Fig. 6.
The uncoupling mutation in Gef1 decreases the capacity of the cytosol to maintain a reduced EGSH upon oxidative challenge. (A) Staining of Δgef1 strain expressing the indicated constructs with monochlorobimane (MCB) after growth in minimal SC medium lacking uracil and leucine and 7.5 μM BPS where indicated. The accumulation of MCB-GS conjugates was determined spectrophotometrically (n=3, error bars depict s.e.). (B) EGSH as a function of time in cells expressing the indicated construct and the cytosolic Grx1-roGFP2 probe. Cells were grown in minimal SC medium lacking uracil and leucine. At the time indicated, 5 mM H2O2 was added to the cell suspension. Lower panel shows response from cells grown in the presence of 4 μM BPS. (C) Area under the time course from the time of oxidative challenge (t=0) to 30 minutes after treatment calculated from data in B and corresponding data for the indicated constructs. n=3; error bars indicate s.e.