Figure 3. Experimental and simulated DTT titration time courses in wild type, hac1Δ, and Ire1^bipless cells.

(A) Wild type cells expressing the GFP splicing reporter (SR) were treated with doses of DTT spanning the active concentration range, sampled over time, and their fluorescence was measured by flow cytometry. The SR, like the TR, reached dose-dependent plateaus due to the >8 h half life of GFP. (B) hac1Δ cells expressing the SR were treated as above. hac1Δ cells were hypersensitive to DTT and saturate the reporter at all experimental doses. (C) Ire1^bipless cells expressing the SR were treated as above and showed increased sensitivity to DTT compared to the wild type, responding to 0.66 mM DTT and saturating at 1.5 mM DTT. (D) Simulations of the “wild type” model. The architecture of the model, described in the text and depicted in Figure 4A, includes BiP binding to Ire1 and negative feedback. When the model includes a cooperative Ire1 deactivation term (described in text), it recapitulated the wild type DTT titration time course. (E) Simulations of the “hac1Δ” in which the negative feedback terms have been removed captured the hypersensitivity observed experimentally. (F) Simulations of the “Ire1^bipless” model in which the Ire1/BiP interaction terms have been removed revealed the increased DTT sensitivity compared to the wild type.