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. Baceteriol. 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.

Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast

“…A similar pattern of responses was seen at extracellular pH 7.5, except that both the cytosolic and vacuolar pH were higher than at pH 5 in the absence of glucose, and the changes with glucose and KCl addition were less pronounced. As in previous studies (26,52), we find that the cytosolic pH of cells supplied with glucose is relatively insensitive to extracellular pH, ranging from 6.7 at pH ext 5 to pH 7.1 at pH ext ϭ 7.5, and is even more constant if KCl is present (cytosolic pH 7.0 -7.1). Vacuolar pH appears to be more dependent on extracellular conditions (20,30) but is generally higher in yeast vacuoles than in mammalian lysosomes.…”

“…We introduced the fluorescent dye BCECF-AM, which measures vacuolar pH in yeast (11,30), or yeast pHluorin, a green fluorescent protein analog that measures cytosolic pH (31), into wild-type and vma mutant cells and studied the dynamic adjustments of cytosolic and vacuolar pH in response to glucose and KCl addition. As described above, both glucose and potassium ion are strongly implicated in cytosolic and vacuolar pH homeostasis (25,26,32,48). Fig.…”

“…The addition of KCl increased the vacuolar pH of the wild-type cells from 6.5 to 6.7 but had relatively little effect on the vma mutants. In wild-type cells cytosolic pH was somewhat higher at extracellular pH 7.5 than at extracellular pH 5 both before and after glucose addition, again consistent with previous measurements (26). Cytosolic pH in the vma mutants was higher at extracellular pH 7.5 than at extracellular pH 5 and showed less difference from wild type.…”

“…However, it should be noted that under similar conditions of brief (20 min) glucose deprivation in YEP, we have shown that there is a relatively modest decrease in cellular ATP levels during the deprivation and no overall increase in total ATP levels in the first 5 min after glucose readdition (19), so regulatory effects of glucose on the two pumps may be particularly important. The additional increase in cytosolic pH with KCl addition can be explained predominantly by the transport of K ϩ into the cell through the Trk1/Trk2 K ϩ -transporters, which balances the membrane potential generated by Pma1p-mediated H ϩ export (13,22,26). The data in Fig.…”

“…Potassium ion is primarily responsible for balancing the plasma membrane potential in yeast (24). Transport of K ϩ ion into the cell results in a depolarization of the plasma membrane, allowing stimulation of Pma1p (22) and consequent cytosolic alkalinization (25,26), and pH-responsive regulation of the Trk1p potassium transporter via phosphorylation provides additional means of activating Pma1p when cytosolic pH drops. The exact mechanisms for balancing the membrane potential at the vacuolar membrane are less clear; many anion channels and other transporters are present in the vacuolar membrane that could contribute to the final potential, but their individual contributions to membrane potential are not well defined.…”