Fig. 2. Determination of cytosolic (pH_cyt) and peroxisomal (pH_per) pH. Wild-type cells were transformed with plasmids containing the coding sequence of EYFP (A), EYFP(H148G) (B), EYFP-SKL (C) and EYFP(H148G)-SKL (D), and were grown in oleate-containing medium. Calibration pH curves were generated by determining the fluorescence of digitonin-permeabilized transformants incubated in buffers with pH values ranging from 4.2 to 9.2 The emission was determined by confocal microscopy and the median of the fluorescence of at least 100 different cells was calculated (see Materials and Methods). To determine the pH_cyt, the fluorescence was measured in at least 100 non-permeabilized cells expressing EYFP and EYFP(H148G) [green circles in (A) and (B)]. To determine the pH_per, the fluorescence was measured in at least 100 non-permeabilized cells expressing EYFP-SKL and EYFP(H148G)-SKL [green circles in (C) and (D)]. Each experiment was performed at least three times and the standard deviations from the mean values are indicated by error bars.