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. 2020 Aug 7;9:e54880. doi: 10.7554/eLife.54880

Figure 4. Quantitative control of intracellular pH reveals that, in the absence of translation, acidification is required for Ssa4 induction.

(A) Schematic of intracellular pH manipulation experiments. (B) Intracellular pH is accurately manipulated during stress. Intracellular pH distributions were measured to determine the efficacy of pH manipulation before (green), during (red), and after (purple) 42°C heat stress. Dashed lines indicate buffer pH, and the black distribution shows unmanipulated cells for comparison. (C) Manipulation of intracellular pH with ionophore reproduces the acidification-dependent induction of Ssa4. Compare to Figure 2B, right hand side. (D) Fold change in Ssa4 expression following stress at different intracellular pHs and recovery in acidic media. Points represent the median of individual measurements; at least three biological replicates were performed for each condition (see Materials and methods). Lines are sigmoid fits (see Materials and methods for fitting details). (E) pH dependence of the induction delay; points are the midpoint of the sigmoidal fits in D. (F) Dependence of the stress response on media pH, followed by recovery in acidic media, recapitulates the pH dependence of the stress response when ionophore treatment is used; compare to D.

Figure 4.

Figure 4—figure supplement 1. Western blot against Ssa4-mCherry and native Hsp26 after heat shock with and without acidification and stress protein production after acidification only.

Figure 4—figure supplement 1.

(A) Western blot and total protein gels for yeast carrying a the genomic copy of SSA4 tagged with a FLAG tag heat stressed with and without ionophore treatment, as described in Growth Conditions in the Materials and methods section. Samples were taken 1 hr after stress. (B) Induction of Ssa4 during recovery from normal (red) or pH-manipulated (gray, pH 6.8) stress. Thin curves are individual experiments and thick curves are smoothed conditional means (see Materials and methods for details). The red curve is the same data from Figure 1D for comparison. Although pH manipulation causes a delay in Ssa4 production, it does not affect the ultimate level of induction. (C) Growth rate difference in cells treated with ionophore for 35 min at room temperature followed by return to ambient growth conditions. Competitor was untreated cells. The values cluster around zero, indicating little to no loss of fitness due to ionophore treatment. Bottom dashed line shows theoretical minimum of the growth rate difference, which would result if cells completely arrested growth. These data are the same as those in Figure 6B, light-colored points. (D) Comparison of Ssa4-mCherry induction in ionophore treated cells that were either heat stressed (left) or held at room temperature (right). Acidification artificially induced by ionophore treatment does not cause appreciable accumulation of stress protein.