Hgt1p, a High Affinity Glutathione Transporter from the Yeast Saccharomyces cerevisiae

Abstract

A high affinity glutathione transporter has been identified, cloned, and characterized from the yeast Saccharomyces cerevisiae. This transporter, Hgt1p, represents the first high affinity glutathione transporter to be described from any system so far. The strategy for the identification involved investigating candidate glutathione transporters from the yeast genome sequence project followed by genetic and physiological investigations. This approach revealed HGT1 (open reading frame YJL212c) as encoding a high affinity glutathione transporter. Yeast strains deleted in HGT1 did not show any detectable plasma membrane glutathione transport, and hgt1⌬ disruptants were non-viable in a glutathione biosynthetic mutant (gsh1⌬) background. The glutathione repressible transport activity observed in wild type cells was also absent in the hgt1⌬ strains. The transporter was cloned and kinetic studies indicated that Hgt1p had a high affinity for glutathione (K m ‫؍‬ 54 M)) and was not sensitive to competition by amino acids, dipeptides, or other tripeptides. Significant inhibition was observed, however, with oxidized glutathione and glutathione conjugates. The transporter reveals a novel class of transporters that has homologues in other yeasts and plants but with no apparent homologues in either Escherichia coli or in higher eukaryotes other than plants.

Gln-222 in Transmembrane Domain 4 and Gln-526 in Transmembrane Domain 9 Are Critical for Substrate Recognition in the Yeast High Affinity Glutathione Transporter, Hgt1p

“…The S. cerevisiae strain (ABC 817) bearing an HGT1 disruption in BY4741background (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0 hgt1⌬::LEU2) is deficient in glutathione uptake ability and was used in the experimental studies (1). The yeast strain was regularly maintained on yeast extract, peptone, and dextrose medium and grown at 28 -30°C.…”

“…Cells were harvested and washed and put on ice in a MES-buffered medium until the transport was initiated. Transport experiments were carried out with [ 35 S]GSH as described earlier (1). The results were expressed as nmol of glutathione⅐mg⅐protein Ϫ1 min Ϫ1 .…”

“…This has prompted us to initiate a systematic study on the structure-function characterization of Hgt1p as a representative of the OPT family. Not only is Hgt1p the best characterized member of the OPT family in terms of its substrate specificity, being also able to transport some oligopeptides albeit with low affinity (1,7,8), its native host S. cerevisiae is a well established model system and easily amendable for mutagenesis-based structure-function studies. We have recently investigated the role of the 12 native cysteine residues in the structural stability and the transporter activity of the protein where 2 of the cysteines were found to be essential for functionality (9).…”

“…Hgt1p or ScOpt1p, a polytopic membrane protein, from the yeast Saccharomyces cerevisiae, was the first high affinity glutathione transporter to be identified in any system (1). Hgt1p belongs to a relatively novel family of transporters, the oligopeptide transporter (OPT) 3 family, that contains a large number of fungal, plant, and prokaryotic members (2).…”

ABCC1, an ATP Binding Cassette Protein from Grape Berry, Transports Anthocyanidin 3-O-Glucosides

“…To test this hypothesis, cotransport experiments with M3G and radiolabeled GSH were performed. All transport experiments were performed in the presence of 5 mM NH 4 Cl to dissipate the proton gradient, thus inhibiting the endogenous yeast GSH transporters, which were described to depend on a proton gradient (Bourbouloux et al, 2000). No ATP-dependent GSH uptake into microsomes was detected when M3G was absent from the transport mixtures (Table 2).…”