for 18 h at 30 °C in APG growth medium, absorbance readings were taken at 600 nm to measure growth, and cultures were then incubated with 50µM BCECF [2´,7´-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein]-acetoxymethyl ester at 30 °C for 20 min, washed and suspended in APG medium. Normalized background-subtracted fluorescence emission values at 485 nm were calculated [NI485 (normalized intensity at 485 nm)] using fluorescence intensity and absorbance readings taken at 485 and 600 nm respectively. A calibration curve of the ratio of fluorescence intensity values versus pH was obtained for each yeast strain at the end of every experiment and vacuolar pH values were determined by incubating yeast cultures in 200 µl of experimental medium, titrated to five different pH values within the range of 4.0 to 8.0 using 1M NaOH33,38,61.CPY secretion: Yeast cultures were seeded in SC media to a starting OD600 of 0.05 ml−1 and grown at 30 °C for 20 h. 1.5 D600 units of cells were centrifuged for 2 min and 600 µl of the supernatants were applied to Immobilon (Millipore) membranes using a slot-blot apparatus (Schleicher & Schuell Manifold II). After drying the membrane overnight, CPY was detected by immunoblotting using monoclonal anti-CPY antibody (Molecular Probes; 1:1000 dilution).
