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 avt3 Δ cells, but decreased in that of avt3 + -overexpressing avt3 Δ 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.