Starvation induces moderate intracellular ROS production, which is required for secretion of antioxidant enzymes and Acb1. (A) Yap1-GFP–expressing cells were grown to mid-logarithmic phase and incubated in the presence or absence of the indicated amount of H₂O₂ for 20 min before visualization by epifluorescence microscopy. Scale bar = 2 μm. (B) Yap1-GFP–expressing cells were grown to mid-logarithmic phase (growth), washed twice, and cultured in 2% potassium acetate for 2.5 h (starvation). Yap1-GFP was visualized by epifluorescence microscopy. DIC, differential interference contrast. Scale bar = 2 μm. (C) For detection of total protein carbonylation, wild-type cells were grown to mid-logarithmic phase (growth), washed twice, and cultured in 2% potassium acetate for 2.5 h (starvation). Growing cells were also subjected to 1 mM H₂O₂ treatment for 1 h as a positive control. Subsequently, cells were lysed and treated with streptomycin sulfate to remove nucleic acids, and protein carbonyl groups were labeled with FTC for 2 h in the dark. Proteins were precipitated with TCA, unbound FTC was removed, and protein pellets were resuspended and separated by SDS-PAGE. Fluorescence was visualized with the Typhoon scanner, while total protein was visualized separately by silver staining. Molecular weight (MW) marker in kiloDaltons (kDa). (D) Wild-type cells were grown to low-logarithmic phase and either treated with 10 mM NAC for 2 h (+) or not (−). Subsequently, equal cell numbers were washed twice and incubated in 2% potassium acetate for 2.5 h. Cell wall proteins were extracted and precipitated with TCA (secreted). Lysates and secreted proteins were analyzed by Western blot. The loading of the secreted fraction is equivalent to 145× that of the lysate. The ratio of secreted/lysate for each cargo was determined, and +NAC was normalized to −NAC in each experiment. Statistical analyses were performed for each cargo and are represented as fold change upon NAC treatment (paired Student’s t test). Acb1 = 0.32 ± 0.02, P = 0.0335; SOD1 = 0.27 ± 0.05, P = 0.032; Ahp1 = 0.26 ± 0.03, P = 0.0004; Trx1 = 0.17 ± 0.05, P = 0.0597; Trx2 = 0.13 ± 0.04, P = 0.002. Error bars indicate SEM, n = 3. Cof1 is nonsecreted, cytoplasmic protein used to monitor for cell lysis, while Bgl2 is a conventionally secreted cell wall protein. (E) For growth samples, wild-type cells were grown to mid-logarithmic phase and treated (+) or not (−) with 0.1 mM H₂O₂ for 1 h. For starvation (starv.) samples, wild-type cells were grown to mid-logarithmic phase, and equal cell numbers were washed twice and incubated in 2% potassium acetate for 2.5 h in the absence (−) or presence (+) of 0.1 mM H₂O₂. Subsequently, cells and samples were processed as in (A) to analyze secreted versus lysate pools of the indicated cargo proteins. The same statistical analyses were performed to compare the fold change upon H₂O₂ treatment in the growth or starvation conditions. The observed changes were minor and not found to be statistically significant. Error bars indicate SEM, n = 4. Cof1 is a nonsecreted, cytoplasmic protein used to monitor for cell lysis, while Bgl2 is a conventionally secreted cell wall protein. n.s., not significant.