The thioredoxin-thioredoxin reductase system can function in vivo as an alternative system to reduce oxidized glutathione in Saccharomyces cerevisiae

Abstract

Cellular mechanisms that maintain redox homeostasis are crucial, providing buffering against oxidative stress. Glutathione, the most abundant low molecular weight thiol, is considered the major cellular redox buffer in most cells. To better understand how cells maintain glutathione redox homeostasis, cells of Saccharomyces cerevisiae were treated with extracellular oxidized glutathione (GSSG), and the effect on intracellular reduced glutathione (GSH) and GSSG were monitored over time. Intriguingly cells lacking GLR1 encoding the GSSG reductase in S. cerevisiae accumulated increased levels of GSH via a mechanism independent of the GSH biosynthetic pathway. Furthermore, residual NADPH-dependent GSSG reductase activity was found in lysate derived from glr1 cell. The cytosolic thioredoxin-thioredoxin reductase system and not the glutaredoxins (Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in glr1 cells reduced GSSG accumulation, increased GSH levels, and reduced cellular glutathione E(h)'. Conversely, deletion of TRX1 or TRX2 in the glr1 strain led to increased accumulation of GSSG, reduced GSH levels, and increased cellular E(h)'. Furthermore, it was found that purified thioredoxins can reduce GSSG to GSH in the presence of thioredoxin reductase and NADPH in a reconstituted in vitro system. Collectively, these data indicate that the thioredoxin-thioredoxin reductase system can function as an alternative system to reduce GSSG in S. cerevisiae in vivo.

FIGURE 1.

Effect of extracellular GSSG on intracellular glutathione redox homeostasis. The wild-type, hgt1 and glr1 cells were grown to exponential phase (A₆₀₀, 0.5) in SD medium exposed to 100 μm GSSG, and intracellular glutathione was determined over the indicated time course. A, total intracellular glutathione; B, GSH; and C, GSSG of the indicated strains were determined. D, cellular glutathione E_h′ was determined over the time course. Error bars indicate the S.E. of four samples from two independent experiments.

FIGURE 2.

Extracellular GSSG leads to an increase in intracellular GSH in a GSH1- and GLR1-independent manner. A, exponentially growing glr1 cells transformed with the GSH1::lacZ construct were treated with 100 μm GSSG and assayed for β-galactosidase activity. B, chromosomal copy of GSH1 in the glr1 mutant was tagged with HA. Exponentially growing cells were treated with 100 μm GSSG over the indicated time, and the cell lysate analyzed for Gsh1-HAp using anti-HA antibodies. Pgk1p was detected using anti-Pgk1p antibodies as a loading control. C, indicated mutants were grown to A₆₀₀ 0.5 in SD medium supplemented with 25 μm GSH. Cells were harvested and analyzed for intracellular GSH and GSSG levels. D, indicated strains were grown for 2 days and A₆₀₀ adjusted to 1.0 before 5 μl of the diluted cultures were spotted onto SD plates containing no glutathione, 25 μm GSH, or 25 μm GSSG. E–G, cells of gsh1 glr1 were treated with 100 μm GSSG (diamond), 100 μm GSSG treated with 2-vinyl-pyridine (square), or untreated (triangle). E, intracellular total glutathione; F, GSH; and G, GSSG were determined at the indicated time. Error bars indicate the S.E. of four samples of two independent experiments. H, GSSG reductase activity in glr1 cell lysate. Wild-type and glr1 cells were grown to exponential phase, and the cell lysate of each strain was assayed for GSSG reductase activity.

FIGURE 3.

The thioredoxin-thioredoxin reductase system can reduce GSSG in vivo. A and B, overexpression of TRR1, TRX1, and TRX2 in glr1 cells reduces intracellular GSSG. The glr1 mutant was transformed with pGAL1-TRR1–3XHA, pGAL1-TRX1–3XHA, or pGAL1-TRX2–3XHA. The transformed cells were grown to stationary phase in SD minus uracil medium and re-inoculated into SD-GAL minus uracil medium, and the cultures were allowed to grow for 18 h to A₆₀₀ 0.5–1.0 before harvesting. A, Western blot analysis (top) of cell lysate using anti-HA antibody. B, intracellular concentration of GSSG, GSH, and glutathione E_h′ of the indicated strains. Error bars indicate S.E. from six replicates from two independent experiments. Asterisks (*) indicate significant differences (p value <0.05) compared with the glr1 mutant harboring the control vector.

FIGURE 4.

The trx1 glr1 and trx2 glr1 strains have a decreased capacity to convert GSSG to GSH compared with the glr1 strain in vivo. The indicated strains were grown to exponential phase (A₆₀₀, 0.5), in SD medium treated with 100 μm GSSG, and intracellular glutathione was determined over the indicated time. A, GSSG and B, GSH of the indicated strains were determined. C, cellular glutathione E_h′ was determined over the indicated time. Error bars indicate the S.E. of six biological replicates from two independent experiments. Asterisks (*) indicate significant differences (p value < 0.05) compared with the glr1 mutant at the indicated time.

FIGURE 5.

Trx1p and Trx2p can facilitate reduction of GSSG to a similar level in vitro. Purified proteins were reconstituted with the indicated compounds, and the rate of NADPH oxidation to NADP⁺ was monitored at 340 nm. Error bars indicate the S.D. of three replicates.