INFLUENCE OF MONOVALENT CATIONS ON YEAST CYTOPLASMIC AND VACUOLAR PH

The effects of monovalent cations on the internal pH of yeast were stu died. Our former procedure was modified, inducing maximal alkalinizati on of the cells with 100 mM-NH4OH instead of Tris base. The pH values were lower than reported before (Pena et al., J. Bacteriol. 1995 177, 1017-1022). With glucose as substrate, the internal cytoplasmic pH rea ched higher values when incubating at an external pH of 6.0, as compar ed to pH 4.0. Monovalent cations added approximately 5 min after gluco se produced a further increase in the internal pH, which was higher at a previous incubation pH of 4.0 than that observed at pH 6.0. The sel ectivity of the changes followed a similar order to that of the transp ort system for monovalent cations. When incubating cells with glucose for more than 30 min, the initial changes of the internal pH appeared to be regulated by the cell. However, under the fluorescence microscop e, it was observed that pyranine, which was confined to the cytoplasm during the first 15 min, was progressively concentrated in the vacuole . By studying the fluorescence changes of cells electroporated and the n incubated with glucose or glucose plus potassium, we could follow th e internal pH of this organelle, obtaining values within the range rep orted by other authors. Also, in cells preincubated with glucose for 6 0 min, and electroporated afterwards, the fluorescence of pyranine, wh ich only entered the cytoplasm, allowed us to measure the pH of this c ompartment, showing that it was more alkaline than the vacuole. Moreov er, the cytoplasmic pH increased upon addition of glucose or potassium . The vacuolar pH, on the other hand, increased upon addition of potas sium after glucose, but decreased upon addition of glucose. In additio n, incubation of the cells with glucose with or without pyranine produ ced vesiculation of the vacuole. (C) 1998 John Wiles & Sons, Ltd.