The thioredoxin system protects ribosomes against stress-induced aggregation

Abstract

We previously showed that thioredoxins are required for dithiothreitol (DTT) tolerance, suggesting they maintain redox homeostasis in response to both oxidative and reductive stress conditions. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to DTT to identify cell functions involved in resistance to reductive stress. We identified 195 mutants, whose gene products are localized throughout the cell. DTT-sensitive mutants were distributed among most major biological processes, but they particularly affected gene expression, metabolism, and the secretory pathway. Strikingly, a mutant lacking TSA1, encoding a peroxiredoxin, showed a similar sensitivity to DTT as a thioredoxin mutant. Epistasis analysis indicated that thioredoxins function upstream of Tsa1 in providing tolerance to DTT. Our data show that the chaperone function of Tsa1, rather than its peroxidase function, is required for this activity. Cells lacking TSA1 were found to accumulate aggregated proteins, and this was exacerbated by exposure to DTT. Analysis of the protein aggregates revealed that they are predominantly composed of ribosomal proteins. Furthermore, aggregation was found to correlate with an inhibition of translation initiation. We propose that Tsa1 normally functions to chaperone misassembled ribosomal proteins, preventing the toxicity that arises from their aggregation.

Figure 1.

Functional grouping of deletion mutant sensitivity data. (A) Localization was assigned based on the “Component” term of the Saccharomyces Genome Database GO term mapper. Cyt, cytoplasm; Vac, vacuole; Mit, mitochondrion; Nuc, nucleus; Golgi, Golgi apparatus; Wall, cell wall; O/U, other/unknown. (B) Gene products were grouped into functional categories according to the MIPS functional database and Saccharomyces Genome Database, combined with visual inspection. More detailed functional data are available in Table 1. PS, protein synthesis; Transcript, transcription; Secret, secretory pathway; Cell, cell cycle and differentiation; Metab, metabolism; Stress, cell rescue and defense; ion, ion homeostasis; Transport, cellular transport; O/U, other/unknown.

Figure 2.

Mutants affecting the thioredoxin system are sensitive to reductive stress. (A) Stress sensitivity was determined by spotting strains onto YEPD plates containing DTT or SD plates containing cysteine (Cys). Cultures of wild-type, trx1 trx2, gpx3, skn7, tsa1, and yap1 strains were grown into stationary phase and adjusted to an A₆₀₀ of 1.0 before spotting onto appropriate plates. Plates were incubated at 30°C for 3 d before scoring growth. (B) Western blot analysis of thioredoxin protein levels. Thioredoxin protein concentrations were measured in wild-type, trx1 trx2, gpx3, skn7, tsa1, and yap1 mutant strains grown to exponential or stationary phases in YEPD media. Blots were analyzed with antibodies specific for Trx1 or Trx2 as indicated.

Figure 3.

Role of Trx1-2, Tsa1, and the UPR in DTT tolerance. (A) Overexpression of TSA1 increases the resistance of thioredoxin mutants to DTT, whereas overexpression of thioredoxins does not affect the tsa1 mutant. The wild-type, trx1 trx2 and tsa1 mutant strains containing empty vector (V), mcTRX1, mcTRX2, or mcTSA1, as indicated, were grown into stationary phase and adjusted to an A₆₀₀ of 1.0, 0.5, 0.25, or 0.125 before spotting onto SD plates containing 2-4 mM DTT. (B) The active site cysteine residues of Tsa1 are required for resistance to DTT. The wild-type and tsa1 mutant strains containing empty vector (V), pTSA1 expressing wild-type Tsa1 or pTSA1::CCS expressing the active-site mutant of Tsa1p, in which cysteine 47 and 170 have been replaced by serines, were grown into stationary phase and adjusted to an A₆₀₀ of 1.0 before spotting onto SGal plates containing 1 mM H₂O₂ or 8 mM DTT. (C) Overexpression of HAC1 does not rescue the DTT sensitivity of a tsa1 mutant. The wild-type, tsa1, and hac1 mutant strains containing empty vector (v), mcHAC1, or mcGAL4(AD)-HAC1 were grown into stationary phase and adjusted to an A₆₀₀ of 1.0 before spotting onto SD plates containing 2 mM DTT. (D) Overexpression of ERO1 does not rescue the DTT sensitivity of a tsa1 mutant. The wild-type and tsa1 mutant strains containing empty vector (V) or mcERO1 were grown into stationary phase and adjusted to an A₆₀₀ of 1.0 before spotting onto SD plates containing 4 mM DTT. Plates were incubated at 30°C for 3 d before scoring growth.

Figure 4.

DTT toxicity is not caused by the formation of reactive oxygen species. (A) DTT treatment lowers the cellular levels of oxidized GSSG. The wild-type and thioredoxin mutant (trx1 trx2) were grown to exponential phase (A₆₀₀ = 1.0) in YEPD media and treated with DTT for 1 h. GSSG concentrations shown are the means of at least three independent determinations and are given in nanomoles per milliliter per A₆₀₀. (B) DTT treatment lowers intracellular ROS levels. ROS were measured in the wild-type and tsa1 mutant strain grown in SD media using the cell-permeable fluorogenic probe DCFH-DA after treatment with 4 mM DTT for 15 min. Data are means ± SD from three independent experiments. (C) Anaerobic growth conditions do not prevent DTT toxicity. The wild-type and thioredoxin mutant strains (trx1 trx2) were spotted onto YEPD plates containing DTT or diamide (DIA) and grown under aerobic or anaerobic conditions.

Figure 5.

DTT stress induces protein aggregation. Induction of DTT resistance by oxidative stress. Wild-type (A) and tsa1 (B) mutant cells grown in YEPD media were pretreated with 50 μM H₂ O₂ for 1 h before growth in the presence of 2 or 4 mM DTT. Growth was monitored by absorbance at 600 nm. (C) Aggregation of ribosomal proteins in the tsa1 mutant and in response to DTT. Insoluble proteins were extracted from the wild-type and tsa1 mutant strains grown in YEPD media after treatment with 16 DTT or 4 mM H₂ O₂ for 1 h. Proteins were separated by SDS-PAGE and detected by silver staining. The indicated proteins were identified via MALDI-TOF mass spectrometry.

Figure 6.

Thioredoxin system mutants contain elevated levels of aggregated ribosomal proteins. (A) Analysis of insoluble proteins from the wild-type, trx1 trx2, trr1, and tsa1 mutant strains as for Figure 5B. (B) Western blot analysis of Rps3 in the aggregated protein fraction and in a total cell extract from the wild-type, trx1 trx2, trr1, and tsa1 mutant strains.

Figure 7.

Reductive stress inhibits protein synthesis. (A) Cultures of the wild-type and tsa1 mutant strain were grown to exponential phase in minimal SD media. Aliquots of cell culture were used to measure the rate of protein synthesis by pulse labeling with [³⁵S]cysteine/methionine for 5 min. Data are shown for untreated cultures (100%) and after treatment with DTT for 1 h. (B) DTT treatment specifically inhibits translation initiation. Polyribosome traces are shown from the wild-type and tsa1 mutant strains. Yeast were grown in SD medium and treated with the indicated concentrations of DTT for 1 h. Polyribosomes were analyzed as described in Materials and Methods. The peaks that contain the small ribosomal subunit (40S), the large ribosomal subunit (60S), and both subunits (80S) are indicated by arrows. The polysome peaks generated by 2, 3, 4, 5, etc., 80S ribosomes on a single mRNA are bracketed. Asterisks indicate an accumulation of halfmer polysomes.