Mapping the changes in protein expression levels provides insight on how S. cerevisiae adapts to a conventional stress condi tion resulting in activation of Yap1p. Moreover, we were able to elucidate if gene expression in the glycolytic and pyruvate ethanol pathways are primarily regulated at the level of the proteome or of the transcriptome. Import antly, studies of Yap1p using different experimental con ditions may help to further improve our understanding of its effect. Identification of the potential Yap1p targeted proteins and their mapping into cellular pro cesses not only give a global overview of the ubiquitous cellular changes elicited by Yap1p, but also provide the framework for understanding Inhibitors,Modulators,Libraries the mechanisms behind Yap1p regulated protective response in yeast.
Methods Transformants and preparation of inoculums All Inhibitors,Modulators,Libraries yeast transformants that were used in this study were previously constructed and stored in our laboratory. Yeast transformants designated Y and C were streaked on SC Ura agar plates, which were then incubated Batimastat at 30 C for 72 h. Inoculum cultures of the two S. cerevisiae transformants were prepared in 500 ml shake flasks with 140 ml of SC Ura medium. The flasks were inoculated with cells from the agar plates and incubated for approximately 17 h at 30 C with agitation. The cells were Inhibitors,Modulators,Libraries harvested in the exponential growth phase by centrifugation at 1,200 �� g for 10 min at 4 C. The cells were then resuspended in a suitable amount of sterile H2O to yield an inoculum of 0. 1 g l in all bioreactor vessels.
Yeast fermentation in multi bioreactor The cultivation of the two transformants Y and C was Inhibitors,Modulators,Libraries carried out with a multi bioreactor system. Four 350 ml bio reactor vessels equipped with condensers, FermProbe pH electrodes and OxyProbe polaro graphic dissolved oxygen sensors were sterilized through autoclavation and filled with 250 ml modified SC Ura medium. The composition of the medium was, 40 g l glucose, 13. 4 g l yeast nitrogen base without amino acids, 10% amino acid supplement solution �� 10 exclud ing uracil, 0. 1% of an ergosterol Tween 80 mixture, and 8 drops of antifoam. 12 The pH electrodes and the pO2 electrodes were calibrated prior to start up. Two ml of inoculum were added to each bioreactor vessel to an ini tial biomass concentration of 0. 1 g l. Throughout the fermentation, the temperature was kept at 30 C, the stirring was kept at 300 rpm, and the pH was kept at 5.
5 by automatic addition of 0. 5 M NaOH. Nitrogen gas was used to maintain anaerobic conditions. The fermentation was discontinued after seven hours when the cells were in the exponential growth phase and had reached a cell density of 1 g l. The yeast cells were harvested by centrifugation at 3,000 �� g for 5 min at 4 C, and stored at ?80 C before protein extraction. Protein extraction and purification Yeast protein extracts were prepared for analysis with 2 DE using a modified approach of Kolkman et al.