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In yeast cells growing under nutrient-rich condition approximately 50% of total amino acids are accumulated in the vacuoles; however, the composition of amino acids in the cytosol and in the vacuoles is quite different. The vacuoles, like lysosomes, degrade proteins transported into their lumen and produce amino acids. These amino acids should be quickly excreted to the cytosol under nutrient starvation condition and recycled for de novo protein synthesis. These suggest that specific machineries that transport amino acids into and out of the vacuoles operate at the vacuolar membrane. Several families of transporter involved in the vacuolar compartmentalization of amino acids have been identified and characterized using budding yeast Saccharomyces cerevisiae. In this review, we describe the vacuolar amino acid transporters identified so far and introduce recent findings on their activity and physiological function.

DOI： 10.1248/bpb.b18-00165

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Amino acids stored in the vacuoles are exported to the cytosol mainly for protein synthesis; however, the molecular identity of vacuolar amino acid exporters remains obscure in plants. Here, we demonstrate that the heterologous expression of AtAVT3 genes, Arabidopsis homologs of AVT3 and AVT4 encoding vacuolar amino acid exporters in yeast, reduces vacuolar amino acid levels in the avt3 Delta avt4 Delta yeast cells. In vitro experiments revealed that C-14-labeled Ala and Pro are exported from vacuolar membrane vesicles by AtAvt3A in an ATP-dependent manner. In Arabidopsis, AtAvt3A fused with green fluorescent protein localizes to the vacuolar membrane. We propose that AtAVT3 family represents the long sought-for vacuolar amino acid exporters in plants.

DOI： 10.1002/1873-3468.12507

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DOI： 10.1271/kagakutoseibutsu.54.324

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In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4 cells, as well as vba1 and vba2 cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4 cells than in wild type, vba1, and vba2 cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.

DOI： 10.1080/09168451.2015.1083401

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Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3((1-270)) expressed in S. pombe avt3 cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3 cells in spore formation was rescued by the expression of avt3(+) but was not completely rescued by the expression of avt3((1-270)). The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.

DOI： 10.1080/09168451.2016.1220819

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The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2 Delta and ypq3 Delta mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2 Delta and ypq3 Delta. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1 Delta ypq2 Delta ypq3 Delta avt1. completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.

DOI： 10.1080/09168451.2016.1141041

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Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter.

DOI： 10.1080/09168451.2015.1058703

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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 avt3 Delta cells, but decreased in that of avt3(+)-overexpressing avt3. 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.

DOI： 10.1371/journal.pone.0130542

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Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1 increment mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by gamma-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.

DOI： 10.1080/09168451.2014.998621

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Active transport systems for various amino acids operate in the vacuolar membrane of Saccharomyces cerevisiae. The gene families for vacuolar amino acid transporters were identified by reverse genetics experiments. In the AVT transporter family, Avt1p works for active uptake of amino acid into vacuole, and Avt3p, Avt4p, and Avt6p for active extrusion of amino acid from vacuole to cytosol. Here, we found green fluorescent protein-tagged Avt7p, an unidentified member of the AVT family, localized to the vacuolar membrane of S. cerevisiae. Disruption of the AVT7 gene enhanced both vacuolar contents of several amino acids and uptake activities of glutamine and proline by vacuolar membrane vesicles. Efficiency of spore formation was impaired by the disruption of the AVT7 gene, suggesting the physiological importance of Avt7p-dependent efflux of amino acid from vacuoles under nutrient-poor condition.

DOI： 10.1080/09168451.2014.963501

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Saccharomyces cerevisiae Ypq1p is a vacuolar membrane protein of the PQ-loop protein family. We found that ATP-dependent uptake activities of amino acids by vacuolar membrane vesicles were impaired by ypq1 Delta mutation. Loss of lysine uptake was most remarkable, and the uptake was recovered by overproduction of Ypq1p. Ypq1p is thus involved in transport of amino acids into vacuoles.

DOI： 10.1080/09168451.2014.918489

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Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.

DOI： 10.1080/09168451.2014.910095

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Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

DOI： 10.2131/jts.39.311

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Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

DOI： 10.2131/jts.39.311

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A vacuolar membrane protein, Vba2p of Schizosaccharomyces pombe, is involved in basic amino acid uptake by intact cells. Here we found evidence that Vba2p mediated ATP-dependent lysine uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae. Vba2p was also responsible for quinidine sensitivity, and the addition of lysine improved cell growth on quinidine-containing media. These findings should be useful for further characterization of Vba2p.

DOI： 10.1271/bbb.130387

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Vba5p is closest to Vba3p in the vacuolar transporter for basic amino acids (VBA) family of Saccharomyces cerevisiae. We found that green fluorescence protein (GFP)-tagged Vba5p localized exclusively to the plasma membrane. The uptake of lysine and arginine by whole cells was little affected by deletion of the VBA5 gene, but was stimulated by overexpression of the VBA5 gene. The inhibitory effect of 4-nitroquinoline N-oxide on cell growth was accelerated by expression of the VBA5 gene, and was lessened by the addition of arginine. These results suggest that Vba5p is a plasma membrane protein involved in amino acid uptake and drug sensitivity.

DOI： 10.1271/bbb.120455

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Amino acid analysis of Saccharomyces cerevisiae cells indicated that neutral amino acids such as glycine and alanine were probably excluded from the vacuoles, and that vacuolar H+-ATPase (V-ATPase) was involved in the vacuolar compartmentalization of these amino acids. We found that vacuolar membrane vesicles export neutral amino acids in an ATP-dependent manner. This is important in identifying vacuolar transporters for neutral amino acids.

DOI： 10.1271/bbb.120372

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation.

DOI： 10.1083/jcb.201202061

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

ATP-binding cassette (ABC) transporter plays an important role for resistance against xenobiotics. There are eleven ABC transporter genes in the genome of fission yeast Schizosaccharomyces pombe. We examined the role of ABC transporter against the toxicity of tributyltin chloride (TBT), a widespread environmental pollutant, in cell growth. Among individual ABC transporter mutants, the growth of a mutant deficient in Bfr1p, a plasma membrane-embedded transporter, was extremely sensitive to TBT. The lethal TBT concentration inducing 50% of cell death (LC50) was 25 mu M for the parent strain and 10.2 mu m for the bfr1 Delta mutant. Thus, Bfr1p was responsible for TBT resistance in S. pombe.

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The fission yeast Schizosaccharomyces pombe has a homolog of the budding yeast Atg22p, which is involved in spore formation (Mukaiyama H. et al., Microbiology, 155, 3816-3826 (2009)). GFP-tagged Atg22p in the fission yeast was localized to the vacuolar membrane. Upon disruption of atg22, the amino acid levels of the cellular fraction as well as the vacuolar fraction decreased. The uptake of several amino acids, such as lysine, histidine, and arginine, was impaired in atg22 Delta cells. S. pombe Atg22p plays an important role in the compartmentalization of amino acids.

DOI： 10.1271/bbb.100747

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ATP-binding cassette (ABC) transporter plays an important role for resistance against xenobiotics. There are eleven ABC transporter genes in the genome of fission yeast Schizosaccharomyces pombe. We examined the role of ABC transporter against the toxicity of tributyltin chloride (TBT), a widespread environmental pollutant, in cell growth. Among individual ABC transporter mutants, the growth of a mutant deficient in Bfrlp, a plasma membrane-embedded transporter, was extremely sensitive to TBT. The lethal TBT concentration inducing 50% of cell death (LC50) was 25 μM for the parent strain and 10.2 μM for the bfrlΔ mutant. Thus, Bfr1p was responsible for TBT resistance in S. pombe.

DOI： 10.2131/jts.36.117

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A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.

DOI： 10.1271/bbb.100627

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The fission yeast Schizosaccharomyces pombe was sensitive to salinity; cell growth was stopped by 0.5 M NaCl and by 10 mm LiCl. The avt5(+) gene encodes a vacuolar transporter with a broad specificity for amino acids. We found that the avt5 Delta mutant became highly tolerant of Li+ and Na+ in growth. Concanamycin A-sensitive Li+ uptake as well as cellular Li+ content was lower in the avt5 mutant, suggesting a role of Avt5p in cellular uptake of toxic Li+.

DOI： 10.1271/bbb.100259

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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. (C) 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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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. © 2010 Federation of European Biochemical Societies.

DOI： 10.1016/j.febslet.2010.04.012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOL RES FOUNDATION  

Here we examined the significance of Avt6, a vacuolar exporter of glutamate and aspartate suggested by the in vitro membrane vesicle experiment, in vacuolar compartmentalization of amino acids in Saccharomyces cerevisiae cells. Fluorescent microscopic observation of GFP-fused Avt6 revealed it to be exclusively localized to the vacuolar membrane, with the amount of Myc-tagged Avt6 significantly increased under nitrogen starvation. Glutamate uptake by cells was enhanced by deletion of the AVT6 gene, indicating indirect involvement of Avt6 in cellular glutamate accumulation. Differences in acidic amino acid content of both total and vacuolar fractions were insignificant between the parent and avt6 Delta cells when cultured in nutrient-rich conditions. However, in nitrogen-starved conditions, the amount of glutamate and aspartate in the vacuolar fraction was notably increased in the avt6 Delta cells. Avt6 is thus involved in vacuolar amino acid compartmentalization in S. cerevisiae cells, especially under conditions of nitrogen starvation.

DOI： 10.2323/jgam.55.409

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Some members of F-ATP synthase (F-ATPase)/vacuolar type ATPase (V-ATPase) superfamily have been identified as the molecular target of this compound. TBT inhibited the activities of W-transporting or Na+-transporting F-ATPase as well as H+-transporting V-ATPase originated from various organisms. However, the sensitivity to TBT of Na+-transporting V-ATPase has not been investigated. We examined the effect of TBT on Na+-transporting V-ATPase from an eubacterium Enterococus hirae. The ATP hydrolytic activity of E. hirae V-ATPase in purified form as well as in membrane-bound form was little inhibited by less than 10 mu M TBT; IC50 for TBT inhibition of purified enzyme was estimated to be about 35 mu M. Active sodium transport by E. hirae-cells, indicating the in vivo activity of this V-ATPase, was not inhibited by 20 mu M TBT. By contrast, IC50 of H+-transporting V-ATPase of the vacuolar membrane vesicles from Saccharomyces cerevisiae was about 0.2 mu M. E. hirae V-ATPase is thus extremely less sensitive to TBT.

DOI： 10.2131/jts.34.575

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Tributyltin chloride (TBT), an environmental Pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Although it has been reported that TBT induces apoptotic cell death in mammalian, the action of TBT on eukaryotic microorganisms has not yet been fully investigated. In this study we examined the mechanism involved in cell death caused by TBT exposure in Saccharomyces cerevisiae. The median lethal concentration of TBT was 10 mu M for the parent strain BY4741 and 3 mu M for the pdr5A mutant defective in a major multidrug transporter, respectively. Fluorescence microscopic observations revealed nuclear condensation and chromatin fragmentation in cells treated with TBT indicating that cells underwent an apoptosis-like cell dearth. TBT-induced cell death was suppressed by deletion of the ycal gene encoding a homologue of the mammalian caspase. In parallel, reactive oxygen species (ROS) were produced by TBT. These results suggest that TBT induces apoptosis-like cell death in yeast via an Yca1 p-dependent pathway possibly downstream of the ROS production. This is the first report on TBT-induced apoptotic cell death in yeast.

DOI： 10.2131/jts.34.541

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Cu2+-treatment is a useful technique in selectively permeabilizing the fungal plasma membrane. We describe herein a practical application with Schizosaccharomyces pombe. Incubation of cells with 0.5 mM CuCl2 at 30 degrees C for 20 min induced efficient leakage of cytosolic constituents. The kinetic characteristics of the calcium and amino acid flux from Cu2+-treated S. pombe cells suggested that the Cu2+ treatment permeabilized the plasma membrane without loss of vacuolar function. As a further application of the method, the amino acid contents of Cu2+-treated and untreated cells were also determined. The amino acid pool of Cu2+-treated wild-type cells was enriched in basic amino acids but not in acidic amino acids, as is characteristic of the vacuolar amino acid pool of fungi, including Saccharomyces cerevisiae and Neurosporra crassa. The amino acid pool of the S. pombe V-ATPase mutant vma1 Delta was also successfully determined. We conclude that the vacuolar amino acid pool of S. pombe can be measured using Cu2+-treated cells. The method is simple, inexpensive, and rapid relative to the isolation of vacuolar vesicles, making it useful in estimating vacuolar pools and transport across the vacuolar membrane.

DOI： 10.1271/bbb.90319

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Autophagy is a degradation system of cytoplasmic proteins and organelles via formation of double-membrane vesicles called autophagosomes. In the yeast Saccharomyces cerevisiae, autophagosomes are formed via the pre-autophagosomal structure (PAS) in a manner dependent on Atg proteins. Under nutrient-rich condition, Atg9 is recruited to the PAS by binding to Atg11 for the Cvt pathway. However, because Atg9 is recruited to the PAS in atg11 Delta cells in starved condition and autophagy is induced, autophagy-specific mechanism for the Atg9 recruitment to the PAS has been assumed. Here, we demonstrate that, in autophagy-inducing condition, Atg9 is recruited to the PAS in a manner dependent on Atg17. Atg9 physically interacts with Atg17 in the presence of rapamycin. This interaction requires Atg1, a protein kinase essential for autophagy. Consistently, the Atg17-dependent PAS localization of Atg9 requires Atg1. However, its kinase activity is dispensable for this process. It rather regulates the equilibrium of assembly and disassembly of Atg9 at the PAS.

DOI： 10.1111/j.1365-2443.2009.01299.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Atg18 is essential for both autophagy and the regulation of vacuolar morphology. The latter process is mediated by phosphatidylinositol 3,5-bisphosphate binding, which is dispensable for autophagy. Atg18 also binds to phosphatidylinositol 3-phosphate ( PtdIns( 3) P) in vitro. Here, we investigate the relationship between PtdIns( 3) P-binding of Atg18 and autophagy. Using an Atg18 variant, Atg18(FTTG), which is unable to bind phosphoinositides, we found that PtdIns( 3) P binding of Atg18 is essential for full activity in both selective and nonselective autophagy. Atg18( FTTG) formed a complex with Atg2 in a normal manner, and Atg18-Atg2 complex formation occurred in cells in the absence of PtdIns( 3) P, indicating that Atg18-Atg2 complex formation is independent of PtdIns(3)P-binding of Atg18. Atg18 localized to endosomes, the vacuolar membrane, and autophagic membranes, whereas Atg18( FTTG) did not localize to these structures. The localization of Atg2 to autophagic membranes was also lost in Atg18( FTTG) cells. These data indicate that PtdIns( 3) P-binding of Atg18 is involved in directing the Atg18-Atg2 complex to autophagic membranes. Connection of a 2 x FYVE domain, a specific PtdIns(3)P-binding domain, to the C terminus of Atg18( FTTG) restored the localization of Atg18-Atg2 to autophagic membranes and full autophagic activity, indicating that PtdIns(3)P-binding by Atg18 is dispensable for the function of the Atg18-Atg2 complex but is required for its localization. This also suggests that PtdIns( 3) P does not act allosterically on Atg18. Taken together, Atg18 forms a complex with Atg2 irrespective of PtdIns( 3) P binding, associates tightly to autophagic membranes by interacting with PtdIns( 3) P, and plays an essential role.

DOI： 10.1074/jbc.M803180200

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Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed. (C) 2008 IUBMB.

DOI： 10.1002/iub.92

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have identified the Schizosaccharonvees pombe SPBC3E7.06c gene (fnx2(+)) from a homology search with the fiix1(+) gene involving in G(0) arrest upon nitrogen starvation. Green fluorescent protein-fused Fnx1p and Fnx2p localized exclusively to the vacuolar membrane. Uptake of histidine or isoleucine by S. pombe cells was inhibited by concanamycin A, a specific inhibitor of the vacuolar H+-ATPase. Amino acid uptake was also defective in the vacuolar ATPase mutant, suggesting that vacuolar compartmentalization is critical for amino acid uptake by whole cells. In both Delta fnx1 and Delta fnx2 mutant cells, uptake of lysine, isoleucine or asparagine was impaired. These results suggest that Jnx1(+) and fnx2(+) are involved in vacuolar amino acid uptake in S. pombe. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2008.05.017

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Autophagy induced by nutrient depletion is involved in survival during starvation conditions. In addition to starvation-induced autophagy, the yeast Saccharomyces cerevisiae also has a constitutive autophagy-like system, the Cvt pathway. Among 31 autophagy-related (Atg) proteins, the function of Atg17, Atg29, and Atg31 is required specifically for autophagy. In this study, we investigated the role of autophagy-specific (i.e., non-Cvt) proteins under autophagy-inducing conditions. For this purpose, we used atg11 Delta cells in which the Cvt pathway is abrogated. The autophagy-unique proteins are required for the localization of Atg proteins to the pre-autophagosomal structure (PAS), the putative site for autophagosome formation, under starvation condition. It is likely that these Atg proteins function as a ternary complex, because Atg29 and Atg31 bind to Atg17. The Atg1 kinase complex (Atg1-Atg13) is also essential for recruitment of Atg proteins to the PAS. The assembly of Atg proteins to the PAS is observed only under autophagy-inducing conditions, indicating that this structure is specifically involved in autophagosome formation. Our results suggest that Atg1 complex and the autophagy-unique Atg proteins cooperatively organize the PAS in response to starvation signals.

DOI： 10.1091/mbc.E07-10-1048

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Autophagy is a bulk degradation process that is conserved in eukaryotic cells and functions in the turnover of cytoplasmic materials and organelles. When eukaryotic cells face nutrient starvation, the autophagosome, a double-membraned organelle, is generated from the pre-autophagosomal structure (PAS). In the yeast Saccharomyces cerevisiae, 16 ATG (autography-related) genes are essential for autophagosome formation. Most of the Atg proteins are involved in the PAS, leading to autophagosome production. However, the mechanism of PAS organization remains to be elucidated. Here, we performed a systematic and quantitative analysis by fluorescence microscopy to develop a hierarchy map of Atg proteins involved in PAS organization. This analysis suggests that Atg17p is the most basic protein in PAS organization: when it is specifically targeted to the plasma membrane, other Atg proteins are recruited to that location, suggesting that Atg17p acts as a scaffold protein to organize Atg proteins to the PAS.

DOI： 10.1111/j.1365-2443.2007.01050.x

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In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex 11, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.

DOI： 10.1091/mbc.E05-09-0841

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affacted. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCLO69w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.

DOI： 10.1074/jbc.M412617200

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To update sequenced data, we determined the 5' and 3' termini of yeast Hansenula wingei (Pichia canadensis) mitochondrial (mt) large subunit ribosomal RNA (LSU) which is encoded in the mt genome. The 5' end position was mapped downstream from a putative transcription starting site which is homologous to a Saccharomyces cerevisiae mitochondrial promoter sequence. This suggests that the primary transcript of LSU is processed from 5' end and then mature transcript is formed. This processing is different from that of S. cerevisiae mt LSU in which processing on its 5' end does not occur. Based on the sequence data of H. wingei mt LSU, we constructed its secondary structure, and compared it with those of the other fungal organisms. Conserved regions of H. wingei LSU were identified and used for subsequent phylogenetic analysis. In genome structure and gene content, H. wingei mt genome has several characteristics similar to those in filamentous fungi, but the phylogenetic analysis indicates closer kinship to yeast S. cerevisiae. This agrees with previous non-sequencing phylogenies and suggests that extraordinary rearrangements have occurred in yeast mt genomes during divergent evolution.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Activation of retrograde signaling (RS) by mitochondrial dysfunction or by inhibition of TOR kinases in yeast results in nuclear accumulation of the transcription factors, Rtg1p and Rtg3p. This process requires Rtg2p, a novel cytoplasmic protein with an N-terminal ATP binding domain. We show that Rtg2p controls RS by reversibly binding a negative regulator, Mksip. The inhibitory form of Mksip is phosphorylated and complexed with the 14-3-3 proteins, Bmh1p and Bmh2p, which are also negative regulators of RS. A hypophosphorylated form of Mks1p bound to Rtg2p is inactive. Point mutations in the Rtg2p ATP binding domain simultaneously block RS and Mks1p-Rtg2p interaction. We propose that activation of RS via mitochondrial dysfunction and TOR inhibition intersect at the Rtg2pMks1p switch.

DOI： 10.1016/S1097-2765(03)00285-5

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TOR plays a key role in cell growth and cell-cycle progression, but in addition recent studies have shown that TOR is also involved in the regulation of a number of molecular processes associated with nutrient deprivation, such as autophagy. In budding yeast, TOR negatively regulates activation of Apg1 protein kinase, which is essential for the induction of autophagy. This review describes recent research in this field and the mechanism by which TOR mediates induction of autophagy.

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An important function of the RTG signaling pathway is maintenance of intracellular glutamate supplies in yeast cells with dysfunctional mitochondria. Herein, we report that MKS1 is a negative regulator of the RTG pathway, acting between Rtg2p, a proximal sensor of mitochondrial function, and the bHLH transcription factors Rtg1p and Rtg3p. In mks1Delta cells, RTG target gene expression is constitutive, bypassing the requirement for Rtg2p, and is no longer repressible by glutamate. We show further that Mks1p is a phosphoprotein whose phosphorylation pattern parallels that of Rtg3p in response to activation of the RTG pathway, and that Mks1p is in a complex with Rtg2p. MKS1 was previously implicated in the formation of [URE3], an inactive prion form of a negative regulator of the nitrogen catabolite repression pathway, Ure2p. rtgDelta mutations induce [URE3] and can do so independently of MKS1. We find that glutamate suppresses [URE3] formation, suggesting that the Mks1p effect on the formation of [URE3] can occur indirectly via regulation of the RTG pathway.

DOI： 10.1091/mbc.01-09-0473

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In cells with reduced mitochondrial function, RTG1, 2 and 3 are required for expression of genes involved in glutamate synthesis. Glutamate negatively regulates RTG-dependent gene expression upstream of Rtg2p, which, in turn, acts upstream of the bHLH/Zip transcription factors, Rtg1p and Rtg3p. Here we report that some mutations [lst8-(2-5)] in LST8, an essential gene encoding a seven WD40-repeat protein required for targeting of amino acid permeases (AAPs) to the plasma membrane, bypass the requirement for Rtg2p and abolish glutamate repression of RTG-dependent gene expression. The lst8-1 mutation, however, which reduces plasma membrane expression of AAP, cannot bypass the Rtg2p requirement, but still suppresses glutamate repression of RTG target gene expression. We show that Lst8p negatively regulates RTG gene function, acting at two sites, one upstream of Rtg2p, affecting glutamate repression of RTG-dependent gene expression through Ssy1p, an AAP-like sensor of external amino acids, and the other between Rtg2p and Rtg1p-Rtg3p. These data, together with genome-wide transcription profiling, reveal pathways regulated by glutamate, and provide insight into the regulation of cellular responses to mitochondrial dysfunction.

DOI： 10.1093/emboj/20.24.7209

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Cells modulate the expression of nuclear genes in response to changes in the functional state of mitochondria, an interorganelle communication pathway called retrograde regulation. In yeast, expression of the CIT2 gene shows a typical retrograde response in that its expression is dramatically increased in cells with dysfunctional mitochondria, such as in ρ°petites. Three genes control this signaling pathway: RTG1 and RTG3, which encode basic helix-loop-helix leucine zipper transcription factors that bind as heterodimer to the CIT2 upstream activation site, and RTG2, which encodes a protein of unknown function. We show that in respiratory-competent (ρ+) cells in which CIT2 expression is low, Rtg1p and Rtg3p exist as a complex largely in the cytoplasm, and in ρ°petites in which CIT2 expression is high, they exist as a complex predominantly localized in the nucleus. Cytoplasmic Rtg3p is multiply phosphorylated and becomes partially dephosphorylated when localized in the nucleus. Rtg2p, which is cytoplasmic in both ρ+and ρ°cells, is required for the dephosphorylation and nuclear localization of Rtg3p. Interaction of Rtg3p with Rtg1p is required to retain Rtg3p in the cytoplasm of ρ+cells; in the absence of such interaction, nuclear localization and dephosphorylation of Rtg3p is independent of Rtg2p. Our data show that Rtg1p acts as both a positive and negative regulator of the retrograde response and that Rtg2p acts to transduce mitochondrial signals affecting the phosphorylation state and subcellular localization of Rtg3p.

DOI： 10.1091/mbc.11.6.2103

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We have accomplished the nucleotide sequence of the 1537 bp mitochondrial gene coding for small subunit (SSU) rRNA of yeast Hansenula wingei, and also determined the 5'- and 3'-termini by S1 nuclease mapping. Eight universally conserved (U) elements of the SSU rRNA were identified. Comparison of U regions among five fungal mitochondrial SSU rRNAs shows the striking similarity between H. wingei and Saccharomyces cerevisiae. The construction of the secondary structure revealed a core structure similar to the counterpart of Escherichia coli 16S rRNA. The secondary structure also enabled us to specify seven variable (V) regions differing from those of other mitochondrial SSU rRNAs in size, sequence and possible secondary structure. Molecular phylogenetic evaluation based on U regions of five fungi indicates that mitochondria of H. wingei and S. cerevisiae diverged from the same lineage. This suggests that the evolution of mitochondria-encoded genes does not directly correlate with the alteration of mitochondrial genetic system: genome size, gene organization and codon usage.

DOI： 10.1266/ggs.71.69

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For long time, it has been believed that the yeast mitochondrial (mt) genome lacks NADH dehydrogenase subunit genes which are designated ND genes. However, our complete mtDNA sequencing of yeast Hansenula wingei led us to the first finding of seven mitochondrial ND genes. We investigated the distribution of ND genes in mtDNAs of other yeasts including Pichia membranaefaciens, Yarrowia lypolitica, Candida maltosa, Saccharomyces kluyveri and Saccharomyces exiguus. By Southern hybridization with probes of H. wingei's ND1, 2 and 5 genes, we detected positive signals on mtDNAs in P. membranaefaciens, Y. lypolitica, and C. maltosa. To confirm this, we cloned and sequenced DNA fragment of ND5 gene in P. membranaefaciens. We have discussed the sequence homology and genome structure.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

The complete 27 694-bp mitochondrial (mt) DNA sequence of Hansenula wingei, which is a typical budding yeast and contains circular mitochondrial DNA, has been determined. The mt sequence contains genes encoding large and small ribosomal RNAs, 25 tRNAs, three subunits of cytochrome c oxidase (subunits 1, 2 and 3), three subunits of ATPase (subunits 6, 8 and 9), apocytochrome b, seven subunits of NADH dehydrogenase (subunits 1, 2, 3, 4, 4L, 5 and 6), and a ribosomal protein, VAR1. The VAR1 gene is considered to be a typical yeast type. This is consistent with data on DNA and the deduced amino-acid sequence homology comparisons of genes ubiquitous in yeast and fungi. However, we have identified seven genes encoding NADH dehydrogenase subunits, which are not found in other yeast mitochondrial genomes, thus placing the H. wingei mitochondrial genome in a unique position. In addition the H. wingei mitochondrial genome also encodes one tRNA pseudogene and one short unidentified ORF. The genome is compact with only two introns both of which contain an ORF. One intron lies in the large rRNA gene while the other is situated in the cytochrome c oxidase subunit-1 gene. The conserved nonanucleotide motif (A/T)TATAAG (T/A)(A/T), which is a transcription start signal in Saccharomyces cerevisiae mitochondria, has also been found in the H. wingei mitochondrial genome. The codon assignments for ATA and CTN in H. wingei mitochondria are different from those in S. cerevisiae mitochondria. These results indicate a unique and novel structure for the H. wingei mitochondrial genome in terms of characteristics which are typical for both yeast and for filamentous fungi. This is the first complete mt DNA sequence report in yeast.

DOI： 10.1007/BF00311880

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For sequencing, mitochondrial DNA from Hansenula wingei yeast was digested with various restriction enzymes and the resultant DNA fragments were cloned into a pEMBL phasmid vector. Our clone bank consists of 39 overlapping clones which cover the entire 27 694 bp region of the H. wingei mitochondrial genome. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Database with the following Accession Number: D31785. Copyright © 1995 John Wiley &amp
Sons Ltd.

DOI： 10.1002/yea.320111313

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer-Verlag  

Genes homologous to those encoding mitochondrial NADH dehydrogenase subunits ND4L and ND5 in filamentous fungi were identified in the mitochondrial genome of a budding yeast, Hansenula wingei. The structure and expression of these genes were investigated. The H. wingei ND4L gene is 282 by long, and potentially codes for a polypeptide of 94 amino acids. The putative ND4L protein sequence shares about 46% homology with the analogous mitochondrial proteins of filamentous fungi. The H. wingei ND5 gene is 1935 by long, and encodes a 645-residue polypeptide. The derived ND5 protein shares about 38% sequence homology with the analogue in filamentous fungi. The ND4L and ND5 genes have no intervening sequence, and form a gene cluster in the order of 5′-ND4L-ND5-3′. A presumptive mature dicistronic or polycistronic transcript of these genes was detected by Northern blot hybridization. These results strongly indicate that these ND4L and ND5 genes are active. As far as we are aware, this is the first report on the identification of mitochondrially encoded ND genes in yeast. © 1994 Springer-Verlag.

DOI： 10.1007/BF00280478

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

DOI： 10.1093/nar/21.15.3589

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Language：Japanese   Publisher：広島大学生物学会  

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Vacuoles have been often thought to be storage compartments: excess nitrogen is stored as amino acid in vacuoles. However, vacuolar amino acid concentrations in several plant species are estimated to be lower than cytosolic ones, suggesting active export of amino acids from vacuoles. So far, transporters that are responsible for amino acid efflux at the tonoplast have not been identified in plants.<br> Yeast Avt3/Avt4 mediate export of neutral amino acids across the vacuole membrane. We found that <I>AtAVT3a </I>, an Arabidopsis homologue of <I>AVT3/AVT4 </I>, was localized at the tonoplast in plant cells. Furthermore, we showed that <I>AtAVT3a </I>could rescue defects of yeast <I>avt3/avt4 </I>mutant in amino acid transport. This is the first report of a plant functional vacuolar amino acid transporter.

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