Determination of the intracellular pH (pHi) of growing cells of Saccharomyces cerevisiae: the effect of reduced-expression of the membrane H+-ATPase

Abstract This report describes the use of the fluorescent probe, 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), to determine pHi in growing cells of the spoilage yeast Saccharomyces cerevisiae. The technique is based on the pH-dependent intracellular fluorescence of the probe in the cytosol of the cell. The major advantage of this probe over other derivatives is the presence of a succinimidyl group which binds to aliphatic amines in cellular proteins. This allows for the determination of pHi under conditions that either permeabilise the cell membrane, or increase active extrusion, and would otherwise result in loss of the intracellular probe to the external medium. Optimal loading of the probe into cells only occurred after exposure to a mild stress regime of 37°C for 24 h in 100 mM citric/phosphate buffer, pH 4.0. This loading regime was selected because there was no appreciable effect on cell viability or subsequent growth rate in batch culture under optimal conditions. The pHi of yeast cells was measured after incubation in citric/phosphate buffer and gave values comparable to those measured by other techniques in the literature. Also, we were able to detect rapid changes in pHi induced by the addition of a known disruptor of pHi homeostasis in yeast, the weak-acid food preservative, sorbic acid. Finally, successful determination of pHi was made in growing cells of an isogenic parent (PMA1) and a mutant with reduced-expression of the membrane H+-ATPase, pma1-205. As might be expected, the pHi in the mutant strain was reduced compared to the parent. Comparison of growth and pHi in growing cells of either strain revealed no correlation between pHi and exit from lag phase. These results are discussed in relation to previous findings.