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

Abstract

Abstract. 31p NMR spectra were obtained from suspensions of Candida utilis, Saccharomyces cerevisiae and Zygosaccharomyces 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 of the polyphosphate observed by 3 ~p NMR. This suggested that the inorganic phosphate fraction at the lower pH and the polyphosphates originated from the same intracellular compartment, most probably the vacuole. In this vacuolar compartment, pH is sensitive to metabolic conditions. 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 inorganic phosphate across the vacuolar membrane was observed 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 exhaustion.

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

“…Cells were resuspended in buffer D to a concentration of 50-65 g dry weight/l and kept on ice until used (within 60-300 rain). 3tp-NMR spectra, with a time resolution of 30 s, were obtained on a Bruker AM 360 WB NMR-spectrometer essentially as described by Nicolay et al [15]. An NMR tube of 20 nun was used, and pure oxygen was bubbled through the suspension at a flow rate of 370 ml/min as described by Gillies et al [16].…”

A 31P NMR study of the internal pH of yeast peroxisomes

“…31p nuclear magnetic resonance 31p NMR spectra were obtained at 145.8 MHz as described previously (Nicolay et al 1982(Nicolay et al , 1983. Usually, 500-2000 scans were averaged for a total time of 1.7-6.8 min.…”

“…The pH of the cytosol and peroxisomes was determined from the chemical shifts of P~ in the specific compartments according to procedures described elsewhere (Nicolay et al 1982(Nicolay et al , 1983.…”

“…This method, which relies on the pH-dependency of the chemical shifts of titratable intracellular phosphates, has been widely used to measure intracellular Offprint requests to: W. Harder Abbreviations. CCCP, Carbonyl cyanide m-chlorophenylhydrazon; DCCD, N,N'-dicyclohexylcarbodiimide pH in micro-organisms, including yeasts (Nicolay et al 1982;Hofer et al 1985). Recently, it has been demonstrated that 31p NMR enables the simultaneous measurement of cytosolic and vacuolar pH from the (considerably different) chemical shifts of the 31p NMR peaks originating from cytosolic and vacuolar Pi, respectively, in yeast grown to stationary phase on glucose (Nicolay et al 1982(Nicolay et al , 1983.…”

“…CCCP, Carbonyl cyanide m-chlorophenylhydrazon; DCCD, N,N'-dicyclohexylcarbodiimide pH in micro-organisms, including yeasts (Nicolay et al 1982;Hofer et al 1985). Recently, it has been demonstrated that 31p NMR enables the simultaneous measurement of cytosolic and vacuolar pH from the (considerably different) chemical shifts of the 31p NMR peaks originating from cytosolic and vacuolar Pi, respectively, in yeast grown to stationary phase on glucose (Nicolay et al 1982(Nicolay et al , 1983. Separate peaks for the Pi-pools in these two intracellular compartments can be seen in 31p spectra of these cells as a consequence of the fact that: (i) a considerable fraction of the cell volume is occupied by the vacuole; (ii) its Pi concentration is relatively high; (iii) a substantial pH gradient (0.8-1.5 units) exists across the vacuolar membrane.…”