Net phosphate transport in phosphate-starved Candida utilis: relationships with pH and K+.

Phosphate transport was studied in phosphate-starved Candida utilis using 31P-NMR and in situ pH and K(+)-specific electrodes. A transient efflux of K+ was associated with the initial net influx of orthophosphate across the plasma membrane and decrease of both the plasma membrane pH gradient and the cytosol pH. Recovery of cytosol pH, and the plasma membrane pH gradient after phosphate uptake, was slow when extracellular K+ was depleted, but rapid when K+ was added to the suspension either with orthophosphate or after phosphate uptake. Net phosphate transport into the vacuole occurs against its concentration gradient and is associated with an increase of the tonoplast pH gradient. It is proposed that transport of orthophosphate into the vacuole is mediated by an electrical uniport and driven by the tonoplast membrane potential.

A 31P-NMR study of phosphate transport and compartmentation in Candida utilis.

The recovery of Candida utilis from phosphate starvation was studied using 31P-NMR. The phosphate analogue methylphosphonate was found to be a useful indicator of cytosol pH. Added orthophosphate was rapidly accumulated by the cells and stored mainly In a stable pool of polyphosphate of mean chain-length at least 200 units. Observed pH changes in the medium and cytosol during uptake of orthophosphate and methylphosphonate are consistent with the transport of these compounds across the plasma membrane by a proton/phosphate symport. However, transport of phosphate across the vacuole membrane occurs by a mechanism for which methylphosphonate is not a substrate. In the cytosol pH changes are strongly correlated with changes in orthophosphate concentration, however, this is not the case in the vacuole.

Cloning and sequencing of four structural genes for the Na(+)-translocating NADH-ubiquinone oxidoreductase of Vibrio alginolyticus.

Oligonucleotide probes based on the N-terminal amino acid sequences of the NqrA and NqrC subunits were used to clone genes for the Na(+)-dependent NADH-ubiquinone oxidoreductase complex from Vibrio alginolyticus. Four consecutive ORFs were identified encoding subunit proteins of 48.6, 46.8, 27.7 and 22.6 kDa, respectively (NqrA-D). A further ORF, showing 71% homology to the BolA protein of Escherichia coli, was located upstream. From sequence comparisons, we conclude that the Na(+)-dependent NADH-ubiquinone oxidoreductase complex of V. alginolyticus is clearly distinct from the corresponding H(+)-dependent enzymes of both prokaryotes and eukaryotes.

A device for aeration and mixing of cell and organelle suspensions during nuclear magnetic resonance studies.

A device is described which maintains homogeneous aerobic or anaerobic cell and organelle suspensions within an NMR sample tube. Line broadening due to magnetic field inhomogeneity is reduced by elimination of gas bubbles from the area of the probe receiver coils. The linewidth of the extracellular orthophosphate resonance of a yeast suspension in 31P NMR was 0.21 ppm compared with 0.4-0.7 ppm in conventionally aerated suspensions. Recirculation of the sample results in complete mixing within 90 s of addition of aliquots of acid or alkali. The maximum rate of oxygen transfer from the gaseous to the liquid phase was approximately 600 microM min-1 when aerated with 95% oxygen/5% carbon dioxide. A 60% wet weight suspension of yeast cells was recirculated for 20 h without settling of cells occurring. A method for estimating oxygen transfer rate is described.