Figure 11.

ERAD-T is not broadly stress-sensitive. A, cycloheximide chase analysis of WT yeast harboring an empty vector (Vec) or expressing Deg1*-Sec62 cultured in inositol-rich medium in the presence or absence of 6 mm DTT for 1 h or shifted to inositol-free medium for 5 h. Cells also possessed a plasmid encoding GFP (driven by the UPRE). DTT concentration and inositol abundance were maintained during incubation with cycloheximide. B, cycloheximide chase analysis of WT yeast expressing Deg1-Sec62 cultured at 30 °C in the presence or absence of 6 mm DTT or shifted to 42 °C in the absence of 6 mm DTT for 1 h. Temperatures were maintained during incubation with cycloheximide. C, cycloheximide chase analysis of WT yeast harboring an empty vector or expressing Deg1*-Sec62 in the presence or absence of 0.4 mm hydrogen peroxide (H₂O₂) for 1h. H₂O₂ concentrations were maintained during incubation with cycloheximide. D, in parallel to experiment depicted in C, mid-exponential phase yeast expressing oxidant-responsive Rtc3-GFP were analyzed by flow cytometry following incubation in the presence of 0.4 mm H₂O₂ for 1 h. The mean fluorescence intensity for each culture was normalized to the average mean fluorescence intensity of three repeats of untreated cells. Mean fluorescence intensity ± standard error of the mean is presented for three repeats of 10,000 cells for each condition. A–C, Pgk1 served as a loading control. A and C, percentage of Deg1*-Sec62 remaining at each time point (normalized to Pgk1) is indicated below the images. Experiments depicted were performed three times.