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Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae a
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Junichi Tone , Ayumi Yoshimura , Kunio Manabe , Nami Murao , Takayuki Sekito , Miyuki Kawano-Kawada
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& Yoshimi Kakinuma
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Laboratory of Molecular Physiology and Genetics, Faculty of Agriculture, Ehime University, Matsuyama, Japan b
Integrated Center for Sciences (INCS), Ehime University, Matsuyama, Japan Published online: 09 Mar 2015.
Click for updates To cite this article: Junichi Tone, Ayumi Yoshimura, Kunio Manabe, Nami Murao, Takayuki Sekito, Miyuki Kawano-Kawada & Yoshimi Kakinuma (2015): Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.998621 To link to this article: http://dx.doi.org/10.1080/09168451.2014.998621
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Bioscience, Biotechnology, and Biochemistry, 2015
Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae Junichi Tone1, Ayumi Yoshimura1, Kunio Manabe1, Nami Murao1, Takayuki Sekito1, Miyuki Kawano-Kawada1,2 and Yoshimi Kakinuma1,2,* 1 2
Laboratory of Molecular Physiology and Genetics, Faculty of Agriculture, Ehime University, Matsuyama, Japan; Integrated Center for Sciences (INCS), Ehime University, Matsuyama, Japan
Received November 12, 2014; accepted December 2, 2014
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http://dx.doi.org/10.1080/09168451.2014.998621
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 avt1Δ 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 γ-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. Key words:
vacuole; amino acid transporter; Saccharomyces cerevisiae
Vacuoles are the largest organelles in the budding yeast Saccharomyces cerevisiae, occupying about 25% of the cell volume and serve as a storage compartment of a variety of amino acids.1,2) The bulk of cationic amino acids (arginine, histidine, lysine) localizes in vacuoles and, in contrast, glutamic acid, which is the most abundant amino acid in yeast, is almost excluded.1,2) Other amino acids are also compartmentalized in vacuoles although it is less significant.1,2) The imbalance in concentrations of free amino acids between the vacuolar and cytosolic pools implies the existence of active transport systems for amino acids on the vacuolar membrane. ATP-dependent amino acid uptake, which is driven by the proton electrochemical gradient generated by the action of vacuolar H+-ATPase
(V-ATPase), was successfully observed by purified vacuolar membrane vesicles.3,4) The kinetic experiment of amino acid uptake by the vesicles has suggested seven independent uptake systems for amino acids.4) However, no active uptake of glutamic acid, aspartic acid, proline, valine, threonine, or alanine was reported.3,4) The genes for vacuolar amino acid transport have been found by reverse genetics by in vitro transport experiments with vacuolar membrane vesicle. Two gene families, VBA and AVT, were reported.5) The VBA family classified into the major facilitator superfamily (MFS)6) consists of seven genes, five VBA genes (VBA1–VBA5), AZR1, and SGE1.7–10) We found that VBA1, VBA2, and VBA3 genes are involved in uptake of cationic amino acids into vacuoles.7) As a member of the amino acid/auxin permease (AAAP) superfamily,11) the AVT family, which is related to the neuronal γ-aminobutyric acid (GABA)-glycine vesicular transporters, consists of seven AVT genes from AVT1 to AVT7 (Fig. 1(A)). It was reported that AVT1 gene is involved in uptake of glutamine, isoleucine, and tyrosine into vacuoles.12) In the opposite, AVT3 and the closest AVT4 are linked with extrusion of glutamine, isoleucine, and tyrosine from vacuoles.12) AVT6 gene works for extrusion of glutamic acid and aspartic acid.12) Recently, we found that both Avt3p and Avt4p mediate extrusion of various neutral amino acids from vacuoles.13) Interestingly, cationic amino acids (arginine, lysine, and histidine) were recognized as the substrate of Avt4p not Avt3p.13) Both Avt3p and Avt4p-mediated transport were dependent on the generation of a proton gradient, suggesting the mechanism of proton/amino acid symporter. In addition, we found that Avt7p is involved in efflux of amino acids from vacuoles,14) but the function of Avt2p and Avt5p is still unknown. Avt3p, Avt4p, and Avt7p were required for spore formation probably because of releasing vacuolar amino acids into cytosol to be utilized under nutrientlimited condition.14) Very recently, it was reported that the PQ-loop proteins in the transporter/opsin/G protein
*Corresponding author. Email: [email protected] Abbreviations: V-ATPase, vacuolar H+-ATPase; ORF, open reading frame; GABA, γ-aminobutyric acid; MFS, the major facilitator superfamily; AAAP, amino acid/auxin permease superfamily; GFP, green fluorescent protein; CCCP, carbonylcyanide m-chlorophenylhydrazone; MES, 2-(N-morpholino)ethanesulfonic acid. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Materials and methods Strains and media. S. cerevisiae strains used in this study are listed in Table 1. For deletion of AVT1, AVT1 ORF was replaced with a loxP-flanked kanMX or a hphMX cassette by a PCR-mediated method.21,22) Proper gene disruptions were confirmed by PCR analysis of chromosomal DNA from transformants. Yeast cells were grown at 30 °C in either YPD (1% yeast extract, 2% bacto-peptone, 2% glucose) or SD + Cas (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate, 0.5% ammonium sulfate, 0.5% casamino acids, 20 mg/L tryptophan, and 2% glucose) medium.
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Fig. 1. The characteristics of Avt1p as a vacuolar membrane protein. Notes: (A) Phylogenetic position of Avt1p in the AVT family. AVT family proteins were aligned using ClustalW (http://clustalw.ddbj.nig. ac.jp/), and the tree was visualized using TreeView software. (B) Topology model of Avt1p. Three phosphopeptides were identified in the N-terminal hydrophilic region by phosphoproteomics analysis,29) and locations of phosphoserine and phosphothreonine were indicated as [P]. (C) Subcellular localization of GFP-Avt1p. GFP-Avt1p expressed under ADH promoter was observed by fluorescence microscopy. Vacuolar membranes were specifically stained with a red fluorescent dye FM4-64. Colocalization of GFP fluorescence and FM4-64-stained vacuolar membrane is shown in a merged image. Bar, 5 μm.
coupled receptor (TOG) superfamily15) are involved in transport of cationic amino acids in vacuoles.16) We found that Ypq1p in this family is involved in lysine transport by vacuolar membrane vesicles.17) Furthermore, Uga4p and Atg22p, which is involved in autophagy, are the vacuolar membrane proteins belonging to MFS. In the experiments with intact cells, it is suggested that Uga4p and Atg22p are involved in transport of GABA and leucine/isoleucine/tyrosine, respectively, in vacuoles.18,19) Vacuolar compartmentalization of amino acids is thus achieved by a variety of transport systems, but individual transporter has not well been studied. Recently, it was reported that the lifespan of S. cerevisiae is affected by function of Avt1p as well as alteration in vacuolar pH.20) Overexpressing or deleting AVT1 was sufficient to extend or shorten replicative lifespan of S. cerevisiae, respectively. In order to understand the significance of Avt1p in lifespan of this organism, it is the prerequisite to characterize the feature of Avt1p as a transporter. In this paper we examined the catalytic properties of Avt1p, and the results
Plasmid construction. The DNA fragment encoding GFP with a 5′ XbaI and 3′ BamHI site was ligated to an XbaI- and BamHI-digested p416ADH plasmid,23) yielding p416ADH-GFP(N). To construct p416ADHGFP-AVT1, the AVT1 ORF fragment with BamHI sites at both ends was amplified by PCR using yeast genomic DNA as the template, and cloned into BamHIdigested p416ADH-GFP(N). p416ADH-AVT1 and p416GPD-AVT1 were constructed by cloning the AVT1 ORF fragment amplified using primers containing XbaI and HindIII sites, and S. cerevisiae genomic DNA as a template into either p416ADH or p416GPD23) digested with XbaI and HindIII. All constructs were confirmed by DNA sequencing. Chemicals. Carbonylcyanide m-chlorophenylhydrazone (CCCP), nigericin, and valinomycin were purchased from Sigma–Aldrich. Concanamycin A was purchased from Wako Pure Chemicals. Radiolabeled amino acids were purchased from Perkin Elmer or GE Healthcare. Fluorescence microscopy. To visualize vacuolar membrane, cells were labeled with FM4-64 (Invitrogen) as described previously.24) Fluorescence images were captured using an Olympus IX71 fluorescence microscope equipped with a SenSys cooled charge-coupled device camera (Hamamatsu Photonics) and MetaMorph software (Molecular Devices). Transport assay. Vacuolar membrane vesicles were prepared as described previously.3) Protein concentration of vacuolar membrane vesicles was determined by the method of Lowry et al.25) using bovine serum albumin as the standard. The mixture (100 μL) consisted of 25 mM 2-(N-morpholino)ethanesulfonic acid (MES)–Tris (pH 6.9), 4 mM MgCl2, 25 mM KCl, 0.5 mM ATP, and 40 μg of protein of vacuolar membrane vesicles which was incubated at 25 °C for 5 min, and the reaction was started by adding 0.1 mM 14Clabeled amino acid and stopped by diluting the reaction mixture with 5 mL of ice-cold wash buffer consisting
Vacuolar proton/amino acid antiporter in yeast Table 1.
Yeast strains used in this study.
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X2180-1B STY3807 STY3827 STY3828 STY4109
MATα SUC2 mal mel gal2 CUP1 X2180-1B ura3Δ::loxP STY3807 avt1Δ::loxP STY3807 avt3Δ::kanMX avt4Δ::loxP STY3807 avt1Δ::hphMX avt3Δ::kanMX avt4Δ::loxP
Yeast genetic stock center13)
of 25 mM MES–Tris (pH 6.9), 5 mM MgCl2, and 25 mM KCl. Vesicles were recovered onto 0.45 μm cellulose acetate membrane filter (ADVANTEC) and washed with 5 ml of ice-cold wash buffer. The radioactivity remained on the filter was measured using an Aloka LSC-5100 liquid scintillation counter.
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Determination of quinacrine quenching. Proton pumping activity was measured by monitoring quinacrine fluorescence quenching as described previously.26) Vacuolar membrane vesicles (40 μg of protein) were added to the buffer containing 5 mM MES–Tris (pH 7.2), 4 mM MgCl2, 25 mM KCl, and 1 μM quinacrine. After incubation in a quartz cuvette at room temperature for 5 min, 0.4 mM ATP was added to initiate quenching. At the steady level of quenching, 0.1 mM of isoleucine or histidine was added as indicated. CCCP (10 μM) was added for dissipation of pH gradients. Fluorescence was measured using a fluorescence spectrophotometer (RF-1500, SHIMADZU) with an excitation wavelength of 427 nm and an emission wavelength of 495 nm. Amino acid composition analysis of vacuolar fraction in S. cerevisiae. Vacuolar amino acids were extracted by cupric ion treatment method.27) Briefly, 10 OD660 units of logarithmic growing cells were suspended in 3 mL of the buffer containing 2.5 mM potassium phosphate buffer (pH 6.0), 0.6 M sorbitol, 10 mM glucose, and 0.2 mM CuCl2 and incubated at 30 °C for 15 min. After washing once with the same buffer without CuCl2, cells were suspended with 500 μL of water and boiled for 15 min. After centrifugation, resulting supernatants were collected as the vacuolar fraction. The amino acid compositions were measured by an automatic amino acid analyzer (L-8900, Hitachi).
Results Avt1p is involved in uptake of various amino acids by the vacuolar membrane vesicles The 65 kDa (602 amino acids) Avt1p is predicted to have ten transmembrane helices by transmembrane prediction servers, such as TMHMM, TMpred, and DAS, and possesses a long hydrophilic region (about 200 amino acids) at its amino terminal (Fig. 1(B)). Since the data of subcellular localization of Avt1p has not been represented in the previous paper,12) the localization of GFP-tagged Avt1p was investigated by fluorescence microscopy. GFP was tagged to the amino terminus of Avt1p and expressed under the ADH promoter in avt1Δavt3Δavt4Δ cells. GFP-Avt1p localized
This study13) This study
in the vacuolar membrane and merged with FM4-64 fluorescence (Fig. 1(C)). It has been reported that ATPdependent uptake of isoleucine, glutamine, and tyrosine was impaired in the vesicles of a single avt1Δ mutant.12) Uptake activities of these amino acids were accelerated in the vesicles of avt3Δ and/or avt4Δ mutants, suggesting that both Avt3p and Avt4p are involved in extrusion of these amino acids from vacuoles.12) Recently, we reported that both Avt3p and Avt4p have the broad substrate specificity for amino acids. In particular, Avt4p recognizes basic amino acids as the substrate.13) Although it has been reported that no active uptake of glutamic acid, aspartic acid, proline, valine, threonine, or alanine was detected by the vesicles of the wild-type strain,4) uptake of these amino acids could be affected by the activities of Avt3p and Avt4p. The activities of amino acid uptake by the vesicles of avt3Δavt4Δ and avt1Δavt3Δavt4Δ mutants were compared (Fig. 2). Although ATP-dependent uptake of alanine or proline was not clearly observed in the vesicles of avt3Δavt4Δ, uptake of valine or threonine was observed (Fig. 2). ATP-dependent uptake of glutamic acid and aspartic acid was not observed in the vesicles of avt3Δavt4Δ (data not shown). Uptake of isoleucine, valine, threonine, and glutamine observed by the vesicles of avt3Δavt4Δ disappeared in the vesicles of avt1Δavt3Δavt4Δ (Fig. 2). Although uptake of arginine and lysine was only slightly affected by AVT1 mutation, histidine uptake was impaired in the vesicles of avt1Δavt3Δavt4Δ mutant. These results suggest that Avt1p is involved in uptake of various amino acids.
Avt1p-dependent amino acid uptake is driven by the proton electrochemical gradient The AVT1 gene was expressed in the avt1Δavt3Δavt4Δ mutant cells under the control of different constitutive promoters, and ATP-dependent uptake of several amino acids by the vacuolar vesicles was examined (Fig. 3(A)). Uptake of glutamine, tyrosine, alanine, isoleucine, and histidine was observed depending upon the GPD promoter. Uptake of arginine and lysine was stimulated by expression of AVT1. The uptake activity of isoleucine was low by the ADH promoter (Fig. 3(A), closed triangle). By the kinetics of the initial rate of isoleucine uptake, the Km values of Avt1pdependent uptake by GPD and ADH promoters were nearly equal, and the Vmax values changed depending upon the strength of promoter, reflecting the amount of Avt1p in the vacuolar membrane vesicles (Fig. 3(B)). Uptake of isoleucine or histidine was clearly inhibited by the V-ATPase inhibitor: concanamycin A, the protonophore: CCCP and nigericin (Fig. 4), indicating that
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Fig. 2. ATP-dependent uptake of various amino acids by the vacuolar membrane vesicles of avt3Δavt4Δ and avt1Δavt3Δavt4Δ mutants. Notes: Amino acid uptake into vacuolar membrane vesicles isolated from avt3Δavt4Δ (closed circles) and avt1Δavt3Δavt4Δ (open circles) mutants was measured. Preparation of the vacuolar membrane vesicles and amino acid uptake assay were performed as described in “Materials and methods.” Results are the mean ± SD of three independent experiments.
uptake is driven by the proton electrochemical gradient across the vacuolar membrane. Effect of valinomycin was also examined in the presence of 25 mM K+ so as to dissipate membrane potential under the assay condition. Both uptake of isoleucine and histidine were little affected by addition of valinomycin (Fig. 4), suggesting that membrane potential is not involved in active uptake of amino acid. Avt1p-dependent proton flux in the presence of amino acid Since it was expected that Avt1p mediates antiport of proton with amino acid, Avt1p-dependent proton movement was examined by a change in fluorescence of quinacrine as the pH indicator (Fig. 5). In the vesicles of avt1Δavt3Δavt4Δ mutant cells, V-ATPasedependent acidification of the internal pH was little affected by addition of isoleucine or histidine, but it was abolished by CCCP (Fig. 5(A)). In the case of the vesicles of Avt1p-overproducing cells, ATP-dependent quenching of quinacrine fluorescence was declined by addition of isoleucine or histidine (Fig. 5(B)). Although determination of the stoichiometry between fluxes of proton and the corresponding amino acid requires further investigation, these results suggested that Avt1p is an antiporter of proton and amino acid.
Fig. 3. Avt1p-dependent uptake of various amino acids by vacuolar membrane vesicles. Notes: (A) Time course of ATP-dependent uptake of isoleucine. Vacuolar membrane vesicles were isolated from avt1Δavt3Δavt4Δ cells carrying either pGPD-AVT1 (closed circles), pADH-AVT1 (closed triangles), or empty vector (open circles). Preparation of the vesicles and amino acid uptake assay were performed as in Fig. 2. Results are the mean ± SD of three independent experiments. (B) Kinetic parameters of Avt1p-dependent isoleucine uptake. The apparent Km and Vmax values for isoleucine uptake were determined.
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Substrate specificity of Avt1p Fig. 6 shows the effect of various amino acids and GABA on Avt1p-dependent isoleucine uptake by the vesicles. Each amino acid (at the final concentration of 0.5 mM) was added 3 min before initiating isoleucine uptake (at 0.1 mM). Isoleucine uptake was significantly
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inhibited by histidine as well as phenylalanine, tryptophan, and alanine and moderately by methionine, leucine, valine, glutamine, proline, serine, threonine, and glycine. Inhibition by lysine, asparagine, and arginine was insignificant, and uptake was little inhibited by aspartic acid, glutamic acid, and GABA (Fig. 6). The Km (mM) and Vmax (nmol/min/mg protein) values of Avt1p-dependent histidine uptake were 0.5 ± 0.1 and 89.5 ± 13.8, respectively. Isoleucine uptake was competitively inhibited by histidine with Ki (mM) value of 0.6 ± 0.1. These results suggest that Avt1p recognizes various amino acids, except for GABA, glutamate, and aspartate. Role of Avt1p in vacuolar compartmentalization of amino acid in cells As described above, vacuolar amino acid transport has been studied in vitro using vacuolar membrane vesicles,3,4,7,12–14,17) but examination on the in vivo involvement of these transporters in vacuolar compartmentalization of amino acid in cells is limited.14,29) Recently, we reported that the vacuolar contents of various amino acids significantly increased in avt3Δavt4Δ mutant cells; neutral amino acids especially increased.13) Here, we examined the involvement of Avt1p in vacuolar amino acid compartmentalization in cells. First, effect of avt1Δ mutation on the vacuolar contents of amino acid was examined (Fig. 7(A)). The
amounts of glutamine, tyrosine, proline, histidine, and arginine slightly decreased in the vacuolar fraction of avt1Δ mutant in comparison with those of the wildtype strain. Effect of avt1Δ mutation was more prominent in avt3Δavt4Δ mutant as background. Threonine, serine, asparagine, glutamine, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, and proline, all increased in avt3Δavt4Δ mutant, clearly decreased in the vacuoles of avt1Δavt3Δavt4Δ mutant (Fig. 7(A)). Aspartic acid and glutamic acid slightly decreased in avt1Δavt3Δavt4Δ mutant, and a decrease in the vacuolar contents of lysine, histidine, and arginine was not so prominent. We also examined the effect of Avt1p overproduction on the vacuolar contents of amino acid in cells of the wild-type strain and avt3Δavt4Δ mutant cultured in SD + Cas medium (Fig. 7(B)). The AVT1 gene was constitutively expressed under GPD promoter. In both strains, most of amino acids, except for glutamic acid and three cationic amino acids, were affected by Avt1p production. Threonine, serine, asparagine, glutamine, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, and proline increased. In particular, an increase in the contents of threonine and glutamine was remarkable, being close to the contents of lysine and arginine, the most abundant ones in vacuoles (Fig. 7(B)). Although the effect of Avt1p overproduction on a prominent increase in the contents of threonine and glutamine should be further investigated, the
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Fig. 5. Avt1p-dependent proton movement coupled with amino acid. Notes: Vacuolar membrane vesicles were isolated from avt1Δavt3Δavt4Δ cells carrying either empty vector (A) or pGPD-AVT1 (B). Following the addition of quinacrine and stabilization of the fluorescence signal, ATP was added to initiate the reaction. Final concentration of additions: ATP 0.4 mM, amino acid 0.1 mM, and CCCP 10 μM. ΔF/F = ratio of fluorescence quenching: initial fluorescence.
Fig. 6. Effect of various amino acids and GABA on Avt1p-dependent isoleucine uptake. Notes: Vacuolar membrane vesicles isolated from avt1Δavt3Δavt4Δ cells carrying pGPD-AVT1 were incubated with 0.1 mM 14C-labeled isoleucine in the presence of 0.5 mM non-labeled amino acids as indicated (black bars). The initial rates (0.5 min) of uptake were determined. The transport activity relative to the control (no addition of non-labeled amino acids, white bar) is shown. Results are mean ± SD of three independent experiments.
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Fig. 7. Effect of avt1Δ mutation and Avt1p overproduction on the vacuolar contents of amino acids. Notes: Cells grown in YPD (A) or SD + Cas (B) were treated with CuCl2 to extract vacuolar amino acids as described in “Materials and methods.” Results are the mean ± SD of three independent experiments. The contents of basic amino acids are indicated in the right panel. Symbols: (A) wild type (white bar), avt1Δ (light gray bar), avt3Δavt4Δ (dark gray bar), or avt1Δavt3Δavt4Δ (black bar). (B) wild type/empty vector (white bar), wild type/p416GPD-AVT1 (light gray bar), avt3Δavt4Δ/empty vector (dark gray bar), or avt3Δavt4Δ/p416GPD-AVT1 (black bar).
results suggest that Avt1p is important for vacuolar compartmentalization of various amino acids, especially neutral ones, being in cooperation with activities of Avt3p and Avt4p.
Discussion Here, we examined some catalytic properties of Avt1p, the only uptake system for amino acid into vacuoles in the AVT family.12) The results suggested that Avt1p is a proton/amino acid antiporter. Avt1p has a relatively broad substrate specificity for amino acids, and Avt1p prefers histidine as well as neutral amino acids as the substrate (Fig. 6). Avt1p-dependent uptake of histidine and isoleucine was little affected by valinomycin (Fig. 4), suggesting the main involvement of the pH gradient as the driving force. Avt1p-dependent arginine uptake (Fig. 3(A)) was also valinomycin insensitive (data not shown). In considering the dissociation arginine constants of histidine (pK3 = 6.04), (pK3 = 12.48), and lysine (pK3 = 10.54), the results suggest that the unprotonated form of histidine as well as other neutral amino acids is preferred as the substrate under the assay condition (pH 6.9). It is reported that various AAAP transporters have a broad substrate specificity for neutral, anionic, and cationic amino acids.12–14,30) We did not clearly observe ATP-dependent uptake of glutamic acid or aspartic acid (data not shown) by the vesicles of avt3Δavt4Δ mutant overproducing Avt1p and a change in the vacuolar contents of these amino acids (Fig. 7). Since Avt6p is involved in extrusion of these anionic amino acids,12,29) it is important to investigate the involvement of Avt1p in transport of anionic amino acids by the avt6Δ mutant as
background. In any case, we are now going to purify Avt1p so as to investigate directly its catalytic feature with proteoliposome reconstitution system. The vacuolar contents of various neutral amino acids are insignificant in the wild-type cells cultured in YPD medium, although the concentrations of these amino acids in vacuoles are slightly higher than those in cytosol (Kakinuma et al., unpublished data). The roles of Avt3p and Avt4p transporters are critical for vacuolar compartmentalization of amino acid.13) In avt3Δavt4Δ mutant cells, the vacuolar contents of threonine, glutamine, glycine, alanine, and other amino acids extremely increased (Fig. 7). In the avt1Δavt3Δavt4Δ mutant, the amounts of these neutral amino acids decreased to more or less 50–60% of those of avt3Δavt4Δ mutant, indicating that Avt1p plays a major role for uptake of these amino acids into vacuoles. Although active transport of these neutral amino acids was impaired in the vesicles of avt1Δavt3Δavt4Δ mutant (Fig. 2), the results suggest that these amino acids still remained in the vacuoles of avt1Δavt3Δavt4Δ mutant. Although fluorescence microscopic observation on intact cells showed a slight enlargement of vacuoles in avt3Δavt4Δ mutant (Kakinuma et al. unpublished data), the concentrations of these amino acids in vacuoles could be still higher than those in cytosol of avt1Δavt3Δavt4Δ mutant cells. In other words, active accumulation of neutral amino acids into vacuoles could be expected in this mutant. The results may be explained by two possibilities. First, there is some other active transport system for amino acids, driven by proton gradient, was inactivated in the preparation of the vacuolar vesicles. Second, there may be other transport system for amino acid, not depending upon proton gradient as well as ATP. It has been reported that the activity of arginine/
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Vacuolar proton/amino acid antiporter in yeast
histidine exchange is found in the S. cerevisiae vacuoles.31) In avt1Δavt3Δavt4Δ mutant cells, a large concentration gradient of cationic amino acids, lysine and arginine, across the vacuolar membrane is still established by the systems such as VBA and YPQ transporters. These gradients may be the driving force of uptake of neutral amino acid, which will be worthwhile to be investigated as another uptake pathway for amino acids. In any case, it is obvious that vacuolar compartmentalization of various amino acids is embodied by harmonized activities of Avt1p and other transporters. Recently, it was reported that mitochondrial dysfunction in replicatively aged S. cerevisiae arises from altered vacuolar pH.20) Vacuolar acidity declined during the early asymmetric divisions of a mother cell, and preventing this decline suppresses mitochondrial dysfunction and extends lifespan. Interestingly, overexpression or deletion of AVT1 was also sufficient to extend or shorten replicative lifespan of this organism, respectively.20) The authors speculated that changes in vacuolar pH limit mitochondrial function probably by reducing Avt1p-dependent amino acid storage in the vacuolar lumen. Mitochondria-dependent catabolism of excess cytoplasmic amino acids may place a large burden on function of mitochondrial carrier proteins. Since the vacuolar acidity was declined by Avt1p-dependent proton efflux (Fig. 5), Avt1p-mediated amino acid uptake, not proton efflux, should be responsible for the reason for replicative lifespan.20) We examined total cell contents of amino acids in strains used in the experiments of Fig. 7 and estimated the cytosolic contents of amino acids by subtracting the vacuolar contents from the total cell contents. However, we did not find any amino acids, the amount of which increased or decreased by deleting or overexpressing AVT1 (data not shown). Phosphopep database (http://www.phospho pep.org/) has indicated that the amino-terminal hydrophilic region of Avt1p possesses several phosphorylation sites (Fig. 1(B)). Since the amounts of phosphopeptides originated from the amino acid sequence of Avt1p were affected by rapamycin treatment,28) TORC1 kinase system is likely involved in its regulation. Therefore, when considering the role of Avt1p in lifespan, we should pay attention to regulatory behavior of Avt1p in signal transduction network. In any case, based on the catalytic feature of Avt1p as a transporter, further study is required for characterizing the physiological role of Avt1p in yeast. Finally, we should pay attention to the results on the effect of Avt1p production on vacuolar content of histidine (Fig. 7). Although histidine is one of the preferable substrates of Avt1p transporter by vesicles, the vacuolar content of histidine in cells was little influenced by AVT1 expression, being in contrast with those of neutral amino acids. What is the reason for this discrepancy? At the moment, we consider the involvement of polyphosphate in vacuolar accumulation of basic amino acids in cells. A large amount of polyphosphate accumulates in vacuoles of fungi,32) presumably posing Donnan potential across the vacuolar membrane. For the charge compensation at the steady state, polyphosphate could offer the accumulation capacity of basic amino acids not neutral ones, although active transport systems are involved in dynamic movements of various
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amino acids. Vacuolar compartmentalization of amino acids, especially basic ones, in vivo does not only depend upon the function of transport systems. We are also going to investigate the significance of vacuolar polyphosphate in amino acid compartmentalization.
Acknowledgments This work was supported in part by Elizabeth Arnold Fuji Foundation (to M.K-K.) and the Noda Science Foundation (to Y.K. and T.S.).
Funding This work was supported by the Public Foundation of Elizabeth Arnold-Fuji and the Noda Institute for Scientific Research.
References [1] Wiemken A, Durr M. Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae. Arch. Microbiol. 1974;101:45–57. [2] Kitamoto K, Yoshizawa K, Ohsumi Y, Anraku Y. Dynamic aspects of vacuolar and cytosolic amino acid pools of Saccharomyces cerevisiae. J. Bacteriol. 1988;170:2683–2686. [3] Ohsumi Y, Anraku Y. Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. J. Biol. Chem. 1981;256: 2079–2082. [4] Sato T, Ohsumi Y, Anraku Y. 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. 1984;259:11505–11508. [5] Sekito T, Fujiki Y, Ohsumi Y, Kakinuma Y. Novel families of vacuolar amino acid transporters. IUBMB Life. 2008;60:519–525. [6] Paulsen IT, Sliwinski MK, Nelissen B, Goffeau A, Saier MH Jr. Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae. FEBS Lett. 1998;430:116–125. [7] Shimazu M, Sekito T, Akiyama K, Ohsumi Y, Kakinuma Y. A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae. J. Biol. Chem. 2005;280:4851–4857. [8] Shimazu M, Itaya T, Pongcharoen P, Sekito T, Kawano-Kawada M, Kakinuma Y. Vba5p, a novel plasma membrane protein involved in amino acid uptake and drug sensitivity in Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 2012;76:1993–1995. [9] Tenreiro S, Rosa PC, Viegas CA, Sá-Correia I. Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae. Yeast. 2000;16:1469–1481. [10] Ehrenhofer-Murray AE, Würgler FE, Sengstag C. The Saccharomyces cerevisiae SGE1 gene product: a novel drug-resistance protein within the major facilitator superfamily. Mol. Gen. Genet. 1994;244:287–294. [11] Young GB, Jack DL, Smith DW, Saier MH Jr. The amino acid/ auxin: proton symport permease family. Biochim. Biophys. Acta. 1999;1415:306–322. [12] Russnak R, Konczal D, McIntire SL. A family of yeast proteins mediating bidirectional vacuolar amino acid transport. J. Biol. Chem. 2001;276:23849–23857. [13] Sekito T, Chardwiriyapreecha S, Sugimoto N, Ishimoto M, Kawano-Kawada M, Kakinuma Y. Vacuolar transporter Avt4 is involved in excretion of basic amino acids from the vacuoles of Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 2014;78:269–275.
Downloaded by [University of Washington Libraries] at 12:33 23 March 2015
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[14] Tone J, Yamanaka A, Manabe K, Murao N, Kawano-Kawada M, Sekito T, Kakinuma Y. A vacuolar membrane protein Avt7p is involved in transport of amino acid and spore formation in Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. Forthcoming 2014. [15] Yee DC, Shlykov MA, Västermark Åke, Reddy VS, Arora S, Sun EI, Saier MH Jr. The transporter-opsin-G protein-coupled receptor (TOG) superfamily. FEBS J. 2013;280:5780–5800. [16] Jezegou A, Llinares E, Anne C, Kieffer-Jaquinod S, O’Regan S, Aupetit J, Chabli A, Sagne C, Debacker C, ChadefauxVekemans B, Journet A, Andre B, Gasnier B. Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy. Proc. Nat. Acad. Sci. U.S.A. 2012;109:E3434–E3443. [17] Sekito T, Nakamura K, Manabe K, Tone J, Sato Y, Murao N, Kawano-Kawada M, Kakinuma Y. Loss of ATP-dependent lysine uptake in the vacuolar membrane vesicles of Saccharomyces cerevisiae ypq1Δ mutant. Biosci. Biotech. Biochem. 2014;78:1199–1202. [18] Yang Z, Huang J, Geng J, Nair U, Klionsky DJ. Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Mol. Biol. Cell. 2006;17:5094–5104. [19] Uemura T, Tomonari Y, Kashiwagi K, Igarashi K. Uptake of GABA and putrescine by UGA4 on the vacuolar membrane in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 2004;315:1082–1087. [20] Hughes AL, Gottschling DE. An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast. Nature. 2012;492:261–265. [21] Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 1996;24:2519–2524. [22] Goldstein AL, McCusker JH. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast. 1999;15:1541–1553.
[23] Mumberg D, Müller R, Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1995;156:119–122. [24] Vida TA, Emr SD. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 1995;128:779–792. [25] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265–275. [26] Ohsumi Y, Anraku Y. Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. J. Biol. Chem. 1983;258:5614–5617. [27] Ohsumi Y, Kitamoto K, Anraku Y. Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion. J. Bacteriol. 1988;170:2676–2682. [28] Huber A, Bodenmiller B, Uotila A, Stahl M, Wanka S, Gerrits B, Aebersold R, Loewith R. Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis. Genes Dev. 2009;23: 1929–1943. [29] Chahomchuen T, Hondo K, Ohsaki M, Sekito T, Kakinuma Y. Evidence for Avt6 as a vacuolar exporter of acidic amino acids in Saccharomyces cerevisiae cells. J. Gen. Appl. Microbiol. 2009;55:409–417. [30] Schiöth HB, Roshanbin S, Hägglund MG, Fredriksson R. Evolutionary origin of amino acid transporter families SLC32, SLC36 and SLC38 and physiological, pathological and therapeutic aspects. Mol. Aspects Med. 2013;34:571–585. [31] Sato T, Ohsumi Y, Anraku Y. An arginine/histidine exchange transport system in vacuolar-membrane vesicles of Saccharomyces cerevisiae. J. Biol. Chem. 1984;259:11509–11511. [32] Cramer CL, Davis RH. Polyphosphate-cation interaction in the amino acid-containing vacuole of Neurospora crassa. J. Biol. Chem. 1984;259:5152–5157.
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae a
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a
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Junichi Tone , Ayumi Yoshimura , Kunio Manabe , Nami Murao , Takayuki Sekito , Miyuki Kawano-Kawada
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& Yoshimi Kakinuma
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Laboratory of Molecular Physiology and Genetics, Faculty of Agriculture, Ehime University, Matsuyama, Japan b
Integrated Center for Sciences (INCS), Ehime University, Matsuyama, Japan Published online: 09 Mar 2015.
Click for updates To cite this article: Junichi Tone, Ayumi Yoshimura, Kunio Manabe, Nami Murao, Takayuki Sekito, Miyuki Kawano-Kawada & Yoshimi Kakinuma (2015): Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.998621 To link to this article: http://dx.doi.org/10.1080/09168451.2014.998621
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Bioscience, Biotechnology, and Biochemistry, 2015
Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae Junichi Tone1, Ayumi Yoshimura1, Kunio Manabe1, Nami Murao1, Takayuki Sekito1, Miyuki Kawano-Kawada1,2 and Yoshimi Kakinuma1,2,* 1 2
Laboratory of Molecular Physiology and Genetics, Faculty of Agriculture, Ehime University, Matsuyama, Japan; Integrated Center for Sciences (INCS), Ehime University, Matsuyama, Japan
Received November 12, 2014; accepted December 2, 2014
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http://dx.doi.org/10.1080/09168451.2014.998621
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 avt1Δ 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 γ-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. Key words:
vacuole; amino acid transporter; Saccharomyces cerevisiae
Vacuoles are the largest organelles in the budding yeast Saccharomyces cerevisiae, occupying about 25% of the cell volume and serve as a storage compartment of a variety of amino acids.1,2) The bulk of cationic amino acids (arginine, histidine, lysine) localizes in vacuoles and, in contrast, glutamic acid, which is the most abundant amino acid in yeast, is almost excluded.1,2) Other amino acids are also compartmentalized in vacuoles although it is less significant.1,2) The imbalance in concentrations of free amino acids between the vacuolar and cytosolic pools implies the existence of active transport systems for amino acids on the vacuolar membrane. ATP-dependent amino acid uptake, which is driven by the proton electrochemical gradient generated by the action of vacuolar H+-ATPase
(V-ATPase), was successfully observed by purified vacuolar membrane vesicles.3,4) The kinetic experiment of amino acid uptake by the vesicles has suggested seven independent uptake systems for amino acids.4) However, no active uptake of glutamic acid, aspartic acid, proline, valine, threonine, or alanine was reported.3,4) The genes for vacuolar amino acid transport have been found by reverse genetics by in vitro transport experiments with vacuolar membrane vesicle. Two gene families, VBA and AVT, were reported.5) The VBA family classified into the major facilitator superfamily (MFS)6) consists of seven genes, five VBA genes (VBA1–VBA5), AZR1, and SGE1.7–10) We found that VBA1, VBA2, and VBA3 genes are involved in uptake of cationic amino acids into vacuoles.7) As a member of the amino acid/auxin permease (AAAP) superfamily,11) the AVT family, which is related to the neuronal γ-aminobutyric acid (GABA)-glycine vesicular transporters, consists of seven AVT genes from AVT1 to AVT7 (Fig. 1(A)). It was reported that AVT1 gene is involved in uptake of glutamine, isoleucine, and tyrosine into vacuoles.12) In the opposite, AVT3 and the closest AVT4 are linked with extrusion of glutamine, isoleucine, and tyrosine from vacuoles.12) AVT6 gene works for extrusion of glutamic acid and aspartic acid.12) Recently, we found that both Avt3p and Avt4p mediate extrusion of various neutral amino acids from vacuoles.13) Interestingly, cationic amino acids (arginine, lysine, and histidine) were recognized as the substrate of Avt4p not Avt3p.13) Both Avt3p and Avt4p-mediated transport were dependent on the generation of a proton gradient, suggesting the mechanism of proton/amino acid symporter. In addition, we found that Avt7p is involved in efflux of amino acids from vacuoles,14) but the function of Avt2p and Avt5p is still unknown. Avt3p, Avt4p, and Avt7p were required for spore formation probably because of releasing vacuolar amino acids into cytosol to be utilized under nutrientlimited condition.14) Very recently, it was reported that the PQ-loop proteins in the transporter/opsin/G protein
*Corresponding author. Email: [email protected] Abbreviations: V-ATPase, vacuolar H+-ATPase; ORF, open reading frame; GABA, γ-aminobutyric acid; MFS, the major facilitator superfamily; AAAP, amino acid/auxin permease superfamily; GFP, green fluorescent protein; CCCP, carbonylcyanide m-chlorophenylhydrazone; MES, 2-(N-morpholino)ethanesulfonic acid. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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suggest that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids in cells.
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Avt7 Avt2 Avt4
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Materials and methods Strains and media. S. cerevisiae strains used in this study are listed in Table 1. For deletion of AVT1, AVT1 ORF was replaced with a loxP-flanked kanMX or a hphMX cassette by a PCR-mediated method.21,22) Proper gene disruptions were confirmed by PCR analysis of chromosomal DNA from transformants. Yeast cells were grown at 30 °C in either YPD (1% yeast extract, 2% bacto-peptone, 2% glucose) or SD + Cas (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate, 0.5% ammonium sulfate, 0.5% casamino acids, 20 mg/L tryptophan, and 2% glucose) medium.
vacuolar lumen
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Fig. 1. The characteristics of Avt1p as a vacuolar membrane protein. Notes: (A) Phylogenetic position of Avt1p in the AVT family. AVT family proteins were aligned using ClustalW (http://clustalw.ddbj.nig. ac.jp/), and the tree was visualized using TreeView software. (B) Topology model of Avt1p. Three phosphopeptides were identified in the N-terminal hydrophilic region by phosphoproteomics analysis,29) and locations of phosphoserine and phosphothreonine were indicated as [P]. (C) Subcellular localization of GFP-Avt1p. GFP-Avt1p expressed under ADH promoter was observed by fluorescence microscopy. Vacuolar membranes were specifically stained with a red fluorescent dye FM4-64. Colocalization of GFP fluorescence and FM4-64-stained vacuolar membrane is shown in a merged image. Bar, 5 μm.
coupled receptor (TOG) superfamily15) are involved in transport of cationic amino acids in vacuoles.16) We found that Ypq1p in this family is involved in lysine transport by vacuolar membrane vesicles.17) Furthermore, Uga4p and Atg22p, which is involved in autophagy, are the vacuolar membrane proteins belonging to MFS. In the experiments with intact cells, it is suggested that Uga4p and Atg22p are involved in transport of GABA and leucine/isoleucine/tyrosine, respectively, in vacuoles.18,19) Vacuolar compartmentalization of amino acids is thus achieved by a variety of transport systems, but individual transporter has not well been studied. Recently, it was reported that the lifespan of S. cerevisiae is affected by function of Avt1p as well as alteration in vacuolar pH.20) Overexpressing or deleting AVT1 was sufficient to extend or shorten replicative lifespan of S. cerevisiae, respectively. In order to understand the significance of Avt1p in lifespan of this organism, it is the prerequisite to characterize the feature of Avt1p as a transporter. In this paper we examined the catalytic properties of Avt1p, and the results
Plasmid construction. The DNA fragment encoding GFP with a 5′ XbaI and 3′ BamHI site was ligated to an XbaI- and BamHI-digested p416ADH plasmid,23) yielding p416ADH-GFP(N). To construct p416ADHGFP-AVT1, the AVT1 ORF fragment with BamHI sites at both ends was amplified by PCR using yeast genomic DNA as the template, and cloned into BamHIdigested p416ADH-GFP(N). p416ADH-AVT1 and p416GPD-AVT1 were constructed by cloning the AVT1 ORF fragment amplified using primers containing XbaI and HindIII sites, and S. cerevisiae genomic DNA as a template into either p416ADH or p416GPD23) digested with XbaI and HindIII. All constructs were confirmed by DNA sequencing. Chemicals. Carbonylcyanide m-chlorophenylhydrazone (CCCP), nigericin, and valinomycin were purchased from Sigma–Aldrich. Concanamycin A was purchased from Wako Pure Chemicals. Radiolabeled amino acids were purchased from Perkin Elmer or GE Healthcare. Fluorescence microscopy. To visualize vacuolar membrane, cells were labeled with FM4-64 (Invitrogen) as described previously.24) Fluorescence images were captured using an Olympus IX71 fluorescence microscope equipped with a SenSys cooled charge-coupled device camera (Hamamatsu Photonics) and MetaMorph software (Molecular Devices). Transport assay. Vacuolar membrane vesicles were prepared as described previously.3) Protein concentration of vacuolar membrane vesicles was determined by the method of Lowry et al.25) using bovine serum albumin as the standard. The mixture (100 μL) consisted of 25 mM 2-(N-morpholino)ethanesulfonic acid (MES)–Tris (pH 6.9), 4 mM MgCl2, 25 mM KCl, 0.5 mM ATP, and 40 μg of protein of vacuolar membrane vesicles which was incubated at 25 °C for 5 min, and the reaction was started by adding 0.1 mM 14Clabeled amino acid and stopped by diluting the reaction mixture with 5 mL of ice-cold wash buffer consisting
Vacuolar proton/amino acid antiporter in yeast Table 1.
Yeast strains used in this study.
Strain
Genotype
Source
X2180-1B STY3807 STY3827 STY3828 STY4109
MATα SUC2 mal mel gal2 CUP1 X2180-1B ura3Δ::loxP STY3807 avt1Δ::loxP STY3807 avt3Δ::kanMX avt4Δ::loxP STY3807 avt1Δ::hphMX avt3Δ::kanMX avt4Δ::loxP
Yeast genetic stock center13)
of 25 mM MES–Tris (pH 6.9), 5 mM MgCl2, and 25 mM KCl. Vesicles were recovered onto 0.45 μm cellulose acetate membrane filter (ADVANTEC) and washed with 5 ml of ice-cold wash buffer. The radioactivity remained on the filter was measured using an Aloka LSC-5100 liquid scintillation counter.
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Determination of quinacrine quenching. Proton pumping activity was measured by monitoring quinacrine fluorescence quenching as described previously.26) Vacuolar membrane vesicles (40 μg of protein) were added to the buffer containing 5 mM MES–Tris (pH 7.2), 4 mM MgCl2, 25 mM KCl, and 1 μM quinacrine. After incubation in a quartz cuvette at room temperature for 5 min, 0.4 mM ATP was added to initiate quenching. At the steady level of quenching, 0.1 mM of isoleucine or histidine was added as indicated. CCCP (10 μM) was added for dissipation of pH gradients. Fluorescence was measured using a fluorescence spectrophotometer (RF-1500, SHIMADZU) with an excitation wavelength of 427 nm and an emission wavelength of 495 nm. Amino acid composition analysis of vacuolar fraction in S. cerevisiae. Vacuolar amino acids were extracted by cupric ion treatment method.27) Briefly, 10 OD660 units of logarithmic growing cells were suspended in 3 mL of the buffer containing 2.5 mM potassium phosphate buffer (pH 6.0), 0.6 M sorbitol, 10 mM glucose, and 0.2 mM CuCl2 and incubated at 30 °C for 15 min. After washing once with the same buffer without CuCl2, cells were suspended with 500 μL of water and boiled for 15 min. After centrifugation, resulting supernatants were collected as the vacuolar fraction. The amino acid compositions were measured by an automatic amino acid analyzer (L-8900, Hitachi).
Results Avt1p is involved in uptake of various amino acids by the vacuolar membrane vesicles The 65 kDa (602 amino acids) Avt1p is predicted to have ten transmembrane helices by transmembrane prediction servers, such as TMHMM, TMpred, and DAS, and possesses a long hydrophilic region (about 200 amino acids) at its amino terminal (Fig. 1(B)). Since the data of subcellular localization of Avt1p has not been represented in the previous paper,12) the localization of GFP-tagged Avt1p was investigated by fluorescence microscopy. GFP was tagged to the amino terminus of Avt1p and expressed under the ADH promoter in avt1Δavt3Δavt4Δ cells. GFP-Avt1p localized
This study13) This study
in the vacuolar membrane and merged with FM4-64 fluorescence (Fig. 1(C)). It has been reported that ATPdependent uptake of isoleucine, glutamine, and tyrosine was impaired in the vesicles of a single avt1Δ mutant.12) Uptake activities of these amino acids were accelerated in the vesicles of avt3Δ and/or avt4Δ mutants, suggesting that both Avt3p and Avt4p are involved in extrusion of these amino acids from vacuoles.12) Recently, we reported that both Avt3p and Avt4p have the broad substrate specificity for amino acids. In particular, Avt4p recognizes basic amino acids as the substrate.13) Although it has been reported that no active uptake of glutamic acid, aspartic acid, proline, valine, threonine, or alanine was detected by the vesicles of the wild-type strain,4) uptake of these amino acids could be affected by the activities of Avt3p and Avt4p. The activities of amino acid uptake by the vesicles of avt3Δavt4Δ and avt1Δavt3Δavt4Δ mutants were compared (Fig. 2). Although ATP-dependent uptake of alanine or proline was not clearly observed in the vesicles of avt3Δavt4Δ, uptake of valine or threonine was observed (Fig. 2). ATP-dependent uptake of glutamic acid and aspartic acid was not observed in the vesicles of avt3Δavt4Δ (data not shown). Uptake of isoleucine, valine, threonine, and glutamine observed by the vesicles of avt3Δavt4Δ disappeared in the vesicles of avt1Δavt3Δavt4Δ (Fig. 2). Although uptake of arginine and lysine was only slightly affected by AVT1 mutation, histidine uptake was impaired in the vesicles of avt1Δavt3Δavt4Δ mutant. These results suggest that Avt1p is involved in uptake of various amino acids.
Avt1p-dependent amino acid uptake is driven by the proton electrochemical gradient The AVT1 gene was expressed in the avt1Δavt3Δavt4Δ mutant cells under the control of different constitutive promoters, and ATP-dependent uptake of several amino acids by the vacuolar vesicles was examined (Fig. 3(A)). Uptake of glutamine, tyrosine, alanine, isoleucine, and histidine was observed depending upon the GPD promoter. Uptake of arginine and lysine was stimulated by expression of AVT1. The uptake activity of isoleucine was low by the ADH promoter (Fig. 3(A), closed triangle). By the kinetics of the initial rate of isoleucine uptake, the Km values of Avt1pdependent uptake by GPD and ADH promoters were nearly equal, and the Vmax values changed depending upon the strength of promoter, reflecting the amount of Avt1p in the vacuolar membrane vesicles (Fig. 3(B)). Uptake of isoleucine or histidine was clearly inhibited by the V-ATPase inhibitor: concanamycin A, the protonophore: CCCP and nigericin (Fig. 4), indicating that
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uptake is driven by the proton electrochemical gradient across the vacuolar membrane. Effect of valinomycin was also examined in the presence of 25 mM K+ so as to dissipate membrane potential under the assay condition. Both uptake of isoleucine and histidine were little affected by addition of valinomycin (Fig. 4), suggesting that membrane potential is not involved in active uptake of amino acid. Avt1p-dependent proton flux in the presence of amino acid Since it was expected that Avt1p mediates antiport of proton with amino acid, Avt1p-dependent proton movement was examined by a change in fluorescence of quinacrine as the pH indicator (Fig. 5). In the vesicles of avt1Δavt3Δavt4Δ mutant cells, V-ATPasedependent acidification of the internal pH was little affected by addition of isoleucine or histidine, but it was abolished by CCCP (Fig. 5(A)). In the case of the vesicles of Avt1p-overproducing cells, ATP-dependent quenching of quinacrine fluorescence was declined by addition of isoleucine or histidine (Fig. 5(B)). Although determination of the stoichiometry between fluxes of proton and the corresponding amino acid requires further investigation, these results suggested that Avt1p is an antiporter of proton and amino acid.
Fig. 3. Avt1p-dependent uptake of various amino acids by vacuolar membrane vesicles. Notes: (A) Time course of ATP-dependent uptake of isoleucine. Vacuolar membrane vesicles were isolated from avt1Δavt3Δavt4Δ cells carrying either pGPD-AVT1 (closed circles), pADH-AVT1 (closed triangles), or empty vector (open circles). Preparation of the vesicles and amino acid uptake assay were performed as in Fig. 2. Results are the mean ± SD of three independent experiments. (B) Kinetic parameters of Avt1p-dependent isoleucine uptake. The apparent Km and Vmax values for isoleucine uptake were determined.
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Substrate specificity of Avt1p Fig. 6 shows the effect of various amino acids and GABA on Avt1p-dependent isoleucine uptake by the vesicles. Each amino acid (at the final concentration of 0.5 mM) was added 3 min before initiating isoleucine uptake (at 0.1 mM). Isoleucine uptake was significantly
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inhibited by histidine as well as phenylalanine, tryptophan, and alanine and moderately by methionine, leucine, valine, glutamine, proline, serine, threonine, and glycine. Inhibition by lysine, asparagine, and arginine was insignificant, and uptake was little inhibited by aspartic acid, glutamic acid, and GABA (Fig. 6). The Km (mM) and Vmax (nmol/min/mg protein) values of Avt1p-dependent histidine uptake were 0.5 ± 0.1 and 89.5 ± 13.8, respectively. Isoleucine uptake was competitively inhibited by histidine with Ki (mM) value of 0.6 ± 0.1. These results suggest that Avt1p recognizes various amino acids, except for GABA, glutamate, and aspartate. Role of Avt1p in vacuolar compartmentalization of amino acid in cells As described above, vacuolar amino acid transport has been studied in vitro using vacuolar membrane vesicles,3,4,7,12–14,17) but examination on the in vivo involvement of these transporters in vacuolar compartmentalization of amino acid in cells is limited.14,29) Recently, we reported that the vacuolar contents of various amino acids significantly increased in avt3Δavt4Δ mutant cells; neutral amino acids especially increased.13) Here, we examined the involvement of Avt1p in vacuolar amino acid compartmentalization in cells. First, effect of avt1Δ mutation on the vacuolar contents of amino acid was examined (Fig. 7(A)). The
amounts of glutamine, tyrosine, proline, histidine, and arginine slightly decreased in the vacuolar fraction of avt1Δ mutant in comparison with those of the wildtype strain. Effect of avt1Δ mutation was more prominent in avt3Δavt4Δ mutant as background. Threonine, serine, asparagine, glutamine, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, and proline, all increased in avt3Δavt4Δ mutant, clearly decreased in the vacuoles of avt1Δavt3Δavt4Δ mutant (Fig. 7(A)). Aspartic acid and glutamic acid slightly decreased in avt1Δavt3Δavt4Δ mutant, and a decrease in the vacuolar contents of lysine, histidine, and arginine was not so prominent. We also examined the effect of Avt1p overproduction on the vacuolar contents of amino acid in cells of the wild-type strain and avt3Δavt4Δ mutant cultured in SD + Cas medium (Fig. 7(B)). The AVT1 gene was constitutively expressed under GPD promoter. In both strains, most of amino acids, except for glutamic acid and three cationic amino acids, were affected by Avt1p production. Threonine, serine, asparagine, glutamine, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, and proline increased. In particular, an increase in the contents of threonine and glutamine was remarkable, being close to the contents of lysine and arginine, the most abundant ones in vacuoles (Fig. 7(B)). Although the effect of Avt1p overproduction on a prominent increase in the contents of threonine and glutamine should be further investigated, the
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Fig. 5. Avt1p-dependent proton movement coupled with amino acid. Notes: Vacuolar membrane vesicles were isolated from avt1Δavt3Δavt4Δ cells carrying either empty vector (A) or pGPD-AVT1 (B). Following the addition of quinacrine and stabilization of the fluorescence signal, ATP was added to initiate the reaction. Final concentration of additions: ATP 0.4 mM, amino acid 0.1 mM, and CCCP 10 μM. ΔF/F = ratio of fluorescence quenching: initial fluorescence.
Fig. 6. Effect of various amino acids and GABA on Avt1p-dependent isoleucine uptake. Notes: Vacuolar membrane vesicles isolated from avt1Δavt3Δavt4Δ cells carrying pGPD-AVT1 were incubated with 0.1 mM 14C-labeled isoleucine in the presence of 0.5 mM non-labeled amino acids as indicated (black bars). The initial rates (0.5 min) of uptake were determined. The transport activity relative to the control (no addition of non-labeled amino acids, white bar) is shown. Results are mean ± SD of three independent experiments.
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results suggest that Avt1p is important for vacuolar compartmentalization of various amino acids, especially neutral ones, being in cooperation with activities of Avt3p and Avt4p.
Discussion Here, we examined some catalytic properties of Avt1p, the only uptake system for amino acid into vacuoles in the AVT family.12) The results suggested that Avt1p is a proton/amino acid antiporter. Avt1p has a relatively broad substrate specificity for amino acids, and Avt1p prefers histidine as well as neutral amino acids as the substrate (Fig. 6). Avt1p-dependent uptake of histidine and isoleucine was little affected by valinomycin (Fig. 4), suggesting the main involvement of the pH gradient as the driving force. Avt1p-dependent arginine uptake (Fig. 3(A)) was also valinomycin insensitive (data not shown). In considering the dissociation arginine constants of histidine (pK3 = 6.04), (pK3 = 12.48), and lysine (pK3 = 10.54), the results suggest that the unprotonated form of histidine as well as other neutral amino acids is preferred as the substrate under the assay condition (pH 6.9). It is reported that various AAAP transporters have a broad substrate specificity for neutral, anionic, and cationic amino acids.12–14,30) We did not clearly observe ATP-dependent uptake of glutamic acid or aspartic acid (data not shown) by the vesicles of avt3Δavt4Δ mutant overproducing Avt1p and a change in the vacuolar contents of these amino acids (Fig. 7). Since Avt6p is involved in extrusion of these anionic amino acids,12,29) it is important to investigate the involvement of Avt1p in transport of anionic amino acids by the avt6Δ mutant as
background. In any case, we are now going to purify Avt1p so as to investigate directly its catalytic feature with proteoliposome reconstitution system. The vacuolar contents of various neutral amino acids are insignificant in the wild-type cells cultured in YPD medium, although the concentrations of these amino acids in vacuoles are slightly higher than those in cytosol (Kakinuma et al., unpublished data). The roles of Avt3p and Avt4p transporters are critical for vacuolar compartmentalization of amino acid.13) In avt3Δavt4Δ mutant cells, the vacuolar contents of threonine, glutamine, glycine, alanine, and other amino acids extremely increased (Fig. 7). In the avt1Δavt3Δavt4Δ mutant, the amounts of these neutral amino acids decreased to more or less 50–60% of those of avt3Δavt4Δ mutant, indicating that Avt1p plays a major role for uptake of these amino acids into vacuoles. Although active transport of these neutral amino acids was impaired in the vesicles of avt1Δavt3Δavt4Δ mutant (Fig. 2), the results suggest that these amino acids still remained in the vacuoles of avt1Δavt3Δavt4Δ mutant. Although fluorescence microscopic observation on intact cells showed a slight enlargement of vacuoles in avt3Δavt4Δ mutant (Kakinuma et al. unpublished data), the concentrations of these amino acids in vacuoles could be still higher than those in cytosol of avt1Δavt3Δavt4Δ mutant cells. In other words, active accumulation of neutral amino acids into vacuoles could be expected in this mutant. The results may be explained by two possibilities. First, there is some other active transport system for amino acids, driven by proton gradient, was inactivated in the preparation of the vacuolar vesicles. Second, there may be other transport system for amino acid, not depending upon proton gradient as well as ATP. It has been reported that the activity of arginine/
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histidine exchange is found in the S. cerevisiae vacuoles.31) In avt1Δavt3Δavt4Δ mutant cells, a large concentration gradient of cationic amino acids, lysine and arginine, across the vacuolar membrane is still established by the systems such as VBA and YPQ transporters. These gradients may be the driving force of uptake of neutral amino acid, which will be worthwhile to be investigated as another uptake pathway for amino acids. In any case, it is obvious that vacuolar compartmentalization of various amino acids is embodied by harmonized activities of Avt1p and other transporters. Recently, it was reported that mitochondrial dysfunction in replicatively aged S. cerevisiae arises from altered vacuolar pH.20) Vacuolar acidity declined during the early asymmetric divisions of a mother cell, and preventing this decline suppresses mitochondrial dysfunction and extends lifespan. Interestingly, overexpression or deletion of AVT1 was also sufficient to extend or shorten replicative lifespan of this organism, respectively.20) The authors speculated that changes in vacuolar pH limit mitochondrial function probably by reducing Avt1p-dependent amino acid storage in the vacuolar lumen. Mitochondria-dependent catabolism of excess cytoplasmic amino acids may place a large burden on function of mitochondrial carrier proteins. Since the vacuolar acidity was declined by Avt1p-dependent proton efflux (Fig. 5), Avt1p-mediated amino acid uptake, not proton efflux, should be responsible for the reason for replicative lifespan.20) We examined total cell contents of amino acids in strains used in the experiments of Fig. 7 and estimated the cytosolic contents of amino acids by subtracting the vacuolar contents from the total cell contents. However, we did not find any amino acids, the amount of which increased or decreased by deleting or overexpressing AVT1 (data not shown). Phosphopep database (http://www.phospho pep.org/) has indicated that the amino-terminal hydrophilic region of Avt1p possesses several phosphorylation sites (Fig. 1(B)). Since the amounts of phosphopeptides originated from the amino acid sequence of Avt1p were affected by rapamycin treatment,28) TORC1 kinase system is likely involved in its regulation. Therefore, when considering the role of Avt1p in lifespan, we should pay attention to regulatory behavior of Avt1p in signal transduction network. In any case, based on the catalytic feature of Avt1p as a transporter, further study is required for characterizing the physiological role of Avt1p in yeast. Finally, we should pay attention to the results on the effect of Avt1p production on vacuolar content of histidine (Fig. 7). Although histidine is one of the preferable substrates of Avt1p transporter by vesicles, the vacuolar content of histidine in cells was little influenced by AVT1 expression, being in contrast with those of neutral amino acids. What is the reason for this discrepancy? At the moment, we consider the involvement of polyphosphate in vacuolar accumulation of basic amino acids in cells. A large amount of polyphosphate accumulates in vacuoles of fungi,32) presumably posing Donnan potential across the vacuolar membrane. For the charge compensation at the steady state, polyphosphate could offer the accumulation capacity of basic amino acids not neutral ones, although active transport systems are involved in dynamic movements of various
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amino acids. Vacuolar compartmentalization of amino acids, especially basic ones, in vivo does not only depend upon the function of transport systems. We are also going to investigate the significance of vacuolar polyphosphate in amino acid compartmentalization.
Acknowledgments This work was supported in part by Elizabeth Arnold Fuji Foundation (to M.K-K.) and the Noda Science Foundation (to Y.K. and T.S.).
Funding This work was supported by the Public Foundation of Elizabeth Arnold-Fuji and the Noda Institute for Scientific Research.
References [1] Wiemken A, Durr M. Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae. Arch. Microbiol. 1974;101:45–57. [2] Kitamoto K, Yoshizawa K, Ohsumi Y, Anraku Y. Dynamic aspects of vacuolar and cytosolic amino acid pools of Saccharomyces cerevisiae. J. Bacteriol. 1988;170:2683–2686. [3] Ohsumi Y, Anraku Y. Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. J. Biol. Chem. 1981;256: 2079–2082. [4] Sato T, Ohsumi Y, Anraku Y. 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. 1984;259:11505–11508. [5] Sekito T, Fujiki Y, Ohsumi Y, Kakinuma Y. Novel families of vacuolar amino acid transporters. IUBMB Life. 2008;60:519–525. [6] Paulsen IT, Sliwinski MK, Nelissen B, Goffeau A, Saier MH Jr. Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae. FEBS Lett. 1998;430:116–125. [7] Shimazu M, Sekito T, Akiyama K, Ohsumi Y, Kakinuma Y. A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae. J. Biol. Chem. 2005;280:4851–4857. [8] Shimazu M, Itaya T, Pongcharoen P, Sekito T, Kawano-Kawada M, Kakinuma Y. Vba5p, a novel plasma membrane protein involved in amino acid uptake and drug sensitivity in Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 2012;76:1993–1995. [9] Tenreiro S, Rosa PC, Viegas CA, Sá-Correia I. Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae. Yeast. 2000;16:1469–1481. [10] Ehrenhofer-Murray AE, Würgler FE, Sengstag C. The Saccharomyces cerevisiae SGE1 gene product: a novel drug-resistance protein within the major facilitator superfamily. Mol. Gen. Genet. 1994;244:287–294. [11] Young GB, Jack DL, Smith DW, Saier MH Jr. The amino acid/ auxin: proton symport permease family. Biochim. Biophys. Acta. 1999;1415:306–322. [12] Russnak R, Konczal D, McIntire SL. A family of yeast proteins mediating bidirectional vacuolar amino acid transport. J. Biol. Chem. 2001;276:23849–23857. [13] Sekito T, Chardwiriyapreecha S, Sugimoto N, Ishimoto M, Kawano-Kawada M, Kakinuma Y. Vacuolar transporter Avt4 is involved in excretion of basic amino acids from the vacuoles of Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 2014;78:269–275.
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[14] Tone J, Yamanaka A, Manabe K, Murao N, Kawano-Kawada M, Sekito T, Kakinuma Y. A vacuolar membrane protein Avt7p is involved in transport of amino acid and spore formation in Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. Forthcoming 2014. [15] Yee DC, Shlykov MA, Västermark Åke, Reddy VS, Arora S, Sun EI, Saier MH Jr. The transporter-opsin-G protein-coupled receptor (TOG) superfamily. FEBS J. 2013;280:5780–5800. [16] Jezegou A, Llinares E, Anne C, Kieffer-Jaquinod S, O’Regan S, Aupetit J, Chabli A, Sagne C, Debacker C, ChadefauxVekemans B, Journet A, Andre B, Gasnier B. Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy. Proc. Nat. Acad. Sci. U.S.A. 2012;109:E3434–E3443. [17] Sekito T, Nakamura K, Manabe K, Tone J, Sato Y, Murao N, Kawano-Kawada M, Kakinuma Y. Loss of ATP-dependent lysine uptake in the vacuolar membrane vesicles of Saccharomyces cerevisiae ypq1Δ mutant. Biosci. Biotech. Biochem. 2014;78:1199–1202. [18] Yang Z, Huang J, Geng J, Nair U, Klionsky DJ. Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Mol. Biol. Cell. 2006;17:5094–5104. [19] Uemura T, Tomonari Y, Kashiwagi K, Igarashi K. Uptake of GABA and putrescine by UGA4 on the vacuolar membrane in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 2004;315:1082–1087. [20] Hughes AL, Gottschling DE. An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast. Nature. 2012;492:261–265. [21] Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 1996;24:2519–2524. [22] Goldstein AL, McCusker JH. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast. 1999;15:1541–1553.
[23] Mumberg D, Müller R, Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1995;156:119–122. [24] Vida TA, Emr SD. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 1995;128:779–792. [25] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265–275. [26] Ohsumi Y, Anraku Y. Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. J. Biol. Chem. 1983;258:5614–5617. [27] Ohsumi Y, Kitamoto K, Anraku Y. Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion. J. Bacteriol. 1988;170:2676–2682. [28] Huber A, Bodenmiller B, Uotila A, Stahl M, Wanka S, Gerrits B, Aebersold R, Loewith R. Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis. Genes Dev. 2009;23: 1929–1943. [29] Chahomchuen T, Hondo K, Ohsaki M, Sekito T, Kakinuma Y. Evidence for Avt6 as a vacuolar exporter of acidic amino acids in Saccharomyces cerevisiae cells. J. Gen. Appl. Microbiol. 2009;55:409–417. [30] Schiöth HB, Roshanbin S, Hägglund MG, Fredriksson R. Evolutionary origin of amino acid transporter families SLC32, SLC36 and SLC38 and physiological, pathological and therapeutic aspects. Mol. Aspects Med. 2013;34:571–585. [31] Sato T, Ohsumi Y, Anraku Y. An arginine/histidine exchange transport system in vacuolar-membrane vesicles of Saccharomyces cerevisiae. J. Biol. Chem. 1984;259:11509–11511. [32] Cramer CL, Davis RH. Polyphosphate-cation interaction in the amino acid-containing vacuole of Neurospora crassa. J. Biol. Chem. 1984;259:5152–5157.
