FIG. 2.

Over-expression of proteins in Δtrr1 and in Δtrx1 cells is partially dependent on the transcription factor Pap1. (A) ICAT data representation: cysteine oxidation versus protein expression in the three ICAT studied pairs. Cysteine oxidation from four experimental conditions was analyzed pairwise using the ICAT method depicted in Figure 1A. In each panel, the Log2 ratio of cysteine oxidation is plotted in a scatter diagram versus its Log2 protein ratio (72%–78% of the cysteine-containing peptides displayed protein values by dimethyl labeling). Left panel: 972 treated with 0.2 mM H₂O₂ for 30 s versus 972 untreated (WT H₂O₂/WT unt.). Center panel: NG25 untreated versus 972 untreated (Δtrr1 unt./WT unt.). Right panel: MJ15 untreated versus 972 untreated (Δtrx1 unt./WT unt.). Green circles represent peptides with increased cysteine oxidation and orange circles represent peptides from over expressed proteins. (B–D) The activity of the transcription factor Pap1 explains increased protein levels in some strain backgrounds. (B) In vivo oxidation of Pap1. Cultures of strains IC2 (WT), IC71 (Δtrr1), and MJ2 (Δtrx1) were treated (H) or not (−) with 0.2 mM H₂O₂ for 5 min. TCA extracts were obtained and analyzed by nonreducing electrophoresis. Reduced/inactive (red.) and oxidized/active (ox.) Pap1 forms are indicated with arrows. (C) Protein levels of some Pap1-dependent targets. Cultures of strains expressing or not tagged proteins were treated (H) or not (−) with 0.2 mM H₂O₂ for 5 min. TCA extracts were obtained, and specific Pap1-dependent proteins were analyzed from extracts of strains: 972, SG167 (Δtrr1) and MJ15 (Δtrx1) for Trx1 and Tpx1; MJ8 (trr1-HA WT), SG167 (Δtrr1), and SG202 (trr1-HA Δtrx1) for Trr1-HA; SB50 (srx1-HA WT), SB69 (srx1-HA Δtrr1), and SB68 (srx1-HA Δtrx1) for Srx1-HA; JF17 (ctt1-HA WT), SG200 (ctt1-HA Δtrr1), and SG198 (ctt1-HA Δtrx1) for Ctt1-HA; and SG18 (zwf1-HA WT), in SB32 (zwf1-HA Δtrr1), and in SG37 (zwf1-HA Δtrx1) for Zwf1-HA. Western blots were performed using polyclonal anti-Tpx1 or anti-Trx1 antibodies, or monoclonal anti-HA antibodies. (D) Transcriptional analysis of Pap1-dependent genes. RNA from strains 972 (WT), IC1 (Δpap1), IC71 (Δtrr1), and MJ2 (Δtrx1), untreated (−) or treated with 0.2 mM H₂O₂ for 15 min (H), was obtained and analyzed by Northern blot with probes for trr1, tpx1, srx1, ctt1, zwf1, caf5, p25, and SPCC663.08c. Ribosomal RNA (rRNAs) was used as a loading control.