The yeast Tsa1 peroxiredoxin, like other 2-Cys peroxiredoxins, has dual activities as a peroxidase and as a molecular chaperone. Its peroxidase function predominates in lower-molecular-mass forms, whereas a super-chaperone form predominates in high-molecular-mass complexes. Loss of TSA1 results in aggregation of ribosomal proteins, indicating that Tsa1 functions to maintain the integrity of the translation apparatus. In the present study we report that Tsa1 functions as an antioxidant on actively translating ribosomes. Its peroxidase activity is required for ribosomal function, since mutation of the peroxidatic cysteine residue, which inactivates peroxidase but not chaperone activity, results in sensitivity to translation inhibitors. The peroxidatic cysteine residue is also required for a shift from ribosomes to its high-molecular-mass form in response to peroxide stress. Thus Tsa1 appears to function predominantly as an antioxidant in protecting both the cytosol and actively translating ribosomes against endogenous ROS (reactive oxygen species), but shifts towards its chaperone function in response to oxidative stress conditions. Analysis of the distribution of Tsa1 in thioredoxin system mutants revealed that the ribosome-associated form of Tsa1 is increased in mutants lacking thioredoxin reductase (trr1) and thioredoxins (trx1 trx2) in parallel with the general increase in total Tsa1 levels which is observed in these mutants. In the present study we show that deregulation of Tsa1 in the trr1 mutant specifically promotes translation defects including hypersensitivity to translation inhibitors, increased translational error-rates and ribosomal protein aggregation. These results have important implications for the role of peroxiredoxins in stress and growth control, since peroxiredoxins are likely to be deregulated in a similar manner during many different disease states.
Peroxiredoxins are ubiquitous thiol-specific proteins that have multiple functions in stress protection. In addition to their peroxidase function, they have proposed roles in diverse cellular processes including differentiation, proliferation, modulation of intracellular signalling, apoptosis and gene expression [1,2]. Peroxiredoxins use redox-active cysteine residues to reduce peroxides and have been divided into two categories, the 1-Cys and 2-Cys peroxiredoxins, based on the number of cysteine residues directly involved in catalysis . Typical 2-Cys peroxiredoxins are active as a dimer and contain two redox active cysteine residues that are directly involved in enzyme activity [4,5]. During catalysis, the peroxidatic cysteine is oxidized to a sulfenic acid, which condenses with a resolving cysteine residue (from the other subunit of the dimer) to form a disulfide. This disulfide bond is reduced by thioredoxin to regenerate the active peroxiredoxin. Peroxiredoxins are therefore active in a redox cycle, accepting electrons from NADPH via thioredoxin and thioredoxin reductase.
Three cytoplasmic 2-Cys peroxiredoxins (Tsa1, Tsa2, Ahp1) have been described in yeast. They all display thioredoxin peroxidase activity, but their exact intracellular roles and targets remain unclear . Tsa1 is the major peroxiredoxin and has best been characterized as an antioxidant in the detoxification of hydroperoxides [6,7], but more recently has been shown to act as a molecular chaperone that promotes resistance to heat shock . Its chaperone activity involves a stress-dependant switch from low-molecular-mass species to high-molecular-mass complexes . During normal growth conditions, Tsa1 is present as a mixture of low and oligomeric forms which possess dual activities as a peroxidase and a chaperone. In response to a peroxide stress it shifts to the high-molecular-mass ‘super-chaperone’ form. Thus the peroxidase function predominates in lower-molecular-mass forms of Tsa1, whereas the chaperone function predominates in high-molecular-mass complexes. Like other molecular chaperones, Tsa1 can suppress thermal aggregation in vitro by binding to unfolded proteins via exposed hydrophobic sites . Studies of several typical 2-Cys peroxiredoxins have revealed similar changes in oligomeric state that are linked to changes in redox state  and human PrxII (where Prx is peroxiredoxin) and Helicobacter pylori AhpC have been shown to posses dual functions as peroxidases and as molecular chaperones [9,10].
Yeast mutants lacking TSA1 are sensitive to heat shock and accumulate heat-shock-induced protein aggregates . The levels of aggregation are also increased in response to oxidative (hydrogen peroxide) or reductive [DTT (dithiothreitol)] stress conditions which may contribute to the sensitivity of this mutant to these stress conditions . We recently identified a number of the proteins that aggregate in a tsa1 mutant. Remarkably, the most abundant aggregated proteins were all ribosomal proteins, including proteins of both the small and large ribosomal subunits . Disrupting the thioredoxin system, through loss of both thioredoxins (trx1 trx2) or thioredoxin reductase (trr1), results in a similar pattern of protein aggregation, presumably reflecting the role of the thioredoxin system in maintaining the redox status of Tsa1. Ribosomal protein aggregation was found to correlate with an inhibition of translation initiation, suggesting that Tsa1 normally functions to protect against the damaging consequences of disrupting the protein synthetic machinery . In the present study we have examined whether Tsa1 is associated with actively translating ribosomes which might explain its role in protecting the translation apparatus. We show that a portion of Tsa1 is associated with actively translating ribosomes and this ribosome-associated pool shifts from ribosomes to the high-molecular-mass super-chaperone form in response to oxidative stress conditions. A further link with ribosome function comes from the finding that deregulation of Tsa1 in a thioredoxin reductase mutant promotes hypersensitivity to translation inhibitors, an increased translational error-rate and elevated ribosomal protein aggregation. Thus Tsa1 is the first peroxiredoxin to be shown to function on the translational apparatus, where we propose it acts to protect ribosomal proteins against oxidative damage and non-specific ribosomal protein aggregation.
Yeast strains, plasmids and growth conditions
The Saccharomyces cerevisiae strains used in the present study were isogenic derivatives of W303 (MATa ura3-52 leu2-3 leu2-112 trp1-1 ade2-1 his3-11 can1-100). Strains deleted for thioredoxins (trx1::TRP1 trx2::URA3), thioredoxin reductase (trr1::HIS3) and TSA1 (tsa1::LEU2) have been described previously [6,11,12]. Strains CY524 (trx1::TRP1 trx2::URA3 trr1::HIS3) and CY1025 (trr1::HIS3 tsa1::LEU2) were constructed using standard yeast techniques. The centromeric plasmids pFL-TSA1 and pFL-TSA1C47S, containing wild-type and Cys47 mutant versions of TSA1 respectively, have been described previously .
Strains were grown in rich YEPD (yeast extract, peptone, dextrose) medium [2% (w/v) glucose, 2% (w/v) bactopeptone and 1% (w/v) yeast extract] or minimal SD (selective drop-out) medium [0.17% (w/v) yeast nitrogen base without amino acids, 5% (w/v) ammonium sulfate and 2% (w/v) glucose] supplemented with appropriate amino acids and bases  at 30 °C and 180 rev./min. Media were solidified by the addition of 2% (w/v) agar. Stress sensitivity was determined by growing cells to stationary phase and spotting diluted cultures (A600=1.0, 0.1) on to agar plates containing various concentrations of inhibitor. Growth was monitored after 3 days. The chaperone activity of tsa1 mutants was determined by exposing exponential-phase cultures to 47 °C for 30 min.
Translation fidelity assays
The levels of termination codon readthrough were measured using a β-galactosidase reporter system . Readthrough was quantified using plasmid pUKC817 (which carries the lacZ gene that bears a premature termination codon) and expressed as a proportion of control β-galactosidase levels, measured in transformants carrying the control plasmid pUKC815 (which carries the wild-type lacZ gene). The missense error-rate was measured using an assay based on the misincorporation of arginine at position 218 in luciferase . The assay is based on the replacement of codon AGA (Arg218) with AGC (encoding Ser218) which requires recognition by a near cognate tRNA for luciferase activity.
Protein aggregation and Western blot analysis
For Tsa1 analysis, total protein extracts were electrophoresed on reducing SDS/PAGE (12% gels) minigels or on 4–12% native PAGE gels and electroblotted on to PVDF membrane (Amersham Pharmacia Biotech). Bound antibody (αTsa1) was visualized by ECL® (enhanced chemiluminescence; Amersham Pharmacia Biotech) following incubation of the blot in donkey anti-rabbit immunoglobulin-HRP (horseradish peroxidase) conjugate (Amersham Pharmacia Biotech). The aggregation of soluble proteins was analysed as described previously . Aggregated protein extracts were separated by reducing SDS/PAGE (12% gels) and visualized by silver staining with the Bio-Rad silver stain plus kit.
Analysis of ribosome distribution by sucrose-density gradients
Ribosomal fractions were separated from soluble components by centrifugation through sucrose cushions. Cell extracts (0.5 ml) prepared in CSB buffer [300 mM sorbitol, 20 mM Hepes (pH 7.5), 1 mM EGTA, 5 mM MgCl2, 10 mM KCl, 10% (v/v) glycerol and 10 μg/ml cycloheximide] were layered on to 0.4 ml of 60% (w/v) sucrose in CSB buffer without sorbitol . Samples were centrifuged at 55000 rev./min in a Beckman MLA-130 rotor for 2.5 h at 4 °C. For quantitative comparison, supernatant fractions were precipitated in 10% (w/v) TCA (trichloroacetic acid) and pellets were washed in acetone before resuspension in SDS sample buffer. For polysome analysis, extracts were prepared in the presence of cycloheximide (100 μg/ml), and layered on to 15–50% sucrose gradients. Gradients were sedimented via centrifugation at 40000 rev./min in a Beckman ultracentrifuge for 2.5 h, and the A254 was measured continuously . Sucrose gradients were fractionated into 1 ml fractions and boiled in SDS sample buffer prior to Western blot analysis. EDTA (15 mM) was added to breakage and gradient buffers to dissociate polysomes.
Tsa1 is associated with translating ribosomes
Polysome profiles were examined to determine whether Tsa1 associates with actively translating ribosomes. Cell lysates were analysed by sedimentation through sucrose-density gradients and the distribution of ribosomes was quantified by measuring the absorbance at 254nm (Figure 1A). Proteins were recovered from fractionated gradients and the presence of Tsa1 and a ribosomal protein (Rps3) were detected by Western blot analysis. Tsa1 was most abundant at the top of the gradient, which is where soluble proteins sediment. However, a signification portion fractionated with polysomes, comparable with ribosomal protein Rps3. Dissociation of polysomes by treatment with EDTA shifted Tsa1 into lower-molecular-mass fractions, confirming that Tsa1 associates with actively translating ribosomes (Figure 1B). Ribosomal fractions were separated from soluble components by centrifugation through sucrose cushions to enable a quantitative assessment of the relative distribution of Tsa1. This analysis revealed that approx. 5% of Tsa1 is associated with the ribosomal fraction compared with soluble components (Figure 1C). This is not a general property of peroxiredoxins, since the cytoplasmic Ahp1 peroxiredoxin could not be detected in the ribosomal fraction (results not shown).
Tsa1 is associated with translating ribosomes
The peroxidase activity of Tsa1 is required for ribosome function
Tsa1 shifts from its low-molecular-mass forms to the high-molecular-mass super-chaperone form in response to peroxide stress . We therefore examined whether oxidative stress affects the ribosome association of Tsa1. Following treatment with 0.2 mM hydrogen peroxide for 10 min, polysome analysis revealed a significant decrease in the ribosome association of Tsa1 (Figure 2). Loss of Tsa1 from polysomes is not simply due to inhibition of translation, since this peroxide treatment had a relatively minor effect on polysome distribution and did not alter the sedimentation of Rps3.
Tsa1 dissociates from ribosomes in response to hydrogen peroxide
For comparison, we examined the effect of this peroxide treatment on the oligomeric structure of Tsa1 using native PAGE (Figure 3A). Wild-type Tsa1 was predominantly detected as low-molecular-mass species, whereas a portion of Tsa1 shifted to the high-molecular-mass form in response to peroxide treatment in agreement with previous observations . The peroxidatic cysteine residue of Tsa1 is known to be required for the switch to the high-molecular-mass super-chaperone form . We confirmed that a Tsa1C47S mutant, which lacks the peroxidatic cysteine residue, was unable to shift to high-molecular-mass complexes in response to hydrogen peroxide (Figure 3A). Similarly, no dissociation of this mutant from ribosomes was observed in response to peroxide treatment indicating that the shift from its ribosome-associated form is dependent on oxidation of the peroxidatic cysteine residue (Figure 2). Thus Tsa1 is predominantly present in low-molecular-mass and ribosome-associated forms during normal growth conditions, but shifts to its super-chaperone form in response to oxidative stress conditions.
The peroxidase activity of Tsa1 is required for ribosome function
Sensitivity to translation inhibitors is often taken to indicate a functional link with the translation apparatus. We therefore examined the sensitivity of a tsa1 mutant to translation inhibitors including hygromycin, paromomycin and cycloheximide. This analysis revealed that a tsa1 mutant is sensitive to all three inhibitors compared with the wild-type control (Figure 3B, compare wt v with tsa1 v). Sensitivity was most pronounced with paromomycin and hygromycin compared with the relatively minor sensitivity observed with cycloheximide. Tsa1 is known to function as a peroxidase in the detoxification of hydroperoxides, and as a molecular chaperone that protects against protein misfolding. To determine which of these activities is important for ribosome function, we examined the sensitivity of the Tsa1C47S mutant to translation inhibitors. The Cys47 residue of Tsa1 is not required for chaperone activity but is essential for peroxidase activity . We first confirmed that the Tsa1C47S mutant lacks peroxidase activity since it does not rescue the hydrogen peroxide sensitivity of a tsa1 deletion strain, whereas it is active as a chaperone since it complements the temperature sensitivity of the tsa1 mutant (Figure 3B). We reasoned that if the chaperone function of Tsa1 is important for tolerance to translation inhibitors, then the Tsa1C47S mutant should still be able to promote resistance. However, the Tsa1C47S mutant was unable to rescue the sensitivity of the tsa1 mutant to paromomycin and hygromycin, indicating that the peroxidase activity is most probably required for tolerance to aminoglycoside antibiotics. In contrast, the Tsa1C47S mutant was able to increase the resistance of the tsa1 mutant to cycloheximide. Taken together, our results indicate that under normal growth conditions, Tsa1 functions predominantly as an antioxidant protecting both the cytoplasm and translation apparatus against endogenous ROS (reactive oxygen species). In response to an oxidative stress, Tsa1 shifts towards a form in which it functions to chaperone stress-denatured proteins.
Ribosome association of Tsa1 in thioredoxin mutants
The redox state of Tsa1 is maintained by the thioredoxin system and so we examined whether thioredoxin reductase (Trr1) or thioredoxins (Trx1/Trx2) influence its ribosome function. This is important since we have previously shown that ribosomal protein aggregation is particularly elevated in a trr1 mutant. Aggregation is actually greater than that observed in a tsa1 mutant indicating that it does not simply arise due to loss of Tsa1 activity . Tsa1 was found to be highly elevated in polysome fractions from a trr1 mutant (Figures 2 and 4A) and this elevated ribosome-association was unaffected by loss of the peroxidatic cysteine residue (Figure 2). Ribosome-associated Tsa1 was also elevated in a trx1/trx2 mutant, although not to the same extent as in the trr1 mutant (Figure 4A). Increased ribosome association presumably arises as a result of the elevated total Tsa1 protein levels which are observed in thioredoxin mutants (Figure 4B) due to the constitutive Yap1 activation which has been described for these mutants [18,19].
Ribosome association of Tsa1 in thioredoxin mutants
Analysis of the oligomeric structure of Tsa1 revealed that a significant portion is constitutively shifted to the high-molecular-mass form in the trr1 mutant (Figure 4C). Given that the oxidant-induced switch from low-molecular-mass species to high-molecular-mass complexes requires thioredoxins, we examined whether thioredoxins are also required for this shift in oligomeric form. No Tsa1 was present in the high-molecular-mass form in a trr1/trx1/trx2 mutant indicating that thioredoxins promote the oligomerization of Tsa1 in the trr1 mutant (Figure 4C). In contrast, polysome analysis revealed that loss of thioredoxins did not affect the elevated Tsa1 ribosome-association observed in the trr1 mutant (Figure 4A). Thus thioredoxins are required for the switch to the high-molecular-mass chaperone form of Tsa1, but not its increased ribosome association in the trr1 mutant. The trr1/trx1/trx2 mutant therefore enabled us to examine the phenotypic consequences of the elevated total and ribosomal Tsa1 in a trr1 mutant in the absence of any complications arising from the shift to the super-chaperone form (see below).
Deregulation of Tsa1 function in a trr1 mutant promotes translation defects
We examined the sensitivity of the trr1 mutant to translation inhibitors to determine the consequences of elevated Tsa1 levels in this mutant. The trr1 mutant displayed little or no sensitivity to cycloheximide compared with a wild-type strain (Figure 5A); however, it was hypersensitive to hygromycin and paromomycin. The trx1/trx2 mutant was also sensitive to hygromycin and paromomycin, but not to the same extent as the trr1 mutant. To determine whether the sensitivity of the trr1 mutant to aminoglycoside antibiotics is influenced by Tsa1 activity, we examined the effect of deleting TSA1 in the trr1 mutant. Interestingly, loss of TSA1 partially rescued its sensitivity to hygromycin and paromomycin indicating that Tsa1 promotes sensitivity to these inhibitors in a trr1 mutant. In comparison, loss of thioredoxins did not affect the sensitivity of the trr1 mutant to aminoglycosides (Figure 5A). These results indicate that it is most probably the ribosome-associated form of Tsa1, rather than the increased total Tsa1 levels themselves, which causes hypersensitivity in this mutant. Similar to the deletion of TSA1, mutation of its peroxidatic cysteine residue also rescued the sensitivity of the trr1 mutant to paromomycin and hygromycin (Figure 5B). Thus deregulation of the active ribosomal form of Tsa1 causes sensitivity to aminoglycoside antibiotics in a trr1 mutant.
Deregulation of Tsa1 promotes translation defects
Aminoglycosides have well-characterized effects on translational fidelity [20,21]. They can act on the ribosome to cause mistranslation, and hence mutants which are affected in the accuracy of the decoding process often show enhanced sensitivity. We therefore examined the fidelity of translation in strains lacking TRR1. Readthrough of termination codons was quantified using a plasmid which carries the lacZ gene with a premature UAA termination codon . The wild-type and tsa1 mutant displayed a similar level of termination codon readthrough (Table 1). In contrast, readthrough was elevated approx. 15-fold in the trr1 mutant. This high level of readthrough was completely abrogated in the trr1/tsa1 mutant consistent with the finding that deletion of TSA1 rescues the aminoglycoside sensitivity of a trr1 mutant. In contrast, readthrough was unaffected by loss of thioredoxins and the trr1/trx1/trx2 mutant still displayed a high translational error-rate. The missense error-rate was measured using a luciferase assay which requires misincorporation of arginine (AGC) at an AGA codon (encoding a serine residue), by a near cognate tRNA, for activity . The missense error-rate was increased by approx. 1.6-fold in the trr1 mutant and this increased error-rate was again abrogated by loss of TSA1 (Table 1).
|Mutant||Nonsense (%)||Missense (%)|
|Mutant||Nonsense (%)||Missense (%)|
Tsa1 promotes ribosomal protein aggregation in a trr1 mutant
To determine whether deregulated Tsa1 accounts for the high levels of protein aggregation in a trr1 mutant, we examined protein aggregation in the trr1/tsa1 mutant. Significantly, deleting TSA1 in the trr1 mutant reduced the high basal levels of aggregated proteins normally detected in the trr1 mutant (Figure 5C). In fact, aggregation was somewhat lower in the trr1/tsa1 than in a tsa1 mutant, which is surprising given that the trr1/tsa1 mutant completely lacks functional Tsa1. We presume that other peroxidases may be able to substitute for Tsa1 in a trr1/tsa1 mutant and, for example, TSA2 is transcriptionally induced in trr1 and trr1/tsa1 mutants (E. W. Trotter and C. M. Grant, unpublished work). It is unclear at present whether Tsa2 has any ribosomal function, but it is highly homologous with Tsa1 and possesses a similar chaperone activity, although it is normally expressed at significantly lower levels compared with Tsa1 . Immunoblot analysis with an anti-ubiquitin antibody revealed the presence of a number of ubiquitin-modified proteins in the trr1 mutant, presumably reflecting aberrant proteins marked for degradation (Figure 5D). Ubiquitination was abrogated in the trr1/tsa1 mutant confirming that Tsa1 propagates protein damage in the trr1 mutant.
The production of ribosomes is a complex, highly regulated energy-requiring process . It must be able to respond to environmental conditions and several conserved signalling pathways [TOR (target of rapamycin), RAS/protein kinase A and protein kinase C] are known to regulate ribosome biosynthesis [23,24]. For example, transcriptional co-regulation of genes involved in ribosome biogenesis has been linked to cellular proliferation, and ribosome synthesis may serve as a measure for cell-cycle progression [24,25]. Little is known regarding how cells maintain ribosome homoeostasis once they have been assembled and are active in cytoplasmic translation. This is surprising, since ribosome synthesis represents greater than 50% of total transcription in growing eukaryotic cells (reviewed in ). The results of the present study indicate that Tsa1 functions as an antioxidant on actively translating ribosomes, protecting the protein synthetic machinery against oxidative damage.
Ribosomal proteins are major targets of aggregation in a tsa1 mutant and aggregation correlates with an inhibition of protein synthesis . We previously attributed ribosomal protein aggregation to a loss of Tsa1 chaperone activity. However, given the functional link with the translation apparatus, it is more probable that the loss of Tsa1 ribosomal activity accounts for ribosomal protein aggregation and the translation effects reported in the present study. Tsa1 was originally identified as a thiol-specific antioxidant which could provide protection against a thiol-containing oxidation system . The purified protein was subsequently shown to possess peroxidase activity towards hydrogen peroxide and alkyl hydroperoxides in the presence of the thioredoxin and thioredoxin reductase reducing system . It is this thioredoxin peroxidase activity that appears to be required for Tsa1 ribosomal function, since the Tsa1 peroxidatic cysteine residue is required for resistance to translation inhibitors. These results can be integrated into a model to describe the multiple functions of Tsa1 (Figure 6). Under normal growth conditions, Tsa1 functions predominantly as an antioxidant protecting both the cytoplasm and translational apparatus against endogenous ROS. Over-oxidation of the peroxidatic cysteine residue causes a shift to the high-molecular-mass super-chaperone form. Thus, in response to an acute oxidative stress, a portion of Tsa1 is shifted towards its super-chaperone form which protects against stress-denatured proteins . The Tsa1 peroxidase function appears therefore to be less important during oxidative stress conditions. Presumably, other peroxidases can substitute for Tsa1 during oxidative stress conditions, many of which are induced in response to such conditions . Tsa1 is ideally structured to undergo this kind of functional switch, since its peroxidatic cysteine residue, in common with other 2-Cys peroxiredoxins, can be readily over-oxidized to cysteine-sulfinic acid following exposure to peroxides [3,29].
Model of multiple Tsa1 functions
Tsa1 protein levels are elevated in thioredoxin system mutants (trr1 and trx1/trx2) due to the constitutive activation of the Yap1 transcription factor in these mutants [18,19]. There is no apparent alteration in the portion of Tsa1 associated with ribosomes as a fraction of the total Tsa1 levels present in thioredoxin mutants compared with the wild-type strain. The elevation in total Tsa1 protein levels presumably accounts for the high levels of ribosomal Tsa1 detected in trr1 and trx1/trx2 mutants. We found that thioredoxin mutants are sensitive to aminoglycoside inhibitors and sensitivity is particularly pronounced in a trr1 mutant. The best evidence that translation defects do not simply arise due to elevated levels of total Tsa1 in the trr1 mutant comes form a comparison of the various thioredoxin mutants analysed in the present study. Mutants lacking thioredoxin or thioredoxin reductase contain similar elevated levels of total Tsa1, but only the trr1 mutant is hypersensitive to aminoglycoside antibiotics and displays elevated translational errors. These results indicate that there is not a correlation between elevated Tsa1 levels and translation defects. The high-molecular-mass super-chaperone form of Tsa1 present in the trr1 mutant does not appear to effect translation, since loss of thioredoxins (trr1/trx1/trx2) prevents Tsa1 from shifting to its high-molecular-mass form in the trr1 mutant but does not rescue its sensitivity to aminoglycoside antibiotics. Therefore taken together these results indicate that the deregulation of Tsa1 ribosomal function most probably causes translation defects.
Many protein synthesis mutants which alter the fidelity of translation are sensitive to aminoglycoside inhibitors such as paromomycin and hygromycin . Similarly, misreading is increased in the trr1 mutant as detected by the elevated levels of nonsense suppression and misincorporation. Loss of TSA1 itself does not affect translational fidelity in a wild-type strain. Rather, it appears that deregulation of Tsa1 in the trr1 mutant promotes translation defects including ribosomal protein aggregation, sensitivity to aminoglycosides and elevated translational error-rates. Further studies will be required to determine whether the high translational error-rate in the trr1 mutant promotes aggregation, or alternatively, whether ribosomal protein aggregation physically disrupts the translation process promoting misreading in the trr1 mutant.
Peroxiredoxins have been implicated in many disease processes [31,32]. For example, alterations in the levels of 2-Cys peroxiredoxins have been reported for many cancers [32–35]. A tumour suppressor role has been proposed for Prx1 (where Prx is peroxiredoxin) based on the observations that overexpression inhibits c-Myc-mediated transformation and increased malignancies are observed in mice with inactivated Prx1 [36,37]. Furthermore, the 2-Cys peroxiredoxin, Pag, can interact with the oncoproteins c-Abl and c-Myc and affects cell size and apoptosis [38,39]. Additionally, altered peroxiredoxin expression levels have been observed in different brain regions of patients affected by neurodegenerative disorders (reviewed in ). Up-regulation of peroxiredoxins in these various pathologies may reflect an antioxidant response to oxidative stress. However, our results indicate that, rather than serving a simple protective role, deregulation of peroxiredoxins can promote stress sensitivity via effects on protein aggregation and errors in translation. This finding is important, since many of the disease processes described above are liable to include elevations in ROS levels which could potentially affect the thioredoxin system and peroxiredoxin function, in a similar manner to that described in the present study for yeast thioredoxin mutants.
There is increasing evidence that the translational apparatus is a target of oxidative damage. A recent global analysis of protein carbonylation identified a large number of ribosomal proteins which become oxidized in response to hydrogen peroxide stress, representing approx. 80% of possible ribosomal protein targets . Interestingly, many of these ribosomal proteins were found to form protein–RNA cross-links which may contribute to ribosomal protein aggregation . Oxidative damage to the protein-synthetic machinery has been implicated in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease [43–45]. Furthermore, oxidation of mRNA has been shown to affect translational fidelity, and active translation of oxidized mRNA can result in the production of aberrant proteins . The antioxidant activity of Tsa1 may therefore be important to maintain both the fidelity of translation during potentially error-prone conditions, as well as to protect the translational apparatus itself, against oxidative damage. It appears that disruption of the translation apparatus may be more widespread than previously recognized, with ribosomal proteins representing important targets that must be protected against the formation of non-native protein structures and aggregates during stress conditions.
This work was supported by the BBSRC (Biotechnology and Biological Sciences Research Council). We thank Martin Pool (Faculty of Life Sciences, University of Manchester, Manchester, U.K.), David Eide (Nutritional Sciences, University of Wisconsin, Madison, WI, U.S.A.), Mick Tuite (Department of Biosciences, University of Kent, Canterbury, Kent, U.K.) and Sabine Rospert (Institute of Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany) for the gifts of strains, plasmids and antibodies.
Present address: Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K.
Present address: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE1 7RU, U.K.