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• W2016758813 abstract "Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1–7) that are related to the neuronal γ-aminobutyric acid-glycine vesicular transporters. We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles. One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine. Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle. This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol. Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo. Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions. Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport systemh described in mammalian lysosomes and melanosomes. These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles. Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1–7) that are related to the neuronal γ-aminobutyric acid-glycine vesicular transporters. We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles. One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine. Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle. This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol. Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo. Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions. Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport systemh described in mammalian lysosomes and melanosomes. These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles. γ-aminobutyric acid piperazine-N,N′-bis-2-ethanesulfonic acid hemagglutinin Caenorhabditis elegans UNC-47 (1McIntire S.L. Reimer R.J. Schuske K. Edwards R.H. Jorgensen E.M. Nature. 1997; 389: 870-876Crossref PubMed Scopus (678) Google Scholar) and the vertebrate homologues from rat (VGAT, 1) and mouse (VIAAT, 2) are synaptic vesicular transporters that are expressed exclusively in inhibitory neurons (see also Ref. 3Chaudhry F.A. Reimer R.J. Bellocchio E.E. Danbolt N.C. Osen K.K. Edwards R.H. Storm-Mathisen J. J. Neurosci. 1998; 18: 9733-9750Crossref PubMed Google Scholar) and are specific for the neurotransmitters γ-aminobutyric acid (GABA)1and glycine. These proteins differ in sequence, structure, and bioenergetics from the previously characterized family of vesicular transporters that package monoamines and acetylcholine (for current reviews, see Refs. 4Masson J. Sagné C. Hamon M. Mestikawy S.E. Pharmacol. Rev. 1999; 51: 439-464PubMed Google Scholar and 5Reimer R.J. Fon E.A. Edwards R.H. Curr. Opin. Neurobiol. 1998; 8: 405-412Crossref PubMed Scopus (98) Google Scholar). Common to all of these transporters is the fact that the movement of substrate from the cytosol into synaptic vesicles is driven by a proton electrochemical gradient that is generated by the action of a vacuolar-type H+-ATPase and involves the exchange of lumenal protons. A search for other vertebrate proteins related to the GABA-glycine vesicular transporters has led recently to the isolation and expression of cDNAs that, surprisingly, code for Na+-coupled plasma membrane carriers. The system N transporters from rat (SN1, 6) and mouse (NAT1, 7) are expressed in the liver, kidney, and heart and within brain astrocytes and are specific for glutamine, histidine, and to a lesser degree, asparagine. Amino acid transport by these proteins is unique in that it also involves an exchange of protons (6Chaudhry F.A. Reimer R.J. Krizaj D. Barber D. Storm-Mathisen J. Copenhagen D.R. Edwards R.H. Cell. 1999; 99: 769-780Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 8Fei Y.-J. Sugawara M. Nakanishi T. Huang W. Wang H. Prasad P.D. Leibach F.H. Ganapathy V. J. Biol. Chem. 2000; 275: 23707-23717Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), the mechanism utilized by vesicular neurotransmitter transporters. In addition, two transporters with ∼50% identity to SN1/NAT1 have now been identified and categorized as system A due to their ability to transport α-methylaminoisobutyric acid (9Reimer R.J. Chaudry F.A. Gray A.T. Edwards R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7715-7720Crossref PubMed Scopus (163) Google Scholar, 10Sugawara M. Nakanishi T. Fei Y.-J. Huang W. Ganapathy M.E. Leibach F.H. Ganapathy V. J. Biol. Chem. 2000; 275: 16473-16477Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 11Varoqui H. Zhu H. Yao D. Ming H. Erickson J.D. J. Biol. Chem. 2000; 275: 4049-4054Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 12Yao D. Mackenzie B. Ming H. Varoqui H. Zhu H. Hediger M.A. Erickson J.D. J. Biol. Chem. 2000; 275: 22790-22797Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). One protein (termed SA1, ATA2, or SAT2) is expressed in most tissues and, compared with system N, has a wider substrate specificity that includes glutamine as well as most of the smaller neutral amino acids. The second system A carrier, referred to as GlnT, transports a similar set of amino acids but is highly expressed in the brain, specifically within neurons that release the excitatory neurotransmitter glutamate (11Varoqui H. Zhu H. Yao D. Ming H. Erickson J.D. J. Biol. Chem. 2000; 275: 4049-4054Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Other characterized members of this growing protein family include the group of plant amino acid/auxin permeases, of which 12 proteins have now been identified in Arabidopsis thaliana (13Young G.B. Jack D.L. Smith D.W. Saier Jr., M.H. Biochim. Biophys. Acta. 1999; 1415: 306-322Crossref PubMed Scopus (121) Google Scholar). These permeases actively transport amino acids into the plant cell and presumably utilize an amino acid/H+ symport mechanism. The vacuole of Saccharomyces cerevisiae is a highly complex organelle that is involved in both the enzymatic degradation of proteins and the homeostatic control of a vast assortment of solutes including ions and amino acids (for a review, see Ref. 14Klionsky D.J. Herman P.K. Emr S.D. Microbiol. Rev. 1990; 54: 266-292Crossref PubMed Google Scholar). Like synaptic vesicles, vacuoles maintain an acidic internal environment through the action of a vacuolar-type H+-ATPase that generates a proton electrochemical gradient of 180 mV (ΔpH of 1.7 units) across the vacuolar-membrane (15Kakinuma Y. Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 10859-10863Abstract Full Text PDF PubMed Google Scholar). In mammals, the equivalent to vacuoles and the major site for protein degradation is the lysosome. Both organelles support a variety of carrier-mediated transport systems, many of which rely on the imposed pH gradient as a driving force. Although several amino acid transport systems have been described in purified vacuoles (16Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 2079-2082Abstract Full Text PDF PubMed Google Scholar, 17Sato T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1984; 259: 11505-11508Abstract Full Text PDF PubMed Google Scholar, 18Sato T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1984; 259: 11509-11511Abstract Full Text PDF PubMed Google Scholar) and lysosomes (for a review, see Ref. 19Pisoni R.L. Thoene J.G. Biochim. Biophys. Acta. 1991; 1071: 351-373Crossref PubMed Scopus (94) Google Scholar), the proteins responsible for these activities have yet to be identified. Sequence analysis of the complete S. cerevisiae genome has predicted seven yeast proteins of unknown function that are related to the UNC-47/VGAT/VIAAT family. Because these proteins do not resemble any of the characterized permeases required for the cellular uptake of amino acids, we have investigated the role of this new family of yeast proteins in vacuolar amino acid transport. Genotypes of the S. cerevisiae strains used in this study are given in Table I. Yeast strains were grown at 30 °C in either YPD or selective SD medium (20Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2543) Google Scholar). To knock out each of theAVT genes, the entire coding region was replaced with the appropriate selective marker gene. Marker genes were polymerase chain reaction-amplified from the plasmids pRS423 (HIS3), pRS424 (TRP1), pRS425 (LEU2), or pRS426 (URA3) (gifts of Joachim Lee) with 60-mer oligonucleotides of which 40 nucleotides corresponded to either the 5′- or 3′-flanking portion of the AVT gene to be disrupted. Transformation of the appropriate haploid yeast strains (21Chen D.C. Yang B.C. Kuo T.T. Curr. Genet. 1992; 21: 83-84Crossref PubMed Scopus (586) Google Scholar) was carried out with gel-purified polymerase chain reaction products followed by selection on SD medium. Correct homologous recombination events were confirmed by chromosomal polymerase chain reaction analysis of the isolated transformants.Table IS. cerevisiae strains used in this studyStrainGenotypeYJL306Mata leu2–3,112 ura3–52,trp1–289 his3-Δ200YSM11Δavt1∷URA3 Δavt2∷TRP1 Δavt3∷LEU2 Δavt4∷HIS3 leu2–3,112,ura3–52 trp1–289 his3-Δ200YSM12Mata Δavt1∷HIS3, leu2–3,112 ura3–52 trp1–289 his3-Δ200YSM13Mata Δavt2∷HIS3, leu2–3,112 ura3–52 trp1–289 his3-Δ200YSM14Mata Δavt3∷HIS3, leu2–3,112 ura3–52 trp1–289 his3-Δ200YSM15Mata Δavt4∷HIS3, leu2–3,112 ura3–52 trp1–289 his3-Δ200YSM16Mata Δavt5∷HIS3, leu2–3,112 ura3–52 trp1–289 his3-Δ200YSM17Mata Δavt6∷HIS3, leu2–3,112 ura3–52 trp1–289 his3-Δ200YSM18Mata Δavt7∷HIS3, leu2–3,112 ura3–52 trp1–289 his3-Δ200YSM22Δavt1∷URA3 Δavt3∷LEU2 Δavt4∷HIS3 leu2–3,112 ura3–52 trp1–289 his3-Δ200YSM33Mata Δavt5∷HIS3 Δavt6∷TRP1 leu2–3,112 ura3–52 trp1–289 his3-Δ200YSM35Mata Δavt5∷HIS3 Δavt6∷TRP1 Δavt7∷URA3 leu2–3,112 ura3–52 trp1–289 his3-Δ200YSM36Mata Δavt3∷HIS3 Δavt4∷URA3 leu2–3,112 ura3–52 trp1–289 his3-Δ200 Open table in a new tab Yeast vacuoles were isolated using a modified version of the procedure described by Kakinumaet al. (15Kakinuma Y. Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 10859-10863Abstract Full Text PDF PubMed Google Scholar). Briefly, 500-ml cultures were grown in YPD medium to an A600 of 3.5–4.0, and the cells were collected by centrifugation at 4,400 ×g for 5 min. Cells were washed once with 30 ml of distilled water at room temperature and converted to spheroplasts by resuspending them in 30 ml of 1 m Sorbitol and adding 500 μl of a 4 mg/ml zymolyase 100T (ICN) solution in 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, and 50% glycerol. The culture was shaken gently for 1 h at 30 °C, and spheroplasts were collected at 2,200 × g for 5 min at 4 °C and washed twice in 20 ml of ice-cold 1 m Sorbitol. All subsequent manipulations were carried out at 4 °C. Spheroplasts were lysed by resuspension in 16 ml of Buffer A (10 mm PIPES, pH 6.9, 0.1 mmMgCl2, 12% Ficoll 400, and 0.42 mm Pefabloc SC) and homogenized by 15 strokes in a 15 ml Dounce with tight pestle. The lysate was cleared by centrifugation at 2,200 ×g for 10 min, and 12 ml of the supernatant was divided between two 89 × 14-mm polyallomer tubes. Each sample was overlaid with another 6 ml of Buffer A and centrifuged at 60,000 × g for 30 min in a Beckman SW41 Ti rotor. The floating vacuoles were collected and completely suspended in 400 μl of 2× Buffer C (1× Buffer C = 10 mm PIPES, pH 6.9, 5 mm MgCl2, and 12.5 mm KCl) and 0.42 mm Pefabloc SC before diluting with an equal volume of 1× Buffer C and 0.42 mm Pefabloc SC. The final vacuole suspension, which was typically at a concentration of 1.25–1.5 μg/μl as determined by the Bradford assay, was either used immediately or frozen at −80 °C in 200-μl aliquots. Standard reaction conditions included 10 mm PIPES, pH 6.9, 4 mmMgCl2, 4 mm KCl, 4 mm ATP, and 50 μm unlabeled amino acid with 1 μCi of3H-amino acid and 10–20 μg of purified vacuoles per 100 μl of reaction volume. Where indicated, nigericin was also included at a final concentration of 6.5 μm. Generally, reaction mixtures minus vacuoles were preincubated for 1 min at 30 °C. After the addition of vacuoles, duplicate (or quadruplicate in the case of aspartate and glutamate) 100-μl samples were removed at the appropriate times. Vacuoles were recovered quickly by suction on a membrane filter (Millipore HA; 0.45 μm) and washed immediately three times with 2 ml of ice-cold 1× Buffer C. Filters were dried, and the incorporated radioactivity was measured by scintillation counting in 5 ml of Filtron-X (National Diagnostics). All radiolabeled amino acids were obtained from New England Nuclear Laboratories. A homology search of the complete S. cerevisiae genome with VGAT sequences identified seven open reading frames that we have designated AVT1–7 foramino acid vacuolartransport (see below). Along with VGAT and its orthologues, the yeast AVT proteins belong to a large family of proteins that includes the amino acid/auxin permease family of plants (13Young G.B. Jack D.L. Smith D.W. Saier Jr., M.H. Biochim. Biophys. Acta. 1999; 1415: 306-322Crossref PubMed Scopus (121) Google Scholar), ∼12 other predicted open reading frames from both C. elegans and Drosophila, and the mammalian system A and system N Na+-dependent plasma membrane transporters (6Chaudhry F.A. Reimer R.J. Krizaj D. Barber D. Storm-Mathisen J. Copenhagen D.R. Edwards R.H. Cell. 1999; 99: 769-780Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 7Gu S. Roderick H.L. Camacho P. Jiang J.X. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3230-3235Crossref PubMed Scopus (84) Google Scholar, 8Fei Y.-J. Sugawara M. Nakanishi T. Huang W. Wang H. Prasad P.D. Leibach F.H. Ganapathy V. J. Biol. Chem. 2000; 275: 23707-23717Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 9Reimer R.J. Chaudry F.A. Gray A.T. Edwards R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7715-7720Crossref PubMed Scopus (163) Google Scholar, 10Sugawara M. Nakanishi T. Fei Y.-J. Huang W. Ganapathy M.E. Leibach F.H. Ganapathy V. J. Biol. Chem. 2000; 275: 16473-16477Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 11Varoqui H. Zhu H. Yao D. Ming H. Erickson J.D. J. Biol. Chem. 2000; 275: 4049-4054Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 12Yao D. Mackenzie B. Ming H. Varoqui H. Zhu H. Hediger M.A. Erickson J.D. J. Biol. Chem. 2000; 275: 22790-22797Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Like other members of this family, the yeast proteins contain a core region of ∼400–450 amino acids that harbors 9–11 predicted hydrophobic transmembrane domains. In addition to this apparent conservation in topological features, a sequence alignment (see Fig. 1 C) reveals a number of amino acid positions (usually within transmembrane domains) where invariant or highly conserved amino acid residues are utilized. Sizes of the predicted yeast proteins range from 448 to 713 amino acids, with the greatest disparity due to extended and divergent hydrophilic N-terminal sequences in the case of AVT1, AVT3, and AVT4. The yeast AVT proteins can be further subdivided into four main branches defined by AVT1, AVT2, AVT3/4, and AVT5/6/7 (Fig. 1 A). As shown, some of these yeast proteins are more closely related to known transporters from distant species such as worms and mammals than they are to other yeast branch members. Interestingly, AVT3 and AVT4 appear to be more highly evolutionarily conserved because these proteins show significant homology (greater than 25% identity) to a number of predicted proteins from C. elegans and Drosophila melanogaster, two organisms whose entire genome sequences have now been determined. The relationship of these predicted proteins to AVT3 and AVT4 as compared with UNC-47 is shown in Fig. 1 B, whereas their amino acid sequences have been aligned in Fig. 1 D.Figure 1Seven yeast proteins are related to the UNC-47 family of amino acid transporters. A, dendrogram of the AVT proteins from S. cerevisiae (see Table II for specific gene loci) with the corresponding known homologues fromSchizosaccharomyces pombe (GenBank™ accession numbers are shown). Also included are the GABA vesicular transporters from C. elegans (UNC-47; GenBank™ accession number AF031935) and rat (VGAT; GenBank™ accession number AF030253), as well as the system N (SN1; GenBank™ accession number AF273025) and system A (GlnT; GenBank™ accession number AF075704) plasma membrane transporters. Overall identities for the more closely related pairs are 52% for AVT5 and AVT6, 50% for SN1 and GlnT, 38% for AVT3 and AVT4, and 35% for UNC-47 and VGAT. AVT7 shares 35% sequence identity with both AVT5 and AVT6. B, dendrogram of proteins from C. elegans and Drosophila that show the highest BLAST alignment scores to both AVT3 and AVT4, compared with UNC-47. The GanBank™ accession numbers for hypothetical nematode proteins T27A1.5 and Y43F4B.7 are AAB71045.1 and T26845, respectively, whereas those for the predicted fly proteins are indicated.C, sequence alignment of AVT1–7 with VGAT showing identical (black) and conserved (gray) residues.D, sequence alignment of the proteins shown in B,excluding UNC-47.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The identification of several genes closely related to the GABA vesicular transporters was interesting, given that many proteins in S. cerevisiae, including UGA4, a GABA-specific permease (22André B. Hein C. Grenson M. Jauniaux J.C. Mol. Gen. Genet. 1993; 237: 17-25Crossref PubMed Scopus (96) Google Scholar), are involved in the use of GABA as a nitrogen source. To investigate the potential role of theAVT genes in GABA flux and to establish a genetic system to study AVT protein function, we decided to test strains carrying deletedAVT genes for GABA-specific growth defects. Preliminary knockout experiments in diploid strains followed by sporulation and tetrad analysis indicated that the AVT genes were not essential for growth. This allowed the creation, by direct transformation (see “Experimental Procedures” for details), of a variety of isogenic haploid strains carrying both the single and multiple gene deletions shown in Table I. We tested these strains for their ability to grow on 1 mmGABA as the sole nitrogen source and found no discernible difference from the wild type. Furthermore, we disrupted theUGA1 gene (23André B. Jauniaux J.C. Nucleic Acids Res. 1990; 18: 3049Crossref PubMed Scopus (32) Google Scholar), which codes for GABA transaminase that converts GABA to glutamate, in each of the single AVTdeletion strains and observed no differential growth on YPD medium containing high levels (50 mm) of GABA compared with the Δuga1 strain alone. Finally, we have observed that GABA is not actively taken up by purified yeast vacuoles, even at high (500 μm) concentrations (Fig.2 A, see below). Seven amino acid uptake systems have been described in yeast vacuoles (16Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 2079-2082Abstract Full Text PDF PubMed Google Scholar, 17Sato T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1984; 259: 11505-11508Abstract Full Text PDF PubMed Google Scholar, 18Sato T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1984; 259: 11509-11511Abstract Full Text PDF PubMed Google Scholar), including three that are specific for basic amino acids, the most abundant amino acids found in this organelle (24Kitamoto K. Yoshizawa K. Ohsumi Y. Anraku Y. J. Bacteriol. 1988; 170: 2683-2686Crossref PubMed Scopus (145) Google Scholar, 25Wiemken A. Dürr M. Arch. Microbiol. 1974; 101: 45-57Crossref PubMed Scopus (135) Google Scholar). To determine whether any of the AVT proteins were involved in these processes, we tested vacuoles purified from the created Δavt strains for the uptake of radiolabeled amino acids. Shown in Fig. 2 A is the ATP-dependent uptake of lysine (10-fold) and arginine (5-fold) in 4 min by vacuoles purified from wild type yeast. Both activities are extremely sensitive to nigericin, a K+/H+ antiporter that disrupts the pH gradient without affecting the electrical gradient. However, robust lysine, arginine, and histidine transport were also observed in vacuoles obtained from any of the AVT gene-deleted strains tested. The results obtained for lysine and arginine with theΔavt1, Δavt2, Δavt3,Δavt4 quadruple mutant and the Δavt5,Δavt6, Δavt7 triple mutant are shown in Fig.2 B. However, the finding that lysine influx is unaffected by AVT protein function allowed us to accurately quantify the activity of purified vacuolar fractions, greatly facilitating the comparative analysis described below. Distinct transport systems have also been described for tyrosine, glutamine/asparagine, isoleucine/leucine, and phenylalanine/tryptophan (17Sato T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1984; 259: 11505-11508Abstract Full Text PDF PubMed Google Scholar). Like lysine, tyrosine was also concentrated ∼10-fold in the presence of ATP (Fig. 3 B). This uptake was decreased dramatically in the presence of nigericin and was not further reduced by the addition of valinomycin, a K+ ionophore that would dissipate any remaining electrical gradients (data not shown). Our first indication that some of the AVT proteins might be involved in vacuolar amino acid transport came from the observation that tyrosine uptake was completely eliminated in theΔavt1 mutant (Fig. 3 B), whereas high lysine activity was retained (Fig. 3 A). Although tyrosine uptake was shown to be blocked in these vacuoles in vitro, high levels of tyrosine (0.5 and 5 mm) were not toxic to theΔavt1 strain (data not shown). Surprisingly, AVT1 was also required for the active uptake of both glutamine and isoleucine, which were previously thought to be transported by separate carriers. The accumulation of these amino acids was reduced ∼5-fold (Fig.4 A) and 8-fold (Fig.4 B), respectively, in vacuoles purified fromΔavt1 compared with the wild type.Figure 4Both AVT1-dependent influx and synergistic efflux by AVT3 and AVT4 determine steady-state levels of tyrosine, glutamine, and isoleucine. Glutamine (A), isoleucine (B), and tyrosine (C) uptake assays in the presence of ATP by vacuoles purified from the single and double deletion strains shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Further testing of vacuoles purified from the other AVT deletion strains resulted in the finding that large neutral amino acids could accumulate more rapidly and to higher levels when compared with wild type. As shown in Fig. 4, tyrosine, glutamine, and isoleucine uptakes were all increased in the Δavt3, Δavt4 double mutant. The effect was more dramatic for tyrosine and isoleucine (greater than 2-fold) but was also significant for glutamine. One interpretation of these observations was that there exists an activity that exports these amino acids to the outside. Thus, AVT3 and AVT4, which share ∼40% sequence identity, appear to act synergistically in this process because single mutants had only partial phenotypes (Fig.4). We note that for each amino acid, the removal of AVT4 appeared to have a more pronounced effect, and, in fact, the uptake of isoleucine was unaltered in vacuoles purified from Δavt3. We conclude from these observations that AVT4 may be a more efficient transporter than AVT3 under the given in vitro assay conditions. One possibility is that AVT4 possesses a higher affinity for the various substrates. As expected, the accumulation of large neutral amino acids in the Δavt3, Δavt4 double mutant was completely eliminated with the further removal of AVT1 (Δavt1, Δavt3, Δavt4 triple mutant; data not shown). Sato et al. (17Sato T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1984; 259: 11505-11508Abstract Full Text PDF PubMed Google Scholar) observed that unlabeled tryptophan or phenylalanine did not compete with tyrosine or leucine for vacuolar uptake and therefore must be actively transported by a protein or proteins other than AVT1. In our hands, uptake assays with [3H]tryptophan appeared to result in some degree of vacuolar accumulation, but over very high background levels due to nonspecific filter binding (data not shown). This has made it difficult to adequately interpret the data and to assess relatively small strain-specific differences. However, we have routinely observed a lack of ATP-dependent tryptophan uptake in vacuoles isolated from Δavt1 but cannot yet offer an explanation for this apparent discrepancy with the previously published competition studies. Previous reports (16Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 2079-2082Abstract Full Text PDF PubMed Google Scholar, 17Sato T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1984; 259: 11505-11508Abstract Full Text PDF PubMed Google Scholar) have also shown that many amino acids, including methionine, serine, glycine, cysteine, proline, valine, threonine, and alanine, are not actively taken up by vacuoles in vitro. With the discovery that the inactivation of the amino acid effluxers AVT3 and AVT4 resulted in higher accumulation of some neutral amino acids, we retested glycine and proline in Δavt3,Δavt4 vacuoles and observed no ATP-dependent uptake (data not shown). Vacuoles purified from wild type cells accumulated less glutamate and aspartate in the presence of ATP (Fig.5, A and B). As could be demonstrated for amino acid uptake, this apparent ATP-dependent efflux of acidic amino acids was sensitive to nigericin. To distinguish between efflux, the actual displacement of substrate from the lumen of the vacuole to the outside, and an alternative phenomenon such as decreased inward diffusion when a proton electrochemical gradient was imposed on the system, we carried out the assays shown in Fig. 6. Vacuoles were preloaded with [3H]glutamate in the absence of ATP and then monitored for amino acid content after the addition of ATP or ATP plus nigericin. Consistent with an active efflux mechanism, glutamate levels decreased steadily in the presence of ATP alone. Again, efflux was not observed in the presence of nigericin (Fig.6 A) nor from vacuoles prepared from the Δavt6mutant (Fig. 6 B, see below).Figure 6AVT6, but not AVT5 or AVT7, is required for glutamate and aspartate transport out of vacuoles. Efflux assays using vacuoles purified from either (A) wild type or (B) Δavt6 strains. Vacuoles were preloaded with [3H]glutamate for 5 min at 30 °C. Samples (0 min) were removed immediately before or 1 min and 4 min after the addition of ATP or ATP plus nigericin. Accumulation in 4 min of (C) glutamate and (D) aspartate in the absence or presence of ATP or ATP plus nigericin in vacuoles isolated from the indicated mutant strains is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We further tested various AVT deletion strains for ATP-dependent efflux of either glutamate (Fig.6 C) or aspartate (Fig. 6 C) and observed that both activities were lost only in vacuoles purified from Δavt6. Even though AVT5 and AVT7 are 50% and 35% identical, respectively, to AVT6, wild type activity was retained in the Δavt5 andΔavt7 mutant strains. In addition, glutamate and aspartate levels were not further altered in a Δavt5,Δavt6 double mutant (Fig. 6) or in a Δavt5,Δavt6, Δavt7 triple mutant (data not shown). The potential inability of the Δavt5, Δavt6mutant strain to pump acidic amino acids from vacuoles in vivo did not alter its growth (data not shown) on medium containing high glutamate (0.5 m, pH 7.0). We have been unable to assign any vacuolar amino acid transport role to proteins AVT2, AVT5, or AVT7. To determine (a) if these proteins are associated with vacuoles and (b) whether transporters with a definite vacuolar function are localized exclusively to this organelle, we have carried out preliminary indirect immunofluorescence using HA-tagged molecules (data not shown). To summarize briefly, AVT1HA, AVT4HA, and AVT6HA (all proteins with vacuolar function) display a staining pattern that is indistinguishable from that of VMA2, the 60-kDa B subunit of the vacuolar ATPase. A similar pattern was obtained with AVT7HA, but in addition, strong immunoreactivity was observed at the plasma membrane in a large fraction of cells. On the other hand, AVT2HA expression was distinct and resulted in" @default.
• W2016758813 created "2016-06-24" @default.
• W2016758813 creator A5060191444 @default.
• W2016758813 creator A5071752372 @default.
• W2016758813 creator A5087493517 @default.
• W2016758813 date "2001-06-01" @default.
• W2016758813 modified "2023-10-14" @default.
• W2016758813 title "A Family of Yeast Proteins Mediating Bidirectional Vacuolar Amino Acid Transport" @default.
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