Activation of the MAPK module downstream of Pkc1 suppresses all the described phenotypic defects of the grx3 grx4 mutant, with the exception of cellular accumulation of iron and ROS. Cultures of wild-type cells and cells of the grx3 grx4 mutant transformed with a plasmid containing SLT2 or with the empty plasmid were grown as described in the legend to Fig. 2 to determine vacuole morphology (A) and function by means of the study of cell survival in sporulation medium (B) and in SD medium at low or high pH (C). (D) Aliquots from each culture were treated with 1 mM hydrogen peroxide for the indicated times and processed for actin staining. Five hundred cells were counted to determine the percentage of depolarized budded cells. Histograms represent the results of an average of three independent experiments. (E) Cultures of the same strains growing exponentially were serially diluted, and 1,000 cells were plated on the indicated media containing calcofluor white (20 μg/ml), Congo red (6.5 μg/ml), caffeine (3 mM), or H2O2 (1 mM) or were heat shocked at 38°C. All the cultures were grown at 30°C for 3 days. These experiments were performed in triplicate, as described in Materials and Methods. Error bars represent standard deviations. (F) Cultures of wild-type, wild-type(pSlt2), wild-type(pBck1-20), grx3 grx4 mutant, grx3 grx4(pSlt2) mutant, and grx3 grx4(pBck1-20) mutant cells were grown exponentially. Aliquots (1 ml) from each culture were collected to measure ROS levels using the dihydroethidine method, as described in Materials and Methods. The histograms represent arbitrary units of fluorescence, calculated as the slope of the graph registered, and represent the results of an average of three independent experiments.