Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae

Abstract

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1∆ 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.

Vacuolar amino acid transporters upregulated by exogenous proline and involved in cellular localization of proline in <i>Saccharomyces cerevisiae</i>

“…A recent report on the extension of replicative lifespan via Avt1 (Hughes and Gottschling, 2012) was shown to spotlight a general significance of vacuolar amino acid uptake. Although previous reports have not detected the in vitro ATPdependent import activity at the vacuolar membrane (Ohsumi and Anraku, 1981;Sato et al, 1984;Tone et al, 2015b), our results showed that proline is significantly accumulated in the vacuole in vivo at least in the presence of exogenous proline (Fig. 4).…”

“…5 and 6), as well as of other large neutral amino acids, such as glutamine, asparagine, leucine, isoleucine, and tyrosine (Russnak et al, 2001;Tone et al, 2015b). We previously reported that the vacuolar function is essential for maintaining cell viability under stress conditions in a prolineaccumulating mutant (Matsuura and Takagi, 2005).…”

“…Among seven vacuolar amino acid transporters, which belong to the amino acid vacuolar transport (AVT)-family, Avt1 imports several neutral amino acids including glutamine, asparagine, leucine, isoleucine, and tyrosine into the vacuole, whereas the closely related Avt3 and Avt4 proteins redundantly export them out of the vacuole (Russnak et al, 2001;Tone et al, 2015b). Avt6 mediates the extrusion of acidic amino acids, glutamate and aspartate (Russnak et al, 2001).…”

“…More recently, Avt3, Avt4, and Avt7 were shown to mediate the efflux of proline under nitrogen starvation conditions (Sekito et al, 2014;Tone et al, 2015a). Although the proline import activity of the purified vacuolar membrane has not been detected (Ohsumi and Anraku, 1981;Sato et al, 1984;Tone et al, 2015b), the in vitro export of proline from the vacuole occurs in an ATP-dependent manner (Ishimoto et al, 2012). Atg22 mediates the efflux of leucine and other amino acids resulting from autophagic degradation, and also redundantly functions with Avt3 and Avt4 (Yang et al, 2006).…”

Vba4p, a vacuolar membrane protein, is involved in the drug resistance and vacuolar morphology of Saccharomyces cerevisiae

“…14) Uptake of arginine was assayed as described. 29) Vacuolar membrane vesicles (200 μg of protein) were incubated for 5 min at 25°C in the assay mixture (500 μL) consisting of 25 mM MES-Tris (pH 7.0), 4 mM MgCl 2 , and 25 mM KCl. ATP (2 mM) was added to the mixture and incubated for 1 min at 25°C.…”