Identification and Functional Characterization of a Novel Mitochondrial Thioredoxin System in Saccharomyces cerevisiae*

José R. Pedrajas, Effie Kosmidou, Antonio Miranda-Vizuete, Jan-Åke Gustafsson, Anthony P. H. Wright, and Giannis SpyrouDagger

From the Department of Biosciences, Center for Biotechnology, Karolinska Institutet, Novum, S-141 57 Huddinge, Sweden

    ABSTRACT
Top
Abstract
Introduction
References

The so-called thioredoxin system, thioredoxin (Trx), thioredoxin reductase (Trr), and NADPH, acts as a disulfide reductase system and can protect cells against oxidative stress. In Saccharomyces cerevisiae, two thioredoxins (Trx1 and Trx2) and one thioredoxin reductase (Trr1) have been characterized, all of them located in the cytoplasm. We have identified and characterized a novel thioredoxin system in S. cerevisiae. The TRX3 gene codes for a 14-kDa protein containing the characteristic thioredoxin active site (WCGPC). The TRR2 gene codes for a protein of 37 kDa with the active-site motif (CAVC) present in prokaryotic thioredoxin reductases and binding sites for NADPH and FAD. We cloned and expressed both proteins in Escherichia coli, and the recombinant Trx3 and Trr2 proteins were active in the insulin reduction assay. Trx3 and Trr2 proteins have N-terminal domain extensions with characteristics of signals for import into mitochondria. By immunoblotting analysis of Saccharomyces subcellular fractions, we provide evidence that these proteins are located in mitochondria. We have also constructed S. cerevisiae strains null in Trx3 and Trr2 proteins and tested them for sensitivity to hydrogen peroxide. The Delta trr2 mutant was more sensitive to H2O2, whereas the Delta trx3 mutant was as sensitive as the wild type. These results suggest an important role of the mitochondrial thioredoxin reductase in protection against oxidative stress in S. cerevisiae.

    INTRODUCTION
Top
Abstract
Introduction
References

Thioredoxin (Trx)1 is a small protein (Mr 12,000) with a conserved sequence (Trp-Cys-Gly-Pro-Cys) in its active site. When thioredoxin is in a reduced state, Trx-(SH)2, the two active-site cysteines form a dithiol group that is able to catalyze the reduction of disulfides in a number of proteins. Oxidized thioredoxin (Trx-S2) can be reduced by NADPH through the catalytic action of the flavoenzyme thioredoxin reductase (Trr). Thus, Trx, Trr, and NADPH form a system (the thioredoxin system) that operates as a general disulfide reductase system by the following sequence of reactions (Reactions 1 and 2).
<UP>Trx-S<SUB>2</SUB></UP>+<UP>NADPH</UP>+<UP>H<SUP>+</SUP></UP> <LIM><OP><ARROW>⇄</ARROW></OP><UL><UP>Trr</UP></UL></LIM><UP> Trx-</UP>(<UP>SH</UP>)<SUB><UP>2</UP></SUB>+<UP>NADP<SUP>+</SUP></UP>
<UP>Trx-</UP>(<UP>SH</UP>)<SUB><UP>2</UP></SUB>+<UP>protein-S<SUB>2</SUB> ⇄ Trx-S<SUB>2</SUB></UP>+<UP>protein-</UP>(<UP>SH</UP>)<SUB><UP>2</UP></SUB>
<UP><SC>Reactions</SC> 1 <SC>and</SC> 2</UP>
Thioredoxin was first identified as electron donor for ribonucleotide reductase, an essential enzyme for DNA synthesis, in Escherichia coli (1). Apart from this function, Trx is an electron donor for 3'-phosphoadenosine-5'-phosphosulfate reductase in the sulfate assimilation pathway as well as for methionine-sulfoxide reductase in E. coli (2, 3). It is also involved in the life cycle of some bacteriophages such as T7, M13, and f1 (4-6). In eukaryotic cells, Trx is also an electron donor for ribonucleotide reductase, 3'-phosphoadenosine-5'-phoshosulfate reductase, and methionine-sulfoxide reductase (7). But eukaryotic thioredoxins can also (a) facilitate refolding of disulfide-containing proteins (8); (b) activate the interleukin-2 receptor (9); (c) modulate the DNA binding activity of some transcription factors, e.g. NF-kappa B (10); and (d) stimulate proliferation of lymphoid cells and of a variety of human solid tumors (11, 12). Furthermore, Trx is an efficient antioxidant able to reduce hydrogen peroxide (13), to scavenge free radicals (14), and to protect cells against oxidative stress (15).

Thioredoxin reductases are homodimers, although two classes of Trr may be distinguished: the Trr present in prokaryotes and lower eukaryotes, like yeast (Mr 70,000); and the mammalian one (Mr 116,000), having a dimer interface domain. The active-site cysteine residues of the E. coli thioredoxin reductase are located in the central NADPH domain and are separated by two amino acids, whereas the redox active-site cysteines of the human thioredoxin reductase are located in the FAD domain with a 4-amino acid bridge between them (7, 16). The enzymatic mechanism of thioredoxin reductases involves the transfer of reducing equivalents from NADPH to a disulfide bond via FAD (7). Unlike the E. coli enzyme, the mammalian thioredoxin reductase contains a selenocysteine that is necessary for the enzymatic activity as a penultimate residue (17-19). The E. coli thioredoxin reductase is very specific for the homologous E. coli Trx1 and Trx2 proteins (20), whereas the mammalian thioredoxin reductase has a broader substrate specificity and can reduce thioredoxins from distant species as well as proteins that contain Trx-like domains in their structures, e.g. the protein-disulfide isomerase (21). In addition, mammalian Trr can reduce lipid hydroperoxides as well as low molecular weight metabolites such as 5,5'-dithiobis(2-nitrobenzoic acid), selenite, selenocysteine, selenoglutathione, vitamin K, and alloxan (7, 22).

In Saccharomyces cerevisiae, two thioredoxins (Trx1 and Trx2) and one thioredoxin reductase have been identified (23, 24), all present in the cytoplasm. Deletion of both TRX genes inhibits vacuole inheritance, decreases the rate of DNA synthesis, increases the cell size and generation time, and makes the cells auxotrophic for methionine/cysteine (25-28). In this paper, we report the identification and characterization of a novel complete mitochondrial thioredoxin system in S. cerevisiae. Furthermore, our results indicate that mitochondrial thioredoxin reductase is implicated in the defense against oxidative stress.

    MATERIALS AND METHODS

Strains and Media-- The yeast strain YM4585 (MATa ADE2+ CANS his3Delta 200 lys2-801 leu23,112 trp1-903 tyr1-501) was used for the construction of both Delta trx3 and Delta trr2 mutants. The yeast peptone media (YP) plus 2% glucose (YPD) and YP plus 3% glycerol and 1% ethanol (YPGE) were prepared as described (29). For selecting the Delta trx3 mutant, the plates were supplemented with 200 mg of G418 (Geneticin, Life Technologies, Inc.) per liter of YPD medium.

Cloning of S. cerevisiae TRX3 and TRR2 Genes-- To clone the TRX3 gene, two primers (TRX3-NdeI, 5'-GAAACCAAAGCTCATATGTTGTTCTATAAG-3' (forward); and TRX3-BamHI, 5'-CATGTGCGGATCCATATAAAATTTATAGATC-3' (reverse)) were designed from the sequence of a thioredoxin-like open reading frame (ORF) coding for a 126-amino acid protein (Saccharomyces Genome Data Bank accession number YCR083W). To clone TRR2, two primers (TRR2-NdeI, 5'-ATATAAATAACATATGATAAAACATATAG-3' (forward); and TRR2-BamHI, 5'-ATAAACGGATCCTTTCACGTTACTCTTG-3' (reverse)) were designed from a thioredoxin reductase-like ORF coding for a 342-amino acid protein (Saccharomyces Genome Data Bank accession number YHR106W). The forward primers introduce an NdeI site at position +1 of TRX3 and TRR2, respectively, and the reverse primers a BamHI site after the stop codon. These primers were used to amplify S. cerevisiae genomic DNA by PCR (30 cycles at 94 °C for 1 min, 50 °C for 1 min, and 68 °C for 3 min) with the ExpandTM Long Template PCR system (Boehringer Mannheim). The PCR products were cloned into the pGEM-T Easy Vector System I (Promega) and sequenced.

Expression and Purification of Recombinant Trx3 and Trr2 Proteins in E. coli-- For expression of recombinant Trx3 and Trr2 proteins in E. coli, two mutagenic forward primers (Delta TRX3-NdeI, 5'-TAAGATTCCAGCATATGTACAACCAGTATTACTA-3'; and Delta TRR2-NdeI, 5'-GTGCTGTCACATATGATTCATCACAAGGTTAC-3') were used, introducing an NdeI site at positions +63 and +69 of TRX3 and TRR2, respectively. The reverse primers and the amplification methods were as described for cloning. The amplified fragments were cloned into the NdeI/BamHI sites of the pET-15b expression vector (AMB Biotechnology), fusing the cloned fragments to a sequence that codes for a polypeptide of 20 amino acids at the N terminus, with six histidine residues (His tag) and a thrombin cleavage site. The recombinant plasmids were designated pET-TRX3 and pET-TRR2. E. coli BL21(DE3) and HMS174(DE3) cells were transformed with the pET-TRX3 and pET-TRR2 constructs, respectively; a positive colony of each transformation was inoculated in LB medium, containing 0.1 mg of ampicillin/ml, and grown at 37 °C until A600 = 0.5. Fusion proteins were then induced by the addition of 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside, and growth was continued at room temperature overnight. The cells were harvested by centrifugation at 5000 × g for 15 min, and the pellet was diluted in cold 20 mM Tris-HCl (pH 8) containing 50 mM NaCl and 1 mM phenylmethylsulfonyl fluoride. Lysozyme was added to a concentration of 0.5 mg/ml, stirring for 30 min on ice. Subsequently, MgCl2 (10 mM), MnCl2 (1 mM), DNase I (19 µg/ml), and RNase (10 µg/ml) were added, and the incubation was continued for another 45 min on ice. The cells were sonicated; the extract was cleared by centrifugation at 15,000 × g for 30 min; and the supernatant was diluted 5-fold with 20 mM Tris-HCl (pH 8) containing 5 mM NaCl (buffer A). The extract was filtered and loaded onto a Talon resin column (CLONTECH) equilibrated with buffer A. The column was washed with buffer A, and the recombinant Trx3 and Trr2 proteins were then eluted with buffer A containing 100 mM imidazole and dialyzed against 100 mM HEPES (pH 7.6). The size and purity of both recombinant Trx3 and Trr2 were determined by SDS-polyacrylamide gel electrophoresis. Protein concentrations were determined from the absorbance at 280 nm using molar extinction coefficients of 10,010 and 23,380 M-1 cm-1 for recombinant Trx3 and Trr2, respectively.

Construction of S. cerevisiae Delta trx3 and Delta trr2 Mutants-- The TRX3 gene was disrupted by the short flanking homology method. Chimeric primers were used to amplify by PCR a gene replacement cassette. The forward primer (5'-AGAAAGCAGGATTGGTTAGTACATAGAGAAACCAAAGCTGTTATGCGTACGCTGCAGGTCGAC-3') contained 18 nucleotides homologous to the replacement cassette and a 45-nucleotide sequence homologous to the region upstream of the start codon of TRX3. In the same way, the reverse primer (5'-CTACATGTGCTCATGAATATAAAATTTATAGATCTTTGATTCCCTATCGATGAATTCGAGCTCG-3') was composed of 18- and 45-nucleotide sequences homologous to the replacement cassette and to the region downstream of stop codon, respectively. The replacement cassette used to construct the Delta trx3 mutant was KanMX4, which contained the kanr ORF of E. coli, conferring resistance to Geneticin, and was amplified from the pFA6a-KanMX4 vector (30). S. cerevisiae YM4585 cells were transformed, and Delta trx3 mutant cells were selected for kanr recombinants on YPD plus G418 plates. The correct gene deletion was verified by analytical PCR using primers (5'-AATTGCCCCGATGAGATATA-3' (forward) and 5'-GCGGTTCATTATTCTTTGATA-3' (reverse)) homologous to the flanking regions of the TRX3 ORF and by Southern blot hybridization using KanMX4 as a probe.

The S. cerevisiae TRR2-disrupted strain (Delta trr2) was a gift from M. Johnston (University of Washington, St. Louis, MO), and it was constructed as described (31). To re-express Trr2 protein, the Delta trr2 strain was transformed with a plasmid containing the TRR2 gene. A fragment containing the TRR2 gene was amplified from the Saccharomyces genome using specific primers (5'-TTACGCGGATCCGAGAAACTTAAC-3' (forward) and 5'-TTACGCGGATCCGAAAGAATCAGAC-3' (reverse)) and cloned in the SmaI site of the pRS425 plasmid (32).

Exposure of S. cerevisiae to H2O2-- S. cerevisiae cells were inoculated from stationary phase cultures in liquid YPD medium at A600 = 0.1 and grown at 30 °C until A600 = 1 was reached. The cells were then harvested and diluted with phosphate-buffered saline until A600 = 0.1, and H2O2 was added for 30 min at 30 °C with shaking. Finally, aliquots were plated on solid YPD medium, and the colonies were counted after a 2-day incubation at 30 °C. When indicated, cells were diluted in YPD medium until A600 = 0.5 and incubated with 1 mM CDNB at 30 °C for 30 min before exposure to H2O2.

Glutathione Determination-- Cells in log phase growing in YPD medium were diluted to A600 = 0.5 and incubated with 1 mM CDNB at 30 °C. At different times, cell aliquots were harvested by centrifugation, diluted in 0.05 volume of 10% metaphosphoric acid, and incubated at 50 °C for 10 min. The samples were spun, and the supernatant was neutralized with 4 M triethanolamine. Glutathione was determined by an enzymatic recycling method using a glutathione assay kit (Cayman Chemical Co., Inc.).

Antibodies-- For antibody production, female chickens were injected subcutaneously at multiple sites with 250 µg of either recombinant Trx3 or Trr2 protein, mixed with complete Freund's adjuvant (Difco), as described (20). Affinity-purified antibodies were prepared using a cyanogen bromide-activated Sepharose 4B column (Amersham Pharmacia Biotech) to which 1.5 mg of either recombinant Trx3 or Trr2 protein had been coupled following the procedure recommended by the manufacturer.

For immunoblotting, samples were subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to nitrocellulose membranes (Hybond-C Super, Amersham Pharmacia Biotech). The membranes were blocked with phosphate-buffered saline containing 5% dry fat-free milk powder and 0.1% Tween 20 and further incubated with the affinity-purified antibodies. Immunodetection was performed with horseradish peroxidase-conjugated rabbit anti-chicken IgG (Sigma) diluted 1:5000 following the ECL protocol (Amersham Pharmacia Biotech).

Preparation of Subcellular Fractions from S. cerevisiae-- Cells were grown in YPGE medium. Isolation of crude mitochondria was achieved according to Glick and Pon (33), using 0.6 mg of yeast lytic enzyme (ICN Biomedical Inc., Aurora, OH) per g of cells, dissolved in 20 mM MES (pH 6.0) containing 0.6 M sorbitol and 10 mM EDTA (buffer B), to convert the cells to spheroplasts. Spheroplasts were homogenized in a 50-ml glass Dounce homogenizer, and after removing unbroken cells and nuclei by centrifugation at 1500 × g, the crude mitochondrial pellet was obtained by centrifugation at 12,000 × g, and the supernatant was named cytosolic fraction. Crude mitochondria were diluted in a small volume of buffer B and overlaid on a 14 × 89-mm Ultra-Clear centrifuge tube that contained 5 ml of 14% Nycodenz (Sigma) overlaying 5 ml of 18% Nycodenz in buffer B. After centrifugation at 40,000 × g in an SW 41 rotor for 30 min, purified mitochondria could be recovered as a light-brown band between the 14 and 18% Nycodenz layers. The solution of mitochondria was diluted 5-fold in 20 mM HEPES (pH 7.4) containing 0.6 M sorbitol and 10 mM EDTA (buffer C) and centrifuged at 12,000 × g for 30 min. Finally, the mitochondrial pellet was diluted in a small volume of buffer C.

Thioredoxin Activity Assays-- The kinetics of the Trr2 activity was determined using the 5,5'-dithiobis(2-nitrobenzoic acid) reduction assay as described elsewhere (20). The Trx3 activity with different thioredoxin reductases was assayed by use of the insulin reduction assay according to Spyrou et al. (34). Thioredoxin was incubated with 30 µM DTT for 10 min at room temperature where indicated.

    RESULTS

Identification and Cloning of the TRX3 and TRR2 genes of S. cerevisiae-- An ORF encoding Trx3 was identified by searching in the Saccharomyces Genome Data Bank2 for genes encoding proteins that contain the QWCGPCK amino acid sequence, derived from the rat TRX2 redox active site (34). Three ORFs were identified; two of them coded for the previously described TRX1 and TRX2 genes (23), whereas the third ORF (accession number YCR083W) coded for an uncharacterized protein of 127 amino acids, which we named Trx3. Similarly, looking for proteins homologous to thioredoxin reductases, we identified another ORF (accession number YHR106W) that encoded a thioredoxin reductase-like protein, which we named Trr2. Both ORFs were isolated by PCR amplification, cloned, and sequenced, confirming the TRR2 and TRX3 sequences described in the data base.

The TRR2 gene is located on chromosome VIII of S. cerevisiae. Trr2 protein has a predicted molecular mass of 37.1 kDa and a pI of 6.7. Trr2 contains the redox active site (Cys-Ala-Val-Cys) characteristic of the thioredoxin reductases of lower organisms. Trr2 is 84% identical (97% similar) to the described S. cerevisiae thioredoxin reductase (24), and both proteins have the same amino acid sequences in the FAD- and NADPH-binding sites (Fig. 1).


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Fig. 1.   Alignment of the amino acid sequences of S. cerevisiae Trr1 and Trr2. The redox-sensitive motif is enclosed in a rectangle. Asterisks indicate the binding sites for FAD, and arrowheads indicate the binding sites for NADPH. The sequence of Trr2 was used as reference for the identity/similarity values.

The TRX3 gene is located on chromosome III of S. cerevisiae and encodes a protein with a predicted mass of 14.4 kDa and a pI of 8.9, containing the characteristic thioredoxin redox active site. Trx3 is larger than both Trx1 and Trx2 of yeast, but it is almost 50% identical (88% similar) to them within the homologous region (Fig. 2A). In contrast to the other Saccharomyces thioredoxins, Trx3 contains two extra cysteine residues, besides the ones in the active site.


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Fig. 2.   A, alignment of the amino acid sequences of S. cerevisiae Trx1, Trx2, and Trx3. The redox active site is enclosed in a rectangle. The sequence of Trx3 was used as reference for the identity/similarity values. B, comparison of potential Yap1-binding sites. The SV40 AP-1-binding site sequence was used as reference (59).

The transcription rates of many yeast genes are increased in response to diverse environmental and physiological insults, such as nitrogen starvation, osmotic shock, heat shock, metals, and oxidative stress. These genes contain in their promoter region a sequence (CCCCT, C4T) described as the stress response element (35). There is such a sequence at position -53 in the promoter region of TRX3. In contrast, the TRX1 and TRX2 genes of S. cerevisiae do not contain any C4T sequence (23). In the TRR2 gene, there is also a C4T sequence 372 nucleotides from the start codon. Although this sequence seems to be located too far upstream to regulate the TRR2 gene, stress response element spacing may be variable, and there are examples that it may control gene expression when situated between 10 and 175 nucleotides from the TATA box (35). Yap1, a c-jun-like transcription factor that binds to AP-1 DNA sites and is activated under oxidative stress conditions in yeast, regulates the expression of Saccharomyces TRX2 (36). In the promoter regions of both TRX3 and TRR2, there are sequences homologous to AP-1 sites (Fig. 2B).

The N-terminal domain of both Trx3 and Trr2 resembles presequence signals for import into mitochondria. Presequences are extremely variable both in length and amino acid sequence, but they do share common features such as the absence of acidic amino acid residues, a predominance of basic and hydroxyl-carrying residues, and an amphiphilic structure (generally an alpha -helix) with polar, positively charged, and apolar surfaces (37). The N-terminal sequences of Trx3 and Trr2 have a high pI (pI 11-12), and they lack acidic residues and may form an amphipathic alpha -helix. When the sequences of Trx3 and Trr2 were analyzed with a program for predictions of protein localization sites (PSORT)3, they showed a high probability to be localized in mitochondria with a predicted cleavage site at amino acids 21 and 23, respectively.

Expression of Trx3 and Trr2 Proteins in E. coli-- We designed primers to amplify TRX3 and TRR2 fragments encoding from amino acid residues 22 and 24, respectively, lacking the predicted N-terminal signal sequence that would be removed in the mature mitochondrial proteins. The amplified fragments were linked to the pET-15b expression vector, and after transforming E. coli cells, the expression of the recombinant proteins was induced by the addition of 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside in an exponentially growing culture. The protein expression was substantially increased when the cells were grown for 12 h at room temperature when compared with growth for 3 h at 37 °C (data not shown). This may be due to the presence of rare codons for Arg (AGG and AGA) and Leu (CTA) in the transcripts for both proteins. Since recombinant Trx3 and Trr2 were His-tagged, their purification was carried out in one chromatographic step, using TalonTM metal affinity columns. After elution with imidazole, we obtained pure Trx3 (14 kDa) and Trr2 (37 kDa), as shown by SDS-polyacrylamide gel electrophoresis (Fig. 3). In addition, Trr2 protein, in native polyacrylamide gel electrophoresis, showed a Mr of 60,400, as expected for a dimeric state of the enzyme (data not shown).


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Fig. 3.   Purification of recombinant Trx3 and Trr2. After induction with isopropyl-1-thio-beta -D-galactopyranoside, the cells were lysed, and the cell-free extract was passed through a TalonTM metal affinity column. Recombinant Trx3 and Trr2 proteins were eluted with 100 mM imidazole.

Activity of Trx3 and Trr2 in Vitro-- The purified recombinant Trx3 and Trr2 proteins were tested for thioredoxin activity in the insulin reduction assay. The insulin disulfides were effectively reduced in the presence of both proteins (Fig. 4A), demonstrating that Trx3 and Trr2 constitute an active thioredoxin system. We also analyzed the activity of Trx3 with both mammalian and E. coli thioredoxin reductases (Fig. 4, B and C). Saccharomyces Trx3 could serve as substrate for the mammalian thioredoxin reductase, but with 7-fold lower affinity than the reduced human TRX1 protein (Fig. 4B). However, Trx3 did not show activity with the E. coli thioredoxin reductase (Fig. 4C), in agreement with the known specificity of this enzyme, which can reduce only E. coli Trx1 and Trx2, whereas mammalian thioredoxin reductase can reduce thioredoxins from other species (7, 20). Trx3 has two cysteine residues at positions 88 and 92, in addition to the two redox active-site cysteine residues in the active site. Cytoplasmic mammalian thioredoxins have at least two extra cysteine residues that can undergo oxidation, leading to inactivation of the enzyme (7), whereas mitochondrial mammalian Trx does not contain extra cysteines and is not inactivated by oxidation (34). Inactivated mammalian TRX1 can be reactivated in vitro after preincubation with a reducing compound such as DTT. We tested whether DTT preincubation of Trx3 has a similar effect as on human TRX1. In contrast to human TRX1, DTT-preincubated Trx3 showed the same activity as the non-preincubated one, assayed with both Saccharomyces and mammalian thioredoxin reductases (Fig. 4, A and B).


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Fig. 4.   Trx activity in different thioredoxin systems, determined by the insulin assay. The assays were performed with a 50 nM concentration of different thioredoxin reductases: A, S. cerevisiae Trr2; B, calf thymus thioredoxin reductase; C, E. coli thioredoxin reductase. , S. cerevisiae Trx3; open circle , human TRX1; triangle , E. coli Trx1. The filled symbols show the assays with Trx preincubated with DTT.

We used the thioredoxin-dependent 5,5'-dithiobis(2-nitrobenzoic acid) reduction assay to determine the affinity of Trr2 for Trx3. To obtain saturation Michaelis-Menten kinetics, we used a low concentration of thioredoxin reductase (25 nM). The Km of Trr2 for recombinant Trx3 at pH 7.0 and 25 °C was 3.5 ± 0.3 µM. Trx3 digested with thrombin to remove the N terminus derived from the pET-15b vector showed a similar value (3.2 ± 1.1 µM), indicating that the His tag does not affect its activity. Trr2 showed a slightly higher Km value for its homologous thioredoxin than the E. coli thioredoxin reductase (1.9 and 2.4 µM for E. coli Trx1 and Trx2, respectively) (20) and the mammalian thioredoxin reductase (2.5 µM for TRX1) (7).

Subcellular Localization of Trx3 and Trr2 in S. cerevisiae-- The subcellular localization of Trx3 and Trr2 was studied by immunoblotting analysis using subcellular yeast fractions. Proteins from cytoplasmic and mitochondrial fractions were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and developed by affinity-purified antibodies.

The anti-Trx3 antibody reacted with a 14-kDa band in the highly purified mitochondrial sample (Fig. 5A). This band was present neither in the cytoplasmic fraction nor in a mitochondrial sample prepared from the Delta trx3 strain.


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Fig. 5.   Western blot analysis of different subcellular fractions with anti-Trx3 (A) and anti-Trr2 (B) antibodies. The first lanein A was loaded with recombinant Trx3 (band a) and thrombin-digested recombinant Trx3 (band b). wt, wild type.

In a total cell extract, the anti-Trr2 antibody reacted with a 37-kDa protein present in the wild-type strain, but not in a Delta trr2 strain (Fig. 5B). The 37-kDa band appeared exclusively in mitochondrial fractions of the wild-type strain. There was a strong cross-reaction of the anti-Trr2 antibody with a 36-kDa protein in the total cell homogenates and cytosolic fractions of both strains that is probably due to the presence of cytoplasmic Trr1, which is 84% identical to Trr2.

Sensitivity to H2O2-- The Delta trr2 and Delta trx3 strains as well as the wild-type strain showed similar growth curves and similar cell size in both fermentative (YPD) and respiratory (YPGE) media. We then checked for growth under oxidative stress conditions by exposing the cells to H2O2. Prior to the oxidant exposure, the cells were grown in YPD medium since cells growing in YPGE medium are very resistant to H2O2 (38). Saccharomyces cells growing in YPD medium show different sensitivity to oxidants at different states of growth. When cultures approach stationary phase, yeast cells become increasingly resistant to H2O2 (38, 39). Yeast cells growing on glucose show changes in cellular respiration paralleled by changes in mitochondrial morphology. During mid- and mid-to-late logarithmic growth, cells contain only few, poorly developed mitochondria; close to the late logarithmic phase, the exhaustion of glucose induces nearly full mitochondrial development (40). Therefore, we checked the sensitivity to H2O2 of Delta trr2 and Delta trx3 strains at different phases of growth: at mid-logarithmic phase, at mid-to-late log phase, and late log phase. The Delta trr2 strain was twice more sensitive to H2O2 than the wild type during mid-logarithmic phase (Fig. 6A). When cells are in the mid-to-late log phase, they become more resistant to H2O2, but the Delta Trr2 mutant was still more sensitive than the wild type (data not shown). In both cases, the Delta trx3 strain behaved as the wild type. When cells reached the late log phase or the stationary phase, all strains became very resistant to H2O2 (100% survival at 20 mM H2O2; data not shown). To confirm that the H2O2 sensitivity is due to the lost Trr2 protein, the Delta trr2 strain was transformed with a yeast expression plasmid encoding the TRR2 gene. The expression of Trr2 protein in the transformed cells was confirmed by Western blotting (Fig. 6B). The transformed cells were even more resistant to H2O2 than the wild type (Fig. 6A). This higher resistance may be due to a larger expression of Trr2 in transformed cells, resulting from the elevated plasmid copy number of the expression vector. Thus, the Trr2 defect can be complemented by expressing Trr2 in trans, suggesting that Trr2 protein contributes to the yeast resistance to H2O2.


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Fig. 6.   A, sensitivity of Delta trr2 and Delta trx3 strains to H2O2. Cells growing in YPD medium at mid-logarithmic phase were exposed to H2O2 for 30 min. open circle , wild type; triangle , Delta trr2; , Delta trx3; black-triangle, Delta trr2 mutant transformed with pRS425-TRR2. Data show the mean of three different experiments. B, Western blot analysis of total cell extracts of the wild type (lane 1), the Delta trr2 mutant (lane 2), and the Delta trr2 mutant transformed with pRS425-TRR2 (lane 3) using the anti-Trr2 antibody.

Glutathione is the main low molecular intracellular thiol compound, and it plays a key role in protecting cells against oxidants. We studied the survival of the mutants exposed to 1 mM CDNB, a treatment that reduces the glutathione levels in S. cerevisiae (41). In our experiments, the glutathione concentration in both the wild-type and mutant strains decreased from 325 ± 48 to 85 ± 13 pmol/106 cells after 15 min of incubation with CDNB, and the glutathione concentrations remained constant for 1 h of incubation. After 30 min of CDNB incubation, all strains showed similar survival close to 100%, but longer CDNB incubation was toxic for the cells, and the effect was more pronounced in the Trr2 mutant (Fig. 7A). We also observed that the CDNB treatment enhanced the H2O2 sensitivity differences between the wild-type and Delta trr2 strains. Cells treated with 1 mM CDNB for 30 min were exposed to different concentrations of H2O2 for an additional 30 min (Fig. 7B). The survival of the Trr2 mutant was further decreased ~35% compared with the survival of the same strain not treated with CDNB. The Delta trx3 strain showed similar resistance to the wild type under these conditions (data not shown).


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Fig. 7.   A, sensitivity of the Delta trr2 mutant to 1 mM CDNB. Cells growing in YPD medium at mid-logarithmic phase were incubated in the presence of 1 mM CDNB, and aliquots were plated on YPD medium to compare the survival at different times with respect to the survival at 0 min. The results show the mean of three different experiments. B, effect of the CDNB preincubation on Delta trr2 and wild-type strain sensitivity to H2O2. Cells in mid-logarithmic phase were incubated or not with 1 mM CDNB for 30 min, harvested by centrifugation, diluted in saline phosphate buffer, and exposed to different concentrations of H2O2 for 30 min. open circle , wild type; triangle , Delta trr2. The survival of the cells not exposed to CDNB is represented with empty symbols and dashed lines,and that of the cells exposed to CDNB with filled symbols and continuous lines.


    DISCUSSION

Trx3 and Trr2 of S. cerevisiae were identified in a search of a yeast genome data bank for proteins homologous to mammalian TRX2 and yeast Trr1, respectively. Trx3 and Trr2 are larger than their respective homologues in yeast due to an extension at the N terminus. These extensions have characteristics of signals that target translocation of proteins into mitochondria. Using affinity-purified antibodies, we have shown that Trx3 and Trr2 are located in mitochondria and not in the cytoplasm. Subsequent studies are needed to establish the submitochondrial localization of both proteins.

Proteins imported into mitochondria are usually cleaved by proteases, thereby losing their N-terminal presequence. Thus, we expressed fragments of these proteins in E. coli lacking the predicted N-terminal signal sequence. The resulting recombinant Trx3 protein shows thioredoxin activity in the presence of recombinant Trr2 and NADPH. Cytoplasmic mammalian thioredoxins have at least two structural or noncatalytic cysteine residues that may undergo oxidation, leading to inactivation by dimerization (7). Their activity may be restored by pretreatment with DTT. Trx3 contains two extra cysteine residues, but its activity is not affected by DTT, suggesting that Trx3 may be resistant to oxidizing agents, e.g. ROS. In a phylogenetic tree analysis, Saccharomyces Trr2 is closer to the prokaryotic thioredoxin reductases than to the mammalian ones. Its subunit Mr is similar to that of the E. coli thioredoxin reductase subunit (35,000), and the redox active-site cysteines are located in the NADPH domain with a two-amino acid bridge between the cysteines (7, 16). The mammalian thioredoxin reductase contains a selenocysteine as the penultimate amino acid, which seems to be essential for the enzymatic activity (17, 19). The expression of mammalian thioredoxin reductase in E. coli has not been successful because the genomic elements that recognize the specific codon for selenocysteine are different between eukaryotic and prokaryotic organisms (42). Trr2 does not contain selenocysteine; thus, we could express and purify active Trr2 from E. coli.

The mitochondrial thioredoxin reductase has an antioxidant function since a TRR2-disrupted yeast strain is more sensitive to H2O2. However, we have not observed increased sensitivity to H2O2 in a Trx3 mutant under the same conditions. Our results suggest that Trr2 would exert its antioxidant function without the participation of its partner, Trx3. Mammalian thioredoxin reductase can act as an antioxidant in a thioredoxin-independent way, e.g. is an electron donor for the plasma glutathione peroxidase (43) and is able to reduce lipid hydroperoxides alone or, more efficiently, in the presence of selenocysteine (22). We have also observed that the Trr2 mutant is more sensitive to a treatment that reduces the glutathione levels. In addition, this treatment enhances the sensitivity of the mutant for H2O2. So, there may exist an antioxidant pathway that both Trr2 and glutathione share in mitochondria. S. cerevisiae does not grow under aerobic conditions when glutathione reductase and cytoplasmic thioredoxins are suppressed because the rate of destruction of peroxides decreases (44). Two additional important enzymes in the cell protection against oxidative aggressions are thioredoxin peroxidase and glutathione peroxidase (45-47). Thioredoxin peroxidase reduces hydroperoxides with thioredoxin as electron donor (24). This enzyme was initially identified in S. cerevisiae (48), and later, a large number of homologous proteins in a wide variety of species were described (49). Glutathione peroxidase has been reported to be present in yeast mitochondria (50), but no yeast mitochondrial thioredoxin peroxidase has been described so far. We have observed that an ORF in the Saccharomyces genome (locus SCYBL064c) codes for a protein homologous to the cytoplasmic thioredoxin peroxidase and with a putative N-terminal mitochondrial signal. Further studies are necessary to corroborate whether this protein is a thioredoxin peroxidase located in mitochondria. In mammalian cells, one specific mitochondrial thioredoxin has already been characterized in rat (34), and one protein with thioredoxin reductase activity has been purified from rat mitochondria (51). In addition, we have cloned a cDNA sequence that codes for a mitochondrial selenoprotein highly homologous to the cytosolic thioredoxin reductase.4

Mitochondria are the sites of vital cellular functions such as lipid metabolism and aerobic respiration (oxidative phosphorylation). In respiration, incomplete reduction of dioxygen results in the formation of ROS, such as hydrogen peroxide (H2O2), the superoxide anion (Obardot 2), and the hydroxyl radical (HO·). Increased levels of ROS, referred as oxidative stress, can result in lipid peroxidation, inactivation of proteins, and DNA breakage (52). ROS can also act as signaling molecules, triggering the activation of protein kinases and phosphatases that regulate gene expression and induce cell proliferation or death. For counteracting the ROS threat, mitochondria have specific antioxidant enzymes such as manganese-superoxide dismutase and glutathione peroxidase (53, 54). Specific mitochondrial thioredoxin peroxidases have been characterized in mammals (47, 55). Therefore, the mitochondrial thioredoxin system would contribute to the defense against ROS as electron donor of the thioredoxin peroxidase. In addition, mitochondrial thioredoxin may act as an antioxidative molecule, scavenging OH·, reducing H2O2, and reactivating proteins inactivated by oxidation (13, 14). Thioredoxin also participates in folding of proteins in a way similar to that of protein-disulfide isomerase (8, 56), and mitochondrial thioredoxin is the only known protein that can reduce protein disulfides in mitochondria. Thus, it may regulate the activity of mitochondrial proteins by redox control. For example, the opening mechanism of the mitochondrial permeability transition pore, an inner membrane complex that is implicated in the Ca2+ efflux from mitochondria, is modulated by a redox-sensitive dithiol group and by the redox state of pyridine nucleotides (57, 58). Considering these conditions, the thioredoxin system would by a good candidate for the regulation of the mitochondrial permeability transition pore opening mechanism.

Redox control provides the cell with a mechanism by which it can respond to changes in its environment through modulation of the activity of certain genes. The thioredoxin system plays a central role in redox control by regulating the activity of transcription factors and enzymes and protecting cells from oxidative stress. We have identified two yeast genes that code for a thioredoxin system in mitochondria. This thioredoxin system may play an important role in controlling the redox status in mitochondria.

    FOOTNOTES

* This work was supported by grants from the Swedish Medical Research Council (Project 13X-10370), the Karolinska Institutet, the Svenska Institutet, and TMR Marie Curie research training grants (Contract ERBFMBICT972824).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 46-8-6089162; Fax: 46-8-7745538; E-mail: giannis.spyrou{at}cbt.ki.se.

2 Available on the World Wide Web at http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.

3 Available on the World Wide Web at http://psort.nibb.ac.jp/.

4 A. Miranda-Vizuete, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Trx, thioredoxin; Trr, thioredoxin reductase; ORF, open reading frame; PCR, polymerase chain reaction; CDNB, 1-chloro-2,4-dinitrobenzene; MES, 4-morpholineethanesulfonic acid; DTT, dithiothreitol; ROS, reactive oxygen species; YP, yeast peptone.

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