Elucidation of the cytoplasmic and vacuolar components in the inorganic phosphate region in the 31P NMR spectrum of yeast

Subcellular compartments, such as the vacuole in yeast, play important roles in cell metabolism and in cell response to external conditions. Concentrations of inorganic phosphate and pH values of the vacuole and cytoplasm were determined for anaerobic Saccharomyces cerevisiae cells based upon (31)P NMR spectroscopy. A new approach allows the determination of these values for the vacuole in cases when the resonance for inorganic phosphate in the cytoplasm overlaps with the resonance for inorganic phosphate in the vacuole. The intracellular inorganic phosphate resonance was first decomposed into two components by computer analysis. The assignments of the components were determined from in vivo correlations of P(i) chemical shift and the chemical shifts of the cytoplasmic sugar phosphates, and the pH dependency of the resonance of pyrophosphate and the terminal phosphate of poly-phosphate (PP(1)) which reside in the vacuole. An in vivo correlation relating PP(1) and P(i) (vac) chemical shifts was established from numerous evaluations of intracellular compositions for several strains of S. cerevisiae. This correlation will aid future analysis of (31)P NMR spectra of yeast and will extend NMR studies of compartmentation to cellular suspensions in phosphate-containing medium. Application of this method shows that both vacuolar and extracellular P(i) were phosphate reserves during glycolysis in anaerobic S. cerevisiae. Net transport of inorganic phosphate across the vacuolar membrane was not correlated with the pH gradient across the membrane.