Phosphorus-31 nuclear magnetic resonance studies of intracellular pH, phosphate compartmentation and phosphate transport in yeasts

Abstract. 31p NMR spectra were obtained from suspensions of Candida utilis, Saccharomyces cerevisiae and Zygo-saccharomyces bailii grown aerobically on glucose. Direct introduction of substrate into the cell suspension, without interruption of the measurements, revealed rapid changes in pH upon addition of the energy source. All 3 ~p NMR spectra of the yeasts studied indicated the presence of two major intracellular inorganic phosphate pools at different pH environments. The pool at the higher pH was assigned to cytoplasmic phosphate from its response to glucose addition and iodoacetate inhibition of glycolysis. After addition of substrate the pH in the compartment containing the second phosphate pool decreased. A parallel response was observed for a significant fraction of the terminal and penultimate phosphates ofthe polyphosphate observed by 3 ~p NMR. This suggested that the inorganic phosphate fraction at the lower pH and the polyphosphates originated from the same in-tracellular compartment, most probably the vacuole. In this vacuolar compartment, pH is sensitive to metabolic on-ditions. In the presence of energy source a pH gradient as large as 0.8 to 1.5 units could be generated across the vacuolar membrane. Under certain conditions net transport of in-organic phosphate across the vacuolar membrane was observ-ed during glycolysis: to the cytoplasm when the cytoplasmic phosphate concentration had become very low due to sugar phosphorylation, and into the vacuole when the former concentration had become high again after glucose ex-haustion