Overlapping roles of the cytoplasmic and mitochondrial redox regulatory systems in the yeast Saccharomyces cerevisiae

Abstract

Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.

FIG. 1.

GSH-glutaredoxin and thioredoxin systems. Yeast contains two gene pairs encoding cytoplasmic glutaredoxins (GRX1 and GRX2) and thioredoxins (TRX1 TRX2). The oxidized disulfide form of thioredoxin is reduced directly by NADPH and thioredoxin reductase (Trr1). In contrast, oxidized glutaredoxin is reduced by GSH, and oxidized GSSG is in turn reduced in an NADPH-dependent reaction catalyzed by GSH reductase (Glr1). Yeast also contains a complete mitochondrial thioredoxin system including a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). Grx2 and Glr1 have been colocalized to the cytosol and mitochondria (32, 35). The redox status of GSH may provide a functional link between the GSH-glutaredoxin and thioredoxin systems, since cytoplasmic thioredoxins function along with Glr1 to maintain the high intracellular GSH/GSSG ratio (9, 29). In addition, the redox state of cytoplasmic Trx1 and Trx2 is maintained independently of the GSH-glutaredoxin system, whereas there is a strong correlation between the redox state of glutaredoxins and the oxidation state of the GSSG-2GSH redox couple (43).

FIG. 2.

Growth and hydrogen peroxide sensitivity of thioredoxin reductase mutants. (A) Wild-type and trr1, trr2, and trr1 trr2 mutant cells were inoculated to the same initial cell density in minimal glucose medium (SD; initial A₆₀₀ = 0.0025) or minimal medium containing glycerol plus ethanol (SGE; initial A₆₀₀ = 0.05), and growth was monitored by A₆₀₀. (B) Sensitivity to hydrogen peroxide was determined by spotting the same strains onto YEPD plates containing 4 mM H₂O₂. Cultures of strains were grown into exponential phase or stationary phase and adjusted to an A₆₀₀ of 1.0 or 0.1 before spotting onto appropriate plates. Plates were incubated at 30°C for 3 days before scoring growth.

FIG. 3.

Growth and hydrogen peroxide sensitivity of thioredoxin mutants. (A) Wild-type and trx1, trx2, trx3, trx1 trx2, trx1 trx3, trx2 trx3, and trx1 trx2 trx3 mutant cells were inoculated to the same initial cell density in minimal glucose medium (SD; initial A₆₀₀ = 0.0025) or minimal medium containing glycerol plus ethanol (SGE; initial A₆₀₀ = 0.05), and growth was monitored by A₆₀₀. (B) Sensitivity to hydrogen peroxide was determined by spotting the same strains onto YEPD plates containing 4 mM H₂O₂. Cultures of strains were grown into exponential phase or stationary phase and adjusted to an A₆₀₀ of 1.0 or 0.1 before spotting onto appropriate plates. Plates were incubated at 30°C for 3 days before scoring growth.

FIG. 4.

Redox state of mitochondrial thioredoxin system mutants. (A) Redox state of cytoplasmic thioredoxins. The indicated strains were grown to exponential phase (A₆₀₀ = 1.0) in SD medium. Proteins were precipitated with TCA, and free thiols were modified by reaction with AMS. Samples were separated using SDS-18% PAGE, and cytoplasmic thioredoxins (Trx1 and Trx2) were detected by Western blot analysis with rabbit anti-yeast thioredoxin antibody (Trx1,2). Fully oxidized and fully reduced proteins are indicated. (B) Western blot analysis of thioredoxin protein levels. Thioredoxin protein concentrations were measured in wild-type and trx3, trr1, trr2, trr1 trr2, and trx1 trx2 mutant strains by Western blot analysis with antibodies specific for thioredoxins (Trx1,2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (C) Trx1 and Trx2 were epitope tagged with a hemagglutinin (HA) tag to determine the redox state of each cytoplasmic thioredoxin isoform. Trx1 and Trx2 are both present in the fully reduced form in a wild-type strain and a strain lacking mitochondrial Trr2.

FIG. 5.

Redox state of the GSH system in mitochondrial thioredoxin mutants. The indicated strains were grown to exponential phase in minimal SD medium. The levels of reduced GSH (A) and oxidized GSH (GSSG) (B) were determined. Values shown are the means of at least three independent determinations and are given in nanomoles per milliliter per A₆₀₀ unit.

FIG. 6.

Redox state of Trx3 in thioredoxin mutants. (A) The indicated strains were grown to exponential phase in SD medium (control) and treated with 2 mM H₂O₂ for 1 h (+H) or 2 mM diamide for 1 h (+D). Proteins were precipitated with TCA, and free thiols were modified by reaction with AMS. Samples were separated using SDS-18% PAGE, and Trx3 was detected by Western blot analysis. Oxidized and reduced proteins are indicated. Trx3 contains two redox active Cys residues, as well as two additional Cys residues, and fully oxidized Trx3 was never detected. (B) Western blot analysis of Trx3 protein levels. Trx3 protein concentrations were measured in wild-type and trx1 trx2, trr1, trr2, and trr1 trr2 mutant strains by Western blot analysis with antibodies specific for c-Myc (Trx3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

FIG. 7.

Growth and hydrogen peroxide sensitivity of GSH reductase mutants. The indicated strains were inoculated to the same initial cell density in minimal glucose medium (SD; initial A₆₀₀ = 0.0025) or minimal medium containing glycerol plus ethanol (SGE; initial A₆₀₀ = 0.05), and growth was monitored by A₆₀₀. (B) Sensitivity to hydrogen peroxide was determined by spotting the same strains onto YEPD plates containing 4 mM H₂O₂. Cultures of strains were grown into stationary phase and adjusted to an A₆₀₀ of 1.0 or 0.1 before spotting onto appropriate plates. Plates were incubated at 30°C for 3 days before scoring growth.

FIG. 8.

Redox state of mitochondrial Trx3 in GSH reductase mutants. The redox state of Trx3 was detected as described for Fig. 5. (A) The indicated strains were grown to exponential phase in SD medium. (B) The glr1 and glr1 trr2 mutants were grown to exponential phase in SD medium (control) and treated with 2 mM H₂O₂ for 1 h (+H) or 2 mM diamide for 1 h (+D). (C) The indicated strains were grown to exponential phase in SGE medium.