Influence of monovalent cations on yeast cytoplasmic and vacuolar pH

Abstract

The effects of monovalent cations on the internal pH of yeast were studied. Our former procedure was modified, inducing maximal alkalinization of the cells with 100 mM‐NH4OH instead of Tris base. The pH values were lower than reported before (Peña et al., J. Bacteriol. 1995 177, 1017–1022). With glucose as substrate, the internal cytoplasmic pH reached higher values when incubating at an external pH of 6·0, as compared to pH 4·0. Monovalent cations added approximately 5 min after glucose 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 selectivity of the changes followed a similar order to that of the transport 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 microscope, 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 then incubated with glucose or glucose plus potassium, we could follow the internal pH of this organelle, obtaining values within the range reported by other authors. Also, in cells preincubated with glucose for 60 min, and electroporated afterwards, the fluorescence of pyranine, which only entered the cytoplasm, allowed us to measure the pH of this compartment, showing that it was more alkaline than the vacuole. Moreover, the cytoplasmic pH increased upon addition of glucose or potassium. The vacuolar pH, on the other hand, increased upon addition of potassium after glucose, but decreased upon addition of glucose. In addition, incubation of the cells with glucose with or without pyranine produced vesiculation of the vacuole. © 1998 John Wiley & Sons, Ltd.

Involvement of Nha1 antiporter in regulation of intracellular pH inSaccharomyces cerevisiae

“…Electroporation of the fluorescent dye pyranine into the cells was performed as previously reported [2]. For cells grown in YPD medium, the pulse voltage was set at 1500 V, and for YNB‐grown cells at 2000 V. Fluorescence measurements were performed in a DMX‐100 SLM spectrofluorometer and the pH values were calculated as described previously [2, 3] using 100 mM NH 4 OH and 100 mM propionic acid to achieve maximum and minimum fluorescence values, respectively [3]. Fluorescence measurements in plasma membrane vesicles were carried out in 10 mM HEPES‐KOH buffer, pH 7.0 according to [11].…”

Effects of amiodarone on K+, internal pH and Ca2+homeostasis inSaccharomyces cerevisiae

“…(2) The effect of the addition of K 1 was also tested; it was found that, as already known (Calahorra et al, 1998), the addition of K 1 in the absence of the drug produced a marked increase in internal pH, but this increase, after the addition of amiodarone, became smaller as its concentration increased.…”

“…The internal pH was measured by the fluorescence changes of pyranine introduced to the cells by electroporation as described previously (Calahorra et al, 1998). The cells (25 mg wet weight) with the internalized pyranine were incubated in 10 mM MES-TEA, pH 6.0, and 10 mM glucose, in a final volume of 2.0 mL.…”

In situ study of K+ transport into the vacuole of Saccharomyces cerevisiae

“…Potassium ion is a fundamental cation for Saccharomyces cerevisiae ; it stimulates fermentation (Rothstein and Demis, 1953) and respiration (Peña et al , 1969; Peña, 1975). It has also been reported that K + is partly accumulated with bicarbonate as a counter‐ion (López et al , 1999) and it participates in the modulation of the internal pH in yeast (Calahorra et al , 1998; Ramírez et al , 1996). The role of the different K + transporters in yeast is being studied in order to get a clearer scheme of its dynamics (Wadskog and Adler, 2003) as well as in the regulation of the internal pH of the cell.…”