditions of nitrogen starvation.
4. Discussion Although phenotypic analysis revealed a role for S. pombe Avt5p in spore formation during nitrogen starvation [16], this protein is phylogenetically related to vacuolar proteins ScAvt5p and ScAvt6 of S. cerevisiae (Fig. 1A). In this study, we have presented evidence that Avt5p localizes to the vacuolar membrane (Fig. 1B) and that plays a critical role in amino acid uptake by S. pombe cells. In the absence of Avt5p the uptake of a variety of amino acids was reduced to that in wild-type cells was sensitive to CCA, indicating that Avt5p is involved in V-ATPase-dependent import of histidine, tyrosine, glutamate, to a lesser extent, lysine, arginine and serine into vacuoles of S. pombe (Figs. 2–4).
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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) in three independent experiments. (B) Effect of CCA. See also the legend for Fig. 2.
In S. cerevisiae, basic amino acids are enriched in vacuoles, whereas the acidic amino acids glutamate and aspartic acid are exclusively localized to the cytosol. The vacuoles of S. pombe retains 85–100% of the cellular content of basic amino acids, and most of aspartic acid and more than 15% of glutamate are accumulated in the vacuole, which suggested the presence of vacuolar importer(s) for these acidic amino acids [6]. Interestingly, accumulation of acidic amino acids by vacuolar membrane vesicles of wild-type cells of S. cerevisiae was low in the presence of ATP, and ScAvt6p was found to contribute to the export of acidic amino acids from vacuoles [11]. Thus one S. cerevisiae homologue of Avt5p appears to be an effluxer of glutamate, but in S. pombe this protein is required for vacuolar glutamate accumulation, which occurs in a V-ATPase-dependent manner (Figs. 3 and 4). Vacuolar compartmentalization of amino acids in S. pombe seems therefore fundamentally similar to that of S. cerevisiae except for the localization of acidic amino acids [6].
Avt5p was also involved in vacuolar transport of amino acids under nitrogen starvation (Fig. 4). Nitrogen starvation has been known to induce a set of cellular responses aimed at maintaining survivability. Many amino acid transporters and permeases operate at the plasma membrane [25,26] and the vacuolar membrane [15,18] and nitrogen starvation induces the expression of these genes [18,27,28]. Our results indicate that nitrogen starvation results in the up-regulation of Avt5p-dependent amino acid uptake by whole cells, and that this stimulation is V-ATPase dependent (Fig. 4). A similar increase has been observed in the case of Fnx1p-dependent lysine uptake (Fig. 4), suggesting that vacuolar amino acid transporters become temporarily more active under nitrogen starvation of S. pombe. It is unlikely that this up-regulation is mediated by an increase in transcription of the genes encoding the subunits of V-ATPase [28]. It cannot be excluded, however, that V-ATPase activity may change as a result of an altered interaction of the integral V0 domain with the peripheral V1 domain, as
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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) as compared 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 of CCA during nitrogen starvation. See also the legend for Fig. 2.
occurs in cells under glucose starvation [29]. Therefore, it needs to be determined whether, and if so how, V-ATPase is activated under nitrogen starvation. It is possible that Avt5p functions as a vacuolar importer with a substrate specificity that is more wide-ranging than that of Fnx1p [15]. It has been reported that transcription of fnx1+ is induced upon nitrogen starvation [18], which parallels activation of the Fnx1p-dependent uptake of lysine (Fig. 4). Northern blot analysis revealed, however, that transcription of avt5+ remained as low during nitrogen starvation as during growth in rich medium (data not shown). In support of this finding, microarray analysis has shown that in Tor2 depleted cells, which have a phenotype very similar to that of wild-type cells starved for nitrogen, expression of many membrane transporters is induced, but putative vacuolar amino acid transporters, except fnx1+, are not up-regulated [27]. Therefore, it remains unclear whether Avt5p is directly or indirectly in-
volved in vacuolar import of amino acids. If, on one hand, Avt5p acts as a vacuolar importer, it could be post-translationally regulated by phosphorylation, or the association or dissociation of a regulatory molecule in response to altered growth conditions. On the other hand, Avt5p may control as a regulatory protein the distribution of other membrane proteins that are directly involved in vacuolar amino acid import. For instance, Avt5p could be important for the organization of a membrane microdomain, analogous to the role of ATP-binding cassette (ABC) transporter Pdr10p, which was recently found to be required for the incorporation of another ABC transporter, Pdr12p, into a detergent-resistant membrane microdomain within the plasma membrane of S. cerevisiae [30]. Ergosterol, a constituent of membrane microdomains, is involved in homotypic vacuolar fusion [31], which suggests that such, functionally important, microdomains also occur in vacuolar membranes.
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Our results demonstrate that Avt5p contributes to amino acid import into vacuoles of S. pombe, in contrast to its S. cerevisiae homologue ScAvt6p, which mediates vacuolar export. It would be interesting to determine the directionality of the other S. cerevisiae homologue and putative amino acid transporter, ScAvt5p. Further work on purification and reconstitution of Avt5p is scheduled to obtain direct evidence for the function of Avt5p in vacuolar amino acid transport. Acknowledgements We thank Shuichi Katoh and Shiro Kajiwara for technical assistance and Dr. Taro Nakamura for providing plasmid pTN197. This work was supported in part by a Grant-in-Aid for Scientific Research (to Y.K.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] Magasanik, B. and Kaiser, C.A. (2002) Nitrogen regulation in Saccharomyces cerevisiae. Gene 290, 1–18. [2] Hinnebusch, A.G. (2005) Translational regulation of Gcn4 and the general amino acid control of yeast. Annu. Rev. Microbiol. 59, 407–450. [3] Klionsky, D.J., Herman, P.K. and Emr, S.D. (1990) The fungal vacuole: composition, function, and biogenesis. Microbiol. Rev. 54, 266–292. [4] Wiemken, A. and Dürr, M. (1974) Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae. Arch. Microbiol. 101, 45– 57. [5] Ohsumi, Y., Kitamoto, K. and Anraku, Y. (1988) Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion. J. Bacteriol. 170, 2676–2682. [6] Chardwiriyapreecha, S., Hondo, K., Inada, H., Chahomchuen, T., Sekito, T., Iwaki, T. and Kakinuma, Y. (2009) A simple and specific procedure to permeabilize the plasma membrane of Schizosaccharomyces pombe. Biosci. Biotechnol. Biochem. 73, 2090–2095. [7] Sato, T., Ohsumi, Y. and Anraku, Y. (1984) Substrate specificities of active transport systems for amino acids in vacuolar-membrane vesicles of Saccharomyces cerevisiae. Evidence of seven independent proton/amino acid antiport systems. J. Biol. Chem. 259, 11505–11508. [8] Sekito, T., Fujiki, Y., Ohsumi, Y. and Kakinuma, Y. (2008) Novel families of vacuolar amino acid transporters. IUBMB life. 60, 519–525. [9] Ohsumi, Y. and Anraku, Y. (1981) Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. J. Biol. Chem. 256, 2079–2082. [10] Shimazu, M., Sekito, T., Akiyama, K., Ohsumi, Y. and Kakinuma, Y. (2005) A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae. J. Biol. Chem. 280, 4851–4857. [11] Russnak, R., Konczal, D. and McIntire, S.L. (2001) A family of yeast proteins mediating bidirectional vacuolar amino acid transport. J. Biol. Chem. 276, 23849–23857. [12] Paulsen, I.T., Sliwinski, M.K., Nelissen, B., Goffeau, A. and Saier Jr., M.H. (1998) Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae. FEBS Lett. 430, 116–125. [13] Saier Jr., M.H. (2000) A functional/phylogenetic classification system for transmembrane solute transporters. Microbiol. Mol. Biol. Rev. 64, 354–411.
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[14] Uemura, T., Tomonari, Y., Kashiwagi, K. and Igarashi, K. (2004) Uptake of GABA and putrescine by UGA4 on the vacuolar membrane in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 315, 1082–1087. [15] Chardwiriyapreecha, S., Shimazu, M., Morita, T., Sekito, T., Akiyama, K., Takegawa, K. and Kakinuma, Y. (2008) Identification of the fnx1+ and fnx2+ genes for vacuolar amino acid transporters in Schizosaccharomyces pombe. FEBS Lett. 582, 2225–2230. [16] Mukaiyama, H., Kajiwara, S., Hosomi, A., Giga-Hama, Y., Tanaka, N., Nakamura, T. and Takegawa, K. (2009) Autophagy-deficient Schizosaccharomyces pombe mutants undergo partial sporulation during nitrogen starvation. Microbiology 155, 3816–3826. [17] Moreno, S., Klar, A. and Nurse, P. (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823. [18] Dimitrov, K. and Sazer, S. (1998) The role of fnx1, a fission yeast multidrug resistance protein, in the transition of cells to a quiescent G0 state. Mol. Cell. Biol. 18, 5239–5246. [19] Nakamura, T., Nakamura-Kubo, M., Hirata, A. and Shimoda, C. (2001) The Schizosaccharomyces pombe spo3+ gene is required for assembly of the forespore membrane and genetically interacts with psy1+-encoding syntaxin-like protein. Mol. Biol. Cell 12, 3955–3972. [20] Lin, H., Kumánovics, A., Nelson, J.M., Warner, D.E., Ward, D.M. and Kaplan, J. (2008) A single amino acid change in the yeast vacuolar metal transporters ZRC1 and COT1 alters their substrate specificity. J. Biol. Chem. 283, 33865– 33873. [21] Dröse, S., Bindseil, K.U., Bowman, E.J., Siebers, A., Zeeck, A. and Altendorf, K. (1993) Inhibitory effects of modified bafilomycins and concanamycins on Pand V-type adenosinetriphosphatases. Biochemistry 32, 3902–3906. [22] Heiland, S., Radovanovic, N., Höfer, M., Winderickx, J. and Lichtenberg, H. (2000) Multiple hexose transporters of Schizosaccharomyces pombe. J. Bacteriol. 182, 2153–2162. [23] Onodera, J. and Ohsumi, Y. (2005) Autophagy is required for maintenance of amino acid levels and protein synthesis under nitrogen starvation. J. Biol. Chem. 280, 31582–31586. [24] Yang, Z., Huang, J., Geng, J., Nair, U. and Klionsky, D.J. (2006) Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Mol. Biol. Cell. 17, 5094–5104. [25] Aspuria, P.J. and Tamanoi, F. (2008) The Tsc/Rheb signaling pathway controls basic amino acid uptake via the Cat1 permease in fission yeast. Mol. Genet. Genomics 279, 441–450. [26] Matsumoto, S., Bandyopadhyay, A., Kwiatkowski, D.J., Maitra, U. and Matsumoto, T. (2002) Role of the Tsc1–Tsc2 complex in signaling and transport across the cell membrane in the fission yeast Schizosaccharomyces pombe. Genetics 161, 1053–1063. [27] Matsuo, T., Otsubo, Y., Urano, J., Tamanoi, F. and Yamamoto, Y. (2007) Loss of TOR kinase Tor2 mimics nitrogen starvation and activates the sexual development pathway in fission yeast. Mol. Cell. Biol. 27, 3154–3164. [28] Wilhelm, B.T., Marguerat, S., Watt, S., Schbert, F., Wood, V., Goodhead, I., Penkett, C.J., Rogers, J. and Bähler, J. (2008) Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Nature 453, 1239– 1243. [29] Parra, K.J. and Kane, P.M. (1998) Reversible association between the V1 and V0 domains of yeast vacuolar H+-ATPase is an unconventional glucose-induced effect. Mol. Cell. Biol. 18, 7064–7074. [30] Rockwell, N.C., Wolfger, H., Kuchler, K. and Thorner, J. (2009) ABC transporter Pdr10 regulates the membrane microenvironment of Pdr12 in Saccharomyces cerevisiae. J. Membr. Biol. 229, 27–52. [31] Wickner, W. (2002) Yeast vacuoles and membrane fusion pathways. EMBO J. 21, 1241–1247.
journal homepage: www.FEBSLetters.org
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 b
Department of Applied Bioresource Science, Faculty of Agriculture, Ehime University, Ehime 790-8566, Japan 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 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
a b s t r a c t 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 vacuolar 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 synthesis. 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 largesize 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 membrane, generated by a vacuolar H+-ATPase (V-ATPase) and likely mediated by an H+/amino acid antiport system [9]. The vacuole is
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).
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 vacuolar 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 vacuolar 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 uptake of neutral amino acids, whereas Avt3p and Avt4p participate in extrusion of these amino acids and Avt6p in efflux of acidic amino 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–polyamine–choline family (APC) [13] and the lysosomal cystine transporter 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 phylogenetically related to Vba2p of S. cerevisiae and involved in the uptake 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
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acids by fractionation after cupric ion-treatment indicated vacuolar compartmentalization of various amino acids, such as threonine, serine, glutamine, glycine, isoleucine, tyrosine, phenylalanine and tryptophan, as well as basic amino acids [6]. It is noteworthy that most of aspartate and more than 15% of glutamate were retained in the vacuole of S. pombe [6]. These results suggested that S. pombe and S. cerevisiae have slightly different vacuolar compartmentalization systems. Here, we focus on S. pombe SPBC1685.07c, designated as avt5+, which is phylogenetically related to the vacuolar amino acid transporters Avt5p and Avt6p of S. cerevisiae [11]. We report that Avt5p is a vacuolar membrane protein involved in acidic, neutral, and basic 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 strain ARC039 (h leu1-32 ura4-C190T), the Dfnx1 mutant (h leu1-32 ura4-C190T fnx1::ura4) [15] and the Davt5 mutant (h leu1-32 ura4-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 were purchased from Wako Pure Chemicals Co. (Osaka, Japan) and Invitrogen Corp. (Carlsbad, CA, USA), respectively, and dissolved in dimethyl sulfoxide (DMSO). L-[14C] labeled materials were purchased from American Radiolabeled Chemical (St. Louis, MO, USA; L-arginine, L-lysine, L-tyrosine, L-asparagine and 2-deoxy-Dglucose), GE healthcare (Buckinghamshire, England; L-histidine, Lisoleucine, L-glutamine, L-valine, L-glutamic acid), and Perkin Elmer Inc. (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 derivative of the thiamine-repressible expression vector pREP41 [19], resulting in pTN197/avt5. Wild-type cells transformed with this plasmid were grown in MM medium without leucine at 30 °C for 20 h and examined with an Olympus BX-60 fluorescence microscope using a U-MGFPHQ filter. Vacuoles were labeled with FM464 as described elsewhere [15]. After exposure to lipophilic dye FM4-64, cells were incubated with sterilized distilled water for 3 h to induce vacuolar fusion. Images were captured with a SenSys cooled 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. In the case of CAA treatment, cells were preincubated for 30 min at 30 °C with 10 lM CCA (in DMSO). For nitrogen starvation conditions, cells cultured in YES medium were harvested during logarithmic phase, washed twice with sterilized water, and then incubated in MM-N medium for the indicated durations. Reactions were initiated by adding specific labeled substrates, (L-[U-14C]histidine (final concentration 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),
14 L-[U- C]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 control, samples were incubated with DMSO. 2.4. Sequence analysis
ClustalW software (http://www.ebi.ac.uk/clustalw/) was used to align multiple sequences and transmembrane domains were predicted by means of the SOSUI algorithm (http://bp.nuap.nagoya-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. pombe proteins, SPBC1685.07c and SPAC3H1.09c, as members of the AVT family. Based on phylogenetic comparison with S. cerevisiae AVT family proteins, which showed that ScAvt5p (YBL089W) and ScAvt6p (YER119C) were the closest relatives of SPBC1685.07c [11], we designated the SPBC1685.07c gene as avt5+. The percentages of amino acid identity of Avt5p with ScAvt5p, and ScAvt6p are 38% and 37%, respectively, with the two S. cerevisiae proteins being 53% identical. All three proteins are predicted to have 11 transmembrane domains. Compared to ScAvt5p and ScAvt6p, the region linking the eighth and ninth transmembrane domains is relatively short in Avt5p (Fig. 1A). Although the function of ScAvt5p is still unknown, ScAvt6p has been found to export aspartic acid and glutamic acid from vacuoles [11]. The phylogenetic relationships between Avt5p and ScAvt6p suggested that Avt5p could be a vacuolar transporter. To determine the subcellular localization of Avt5p, GFP was attached at the carboxyl terminus of Avt5p and the localization of the Avt5p-GFP fusion protein was detected by fluorescence microscopy. Wild-type cells expressing Avt5p-GFP exhibited ring-like fluorescence which largely co-localized with the vacuolar membrane stained with FM4-64 (Fig. 1B), indicating that Avt5p was a vacuolar membrane protein. 3.2. Amino acid uptake in S. pombe involves vacuolar compartmentalization In S. cerevisiae, many vacuolar transporters have been characterized directly with isolated vacuolar membrane vesicles [10,20] and indirectly with whole cells [10]. Since it is difficult to isolate vacuoles from S. pombe, examination of vacuolar transport was performed by indirect observation using whole cells, as described previously [6,15]. We determined the uptake of several amino acids and that of 2-deoxy-D-glucose (2-DG), which is a substrate for the S. pombe glucose/H+ symporter Ght1p on the plasma membrane [22]. Furthermore, the effect of CCA, a specific potent inhibitor of V-ATPase [21], on these activities was studied. We recently reported that treatment with 10 lM CCA significantly decreased the vacuolar uptake of the basic amino acid histidine and the neutral amino acid isoleucine [15]. In this study, similar effects in the case of the basic amino acid arginine, the aromatic amino acid tyrosine, the acidic amino acid glutamate (Fig. 2) and the neutral amino acid glutamine (Fig. 3) were observed. Compared to amino acid uptake by untreated cells, treatment with CCA resulted in a more than two-fold decrease of the uptake of arginine (Fig. 2B), tyrosine (Fig. 2C) and glutamine (Fig. 3B), whereas glutamate uptake was inhibited by 40% (Fig. 2D), reflecting V-ATPase-dependent accumulation of these amino acids [6]. In contrast, uptake of 2-DG was, as expected, little affected by 10 lM CCA (Fig. 2A). These re-
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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 gray boxes indicate similar residues. Black bars indicate transmembrane domains of Avt5p. (B) Fluorescence microscopy of wild-type cells expressing the Avt5p-GFP fusion protein. Vacuoles are visualized with lipophilic dye FM4-64.
sults indicate that amino acid uptake by S. pombe involves V-ATPase-dependent vacuolar compartmentalization. 3.3. Avt5p is involved in vacuolar uptake of a specific set of amino acids In order to establish a role of Avt5p in vacuolar amino acid transport, the uptake of a variety of amino acids and 2-DG by Davt5 mutant cells was examined (Fig. 3). As shown in Fig. 3A, uptake of histidine, tyrosine and glutamate was significantly impaired and after 90 min in Davt5 cells only 50% of radio-labeled amino acid accumulated that was taken up by parental cells. Uptake of basic amino acids arginine and lysine was also affected
(Fig. 3A) and serine uptake decreased by about 30% in Davt5 cells after 90 min (data not shown). In contrast, cellular accumulation of 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 the transport of a particular set of amino acids. We conclude that Avt5p significantly contributes to the uptake of histidine, glutamate or tyrosine and, to a lesser extent, to that of arginine, lysine or serine. To clarify whether reduced uptake of these amino acids in the absence of Avt5p is caused by a deficiency of sequestration into vacuoles, we compared the effect of CCA on the uptake of glutamine and glutamate (Fig. 3B). While the uptake of 2-DG was little
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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-type cells 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 in DMSO (solid bars). The activity without addition (normal uptake) was taken as 100%. The results shown are the means ± standard deviations of three independent experiments.
affected by 10 lM CCA in the case of either mutant or parental cells, glutamine uptake was equally affected, suggesting vacuolar accumulation of this amino acid and confirming that this happens via an Avt5p-independent process. In contrast, glutamate uptake by parental cells was inhibited by CCA to the same extent as when Avt5p was absent in untreated cells, by about 40%. Treatment of Davt5 cells with CCA did not cause a significantly further decrease in glutamate uptake, suggesting that Avt5p mediates or controls VATPase-dependent uptake of glutamate into vacuoles. These results indicate that Avt5p is involved in vacuolar uptake of a specific set of amino acids. 3.4. Vacuolar uptake of amino acids during nitrogen starvation Under nitrogen starvation, cells utilize vacuolar amino acid pool as nitrogen sources [23]. In S. cerevisiae, several vacuolar amino acid transporters respond to nitrogen starvation and have been investigated for understanding the physiological role of vacuolar compartmentalization [11,24].
To define the role of Avt5p in the S. pombe response to nitrogen starvation, we examined the uptake activities of glutamate and lysine by parental cells, Davt5 cells and Dfnx1 cells in nitrogen-poor (MM-N) medium. As described above, Avt5p is involved in vacuolar uptake of glutamate and lysine (Fig. 3A), and, as reported in our previous paper, Fnx1p is a vacuolar transporter for lysine, isoleucine and asparagine, but not glutamate [15]. The glutamate and lysine uptake varied when wild-type cells were subjected to nitrogen starvation: after one hour incubation in MN-N medium the uptake of these amino acids peaked and increased almost three-fold, but upon further incubation this increase reversed and after three hours the uptake was close to normal, i.e. the level observed during growth on rich medium (Fig. 4A). In the absence of Avt5p, however, nitrogen starvation induced a very limited transient increase in the glutamate uptake by about 20%. In contrast, lysine uptake peaked after one hour on MM-N media as this was still two-fold higher than in the case of Davt5 cells grown on rich medium. Compared to the parental cells the overall uptake activities for glutamate and lysine were reduced to 40% and 50%, in line with the expected transport defect caused by lack of Avt5p (Fig. 3A). Despite its role in the uptake of lysine, Avt5p seems not essential for the occurrence of a transient increase in lysine uptake during nitrogen starvation. In Davt5 cells this response could be mediated by Fnx1p and Fnx2p [15], which was confirmed by the finding that upon disruption of fnx1, the transient increase in lysine uptake on MM-N was limited to about 20% when compared to lysine uptake during growth on rich medium. After 1 h nitrogen starvation the lysine uptake of Dfnx1 cells was only about 25% of that of parent cells and about 50% of that of Davt5 cells, suggesting that the fnx1/2 gene products are major determinants of lysine transport. Glutamate uptake, however, seems more under the control of Avt5p and independent of Fnx1p as in Dfnx1 cells this transport was comparable to that observed for the parental strain during growth on rich media as well as under conditions of nitrogen starvation (Fig. 4A, [15]). These results suggest that nitrogen starvation induces Avt5p-dependent glutamate uptake as well as Fnx1p-dependent lysine uptake. The transient increase of amino acid uptake in response to nitrogen starvation also involves vacuolar compartmentalization as it relies on V-ATPase activity. Exposure to CCA reduced the uptake of glutamate or lysine, as measured after one hour incubation in MM-N medium, to a basal level which was about 25% of the uptake observed for untreated wild-type cells (Fig. 4B). Furthermore, all the remaining uptake activities of Davt5 and Dfnx1 cells were inhibited by CCA to the same basal level. In contrast, uptake of 2-DG by parental or mutant strains under nitrogen starvation was constant (Fig. 4A) and not affected by exposure to CAA (Fig. 4B). We conclude that both Avt5p- and Fnx1p-dependent amino acid uptake should be attributed to vacuolar transport occurring during growth on rich medium or under conditions of nitrogen starvation.
4. Discussion Although phenotypic analysis revealed a role for S. pombe Avt5p in spore formation during nitrogen starvation [16], this protein is phylogenetically related to vacuolar proteins ScAvt5p and ScAvt6 of S. cerevisiae (Fig. 1A). In this study, we have presented evidence that Avt5p localizes to the vacuolar membrane (Fig. 1B) and that plays a critical role in amino acid uptake by S. pombe cells. In the absence of Avt5p the uptake of a variety of amino acids was reduced to that in wild-type cells was sensitive to CCA, indicating that Avt5p is involved in V-ATPase-dependent import of histidine, tyrosine, glutamate, to a lesser extent, lysine, arginine and serine into vacuoles of S. pombe (Figs. 2–4).
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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) in three independent experiments. (B) Effect of CCA. See also the legend for Fig. 2.
In S. cerevisiae, basic amino acids are enriched in vacuoles, whereas the acidic amino acids glutamate and aspartic acid are exclusively localized to the cytosol. The vacuoles of S. pombe retains 85–100% of the cellular content of basic amino acids, and most of aspartic acid and more than 15% of glutamate are accumulated in the vacuole, which suggested the presence of vacuolar importer(s) for these acidic amino acids [6]. Interestingly, accumulation of acidic amino acids by vacuolar membrane vesicles of wild-type cells of S. cerevisiae was low in the presence of ATP, and ScAvt6p was found to contribute to the export of acidic amino acids from vacuoles [11]. Thus one S. cerevisiae homologue of Avt5p appears to be an effluxer of glutamate, but in S. pombe this protein is required for vacuolar glutamate accumulation, which occurs in a V-ATPase-dependent manner (Figs. 3 and 4). Vacuolar compartmentalization of amino acids in S. pombe seems therefore fundamentally similar to that of S. cerevisiae except for the localization of acidic amino acids [6].
Avt5p was also involved in vacuolar transport of amino acids under nitrogen starvation (Fig. 4). Nitrogen starvation has been known to induce a set of cellular responses aimed at maintaining survivability. Many amino acid transporters and permeases operate at the plasma membrane [25,26] and the vacuolar membrane [15,18] and nitrogen starvation induces the expression of these genes [18,27,28]. Our results indicate that nitrogen starvation results in the up-regulation of Avt5p-dependent amino acid uptake by whole cells, and that this stimulation is V-ATPase dependent (Fig. 4). A similar increase has been observed in the case of Fnx1p-dependent lysine uptake (Fig. 4), suggesting that vacuolar amino acid transporters become temporarily more active under nitrogen starvation of S. pombe. It is unlikely that this up-regulation is mediated by an increase in transcription of the genes encoding the subunits of V-ATPase [28]. It cannot be excluded, however, that V-ATPase activity may change as a result of an altered interaction of the integral V0 domain with the peripheral V1 domain, as
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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) as compared 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 of CCA during nitrogen starvation. See also the legend for Fig. 2.
occurs in cells under glucose starvation [29]. Therefore, it needs to be determined whether, and if so how, V-ATPase is activated under nitrogen starvation. It is possible that Avt5p functions as a vacuolar importer with a substrate specificity that is more wide-ranging than that of Fnx1p [15]. It has been reported that transcription of fnx1+ is induced upon nitrogen starvation [18], which parallels activation of the Fnx1p-dependent uptake of lysine (Fig. 4). Northern blot analysis revealed, however, that transcription of avt5+ remained as low during nitrogen starvation as during growth in rich medium (data not shown). In support of this finding, microarray analysis has shown that in Tor2 depleted cells, which have a phenotype very similar to that of wild-type cells starved for nitrogen, expression of many membrane transporters is induced, but putative vacuolar amino acid transporters, except fnx1+, are not up-regulated [27]. Therefore, it remains unclear whether Avt5p is directly or indirectly in-
volved in vacuolar import of amino acids. If, on one hand, Avt5p acts as a vacuolar importer, it could be post-translationally regulated by phosphorylation, or the association or dissociation of a regulatory molecule in response to altered growth conditions. On the other hand, Avt5p may control as a regulatory protein the distribution of other membrane proteins that are directly involved in vacuolar amino acid import. For instance, Avt5p could be important for the organization of a membrane microdomain, analogous to the role of ATP-binding cassette (ABC) transporter Pdr10p, which was recently found to be required for the incorporation of another ABC transporter, Pdr12p, into a detergent-resistant membrane microdomain within the plasma membrane of S. cerevisiae [30]. Ergosterol, a constituent of membrane microdomains, is involved in homotypic vacuolar fusion [31], which suggests that such, functionally important, microdomains also occur in vacuolar membranes.
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Our results demonstrate that Avt5p contributes to amino acid import into vacuoles of S. pombe, in contrast to its S. cerevisiae homologue ScAvt6p, which mediates vacuolar export. It would be interesting to determine the directionality of the other S. cerevisiae homologue and putative amino acid transporter, ScAvt5p. Further work on purification and reconstitution of Avt5p is scheduled to obtain direct evidence for the function of Avt5p in vacuolar amino acid transport. Acknowledgements We thank Shuichi Katoh and Shiro Kajiwara for technical assistance and Dr. Taro Nakamura for providing plasmid pTN197. This work was supported in part by a Grant-in-Aid for Scientific Research (to Y.K.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] Magasanik, B. and Kaiser, C.A. (2002) Nitrogen regulation in Saccharomyces cerevisiae. Gene 290, 1–18. [2] Hinnebusch, A.G. (2005) Translational regulation of Gcn4 and the general amino acid control of yeast. Annu. Rev. Microbiol. 59, 407–450. [3] Klionsky, D.J., Herman, P.K. and Emr, S.D. (1990) The fungal vacuole: composition, function, and biogenesis. Microbiol. Rev. 54, 266–292. [4] Wiemken, A. and Dürr, M. (1974) Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae. Arch. Microbiol. 101, 45– 57. [5] Ohsumi, Y., Kitamoto, K. and Anraku, Y. (1988) Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion. J. Bacteriol. 170, 2676–2682. [6] Chardwiriyapreecha, S., Hondo, K., Inada, H., Chahomchuen, T., Sekito, T., Iwaki, T. and Kakinuma, Y. (2009) A simple and specific procedure to permeabilize the plasma membrane of Schizosaccharomyces pombe. Biosci. Biotechnol. Biochem. 73, 2090–2095. [7] Sato, T., Ohsumi, Y. and Anraku, Y. (1984) Substrate specificities of active transport systems for amino acids in vacuolar-membrane vesicles of Saccharomyces cerevisiae. Evidence of seven independent proton/amino acid antiport systems. J. Biol. Chem. 259, 11505–11508. [8] Sekito, T., Fujiki, Y., Ohsumi, Y. and Kakinuma, Y. (2008) Novel families of vacuolar amino acid transporters. IUBMB life. 60, 519–525. [9] Ohsumi, Y. and Anraku, Y. (1981) Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. J. Biol. Chem. 256, 2079–2082. [10] Shimazu, M., Sekito, T., Akiyama, K., Ohsumi, Y. and Kakinuma, Y. (2005) A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae. J. Biol. Chem. 280, 4851–4857. [11] Russnak, R., Konczal, D. and McIntire, S.L. (2001) A family of yeast proteins mediating bidirectional vacuolar amino acid transport. J. Biol. Chem. 276, 23849–23857. [12] Paulsen, I.T., Sliwinski, M.K., Nelissen, B., Goffeau, A. and Saier Jr., M.H. (1998) Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae. FEBS Lett. 430, 116–125. [13] Saier Jr., M.H. (2000) A functional/phylogenetic classification system for transmembrane solute transporters. Microbiol. Mol. Biol. Rev. 64, 354–411.
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