Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3+-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3+ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3+-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles

Predicted topology model and intracellular localization of SpAvt3p.

<p>(A)<i>Top</i>, predicted topology model of SpAvt3p. <i>Bottom</i>, sequence alignments of SpAvt3p (Q10074.1) in TM6 (amino acids 451–477) and analogous regions in the homologs according to CLUSTALW: <i>Saccharomyces cerevisiae</i> Avt3p and Avt4p (P36062 and P50944, respectively), <i>Arabidopsis thaliana</i> At5G65990 (ABH04593), and human hPAT1and hPAT2 (AAI36439 and AAI01104, respectively). Identical and similar residues are denoted by <i>black boxes</i> and <i>gray boxes</i>, respectively. The conserved glutamate residue is indicated by an asterisk. (B) The <i>avt3</i>Δ mutant cells expressing GFP-SpAvt3p fusion protein were subjected to fluorescence microscopy. Vacuolar membranes were stained with FM4-64. BF, bright field; bar, 5 μm.</p

SpAvt3p-dependent extrusion of amino acids by vacuolar membrane vesicles.

<p>(A) Immunoblot analysis of GFP-SpAvt3p and GFP-SpAvt3p^E469A in the vacuolar membrane vesicles isolated from <i>S</i>. <i>cerevisiae avt1</i>Δ<i>avt3</i>Δ<i>avt4</i>Δ mutant cells. Vacuolar membrane vesicles were prepared and analyzed by immunoblotting using anti-GFP serum and anti-Pho8 antibody. Pho8p was detected as the loading control. (B) Alanine and tyrosine export by vacuolar membrane vesicles. [¹⁴C]-labeled amino acids were preloaded into the vacuolar membrane vesicles isolated from <i>avt1</i>Δ<i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid (<i>circles</i>), pGPD-GFP-<i>avt3</i>⁺ (<i>triangles</i>), or pGPD-GFP-<i>avt3</i>^<i>E469A</i> (<i>diamonds</i>). The export assay was performed in the presence (<i>black symbols</i>) or absence (<i>white symbols</i>) of 2 mM ATP. Preloaded vesicles were removed immediately before (0 min) or at 1, 2, 4, and 8 min after the addition of ATP, and collected on cellulose acetate membrane filters. The amount of preloaded [¹⁴C]-labeled amino acids at 0 min was taken as 100%. The relative amounts trapped on the filters are shown. The values represent the mean ± SD based on at least three independent experiments. (C) Effects of CCA on ATP-driven alanine and tyrosine export. The experiments were performed as described above. Vacuolar membrane vesicles were incubated with 1 μM CCA for 10 min before loading with [¹⁴C]-labeled amino acids. The amount of preloaded [¹⁴C]-labeled amino acids at 0 min was taken as 100%. The relative amounts trapped on the filters at 8 min after the addition of ATP are shown. The values represent the mean ± SD based on at least three independent experiments: <i>avt1</i>Δ<i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid without (<i>white bar</i>) or with (<i>black bar</i>) CCA, and pGPD-GFP-<i>avt3</i>⁺ without (<i>light gray bar</i>) or with CCA (<i>dark gray bar</i>).</p

Vacuolar morphology of <i>S</i>. <i>pombe</i> ARC039 (wild-type), <i>avt3</i>Δ, <i>avt3</i>Δ/pTN54, and <i>avt3</i>Δ/pTN54-<i>avt3</i>⁺.

<p>Cells were grown in MM medium without thiamine for 20 h and the vacuolar membranes were then stained with FM4-64. BF, bright field; bar, 5 μm.</p

Effects of <i>avt3</i>⁺ expression on the vacuolar amino acid composition of <i>S</i>. <i>cerevisiae</i>.

<p>The vacuolar pools of <i>S</i>. <i>cerevisiae</i> were prepared and analyzed using an amino acid analyzer. The results represent the mean ± SD based on at least three independent experiments: wild-type cells carrying an empty plasmid (<i>white bar</i>), <i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid (<i>black bar</i>), pGPD-GFP-<i>avt3</i>⁺ (<i>light gray bar</i>), and pGPD-GFP-<i>avt3</i>^<i>E469A</i> (<i>dark gray bar</i>).</p

Effects of <i>avt3</i>⁺ expression on the ATP-dependent uptake of basic amino acids by vacuolar membrane vesicles.

<p>Vacuolar membrane vesicles were isolated from the wild-type cells carrying an empty plasmid (<i>squares</i>), the <i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid (<i>circles</i>), pGPD-GFP-<i>avt3</i>⁺ (<i>triangles</i>), and pGPD-GFP-<i>avt3</i>^<i>E469A</i> (<i>diamonds</i>). The amino acid uptake assay was performed with (<i>black symbols</i>) or without (<i>white symbols</i>) 2 mM ATP. The values represent the mean ± SD based on at least three independent experiments.</p