Interaction of phosphate with monovalent cation uptake in yeast.

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb+ uptake shows saturation kinetics and the phosphate concentration at which half-maximal inhibition is observed is equal to the Km of phosphate for the sodium-independent phosphate uptake mechanism. The kinetic coefficients of Rb+ and TI+ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased. In the case of Na+ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na+ uptake via a sodium-phosphate cotransport mechanism. Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimethylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium uptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

Effect of a brewer's yeast extract on growth and glucose metabolism of cells in culture.

Brewer's yeast preparations influence glucose metabolism in vivo and in isolated tissues. We have studied the effect of a brewer's yeast extract on glucose metabolism and grwoth of rat hepatoma and human embryonic cells. Growth of the rat hepatoma cells was very much stimulated by the extract in a concentration-dependent manner. Glucose uptake was, on the other hand, appreciably inhibited, and lactate uptake completely abolished by the extract. Insulin stimulated cell growth and inhibited lactate uptake but did not affect the glucose level. Insulin and the extract had additive effects on growth and lactate uptake of the hepatoma cells. The inhibition by the brewer's yeast extract of glucose uptake was, however, antagonized by insulin. Niacin or Cr³⁺, which are suggested to be components of a “glucose tolerance factor” of brewer's yeast, did not affect growth or glucose and lactate uptake. The glucose uptake of the human embryonic cells was strongly inhibited by the brewer's yeast extract. Cell growth and lactate production were not influenced by the extract or by insulin; however, when both insulin and extract were present simultaneously, a slight stimulation of growth and inhibition of lactate production was observed. The results indicate that brewer's yeast can have appreciable direct effects on cells and that not all of these effects are “insulin-like”.     

Simultaneous utilization of galactose and glucose bySaccharomyces spp.

Summary The adaptation of yeast to galactose utilization allows the simultaneous uptake of galactose and glucose, during high cell density fermentation. Furthermore, yeast cells grown in galactose show higher rates of glucose uptake than those under derepressing conditions.     

Isolation and Characterization of Pichia heedii Mutants Defective in Xylose Uptake

To investigate the role of xylose uptake in xylose metabolism in yeasts, we isolated a series of mutated strains of the yeast Pichia heedii which are defective in xylose utilization. Four of these demonstrated defects in xylose uptake. Overlaps between the functional or regulatory mechanisms for glucose and xylose uptake may exist in this yeast since some of the mutants defective in xylose uptake were also defective in glucose transport. None of the mutants were defective in xylose reductase or xylitol dehydrogenase activities.     

Interaction of phosphate with monovalent cation uptake in yeast

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb⁺ uptake shows saturation kinetics and the phosphate concentration at which halfmaximal inhibition is observed is equal to the K_m of phosphate for the sodiumindependent phosphate uptake mechanism. The kinetic coefficients of Rb⁺ and Tl⁺ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased.In the case of Na⁺ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na⁺ uptake via a sodium-phosphate cotransport mechanism.Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimenthylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium eptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

Pitfalls in the measurement of membrane potential in yeast cells using tetraphenylphosphonium

The uptake of the lipophilic cation tetraphenylphosphonium (Ph₄P⁺) by Saccharomyces cerevisiae was measured using yeast grown on glucose and harvested either at the logarithmic or at the stationary phase of growth. When yeast was collected at the stationary phase, Ph₄P⁺ uptake proceeded steadily during several hours until an equilibrium was reached. When yeast was collected in the logarithmic phase of growth, a biphasic uptake was observed. The second phase of uptake began when the glucose of the incubation medium had been exhausted. From experiments in the presence of cycloheximide or chloramphenicol it is concluded that the second phase of Ph₄P⁺ uptake is dependent on the synthesis of some protein(s) repressed by glucose but unrelated with the existence of functional mitochondria. The addition of compounds which collapse the membrane potential provokes an efflux from the yeast cells of the Ph₄P⁺ accumulated both during the first phase and the second phase of uptake. It is concluded that accumulation of Ph₄P⁺ in yeast cells is a complex process and that Ph₄P⁺ cannot be used to give a quantitative measure of the yeast plasma membrane potential.     

Accumulation and cellular distribution of zinc by brewing yeast

This study focused on the interactions between yeast and zinc in relation to beer fermentations. Yeast accumulation of zinc from growth media, including malt wort, was found to be rapid following inoculation with a brewing strain of Saccharomyces carlsbergensis. In contrast, at the onset of the fermentation, the uptake of other divalent cations such as magnesium and calcium was not as pronounced compared with zinc. At the end of fermentation, both growth media and yeast cells became zinc-depleted, the latter due to dilution of zinc to daughter cells following growth and cell division. In addition, in brewing fermenters, the levels of intracellular zinc were much higher in suspended yeast cells compared with cells that sedimented in the yeast cone at the end of fermentation. This may result in impaired yeast performance in subsequent fermentations if yeast is recycled into low zinc media and if the sub-population is composed by zinc-depleted daughter cells. Cellular uptake of zinc was mediated by a metabolism-dependent mechanism as evidenced by impaired uptake following heat shock. Zinc was thereafter localised in the yeast cell vacuole. As industrial fermentation processes may occasionally be suppressed due to zinc deficiencies, the findings of this study are pertinent for several yeast-based industries, especially beer production.     

Evaluation of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose, a new fluorescent derivative of glucose, for viability assessment of yeast Candida albicans

A new fluorescent derivative of d-glucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), which had been previously developed for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeast Candida albicans. Lineweaver-Burk plots showed the uptake of 2-NBDG to be competitively inhibited by d-glucose and not by l-glucose, which suggested the involvement of the glucose transporting system of C. albicans in the uptake of 2-NBDG. A good correlation was obtained between the yeast viability, determined by the plate-count method, and the 2-NBDG uptake activity of yeast cells (correlation constant: r=0.97). This is expected to lead to the development of a new fluorescent probe for the determination of yeast cell viability.     

A method for expression cloning of transporter genes by screening yeast for uptake of radiolabelled substrate

A method has been developed for the cloning of plasma membrane transporters by screening yeast transformed with a cDNA library for the accumulation of radiolabelled substrate. The applicability of the method is demonstrated by cloning the amino acid permease AAP1. A yeast mutant defective in proline uptake was transformed with an Arabidopsis thaliana cDNA library and plated on medium supplemented with L-[U-(14)C]proline. Yeast colonies accumulating radiolabelled proline were identified by autoradiography. The plasmids of these colonies were reintroduced into the yeast mutant and restoration of proline uptake was confirmed by L-[U-(14)C]proline uptake measurements. Whereas cloning of transporters by functional complementation requires that the substrate taken up is metabolized by yeast to promote growth, the method described here can be used to isolate transporters of substrates which are not metabolized. The method has great potential for the isolation of transporters of various substrates such as secondary plant products.     

Biosynthesis of the Branched-Chain Amino Acids in Yeast: a Leucine-Binding Component and Regulation of Leucine Uptake

Use of an ion-exchange resin assay has shown that leucine is bound to a component of a dialyzed extract of yeast. Leucine binding may be related to in vivo uptake of the amino acid. A yeast strain with a 30-fold lower affinity for leucine uptake in vivo has a parallel reduction in affinity for in vitro leucine binding; the rate of leucine uptake in wild-type yeast can be increased four- to fivefold by growth on leucine as a sole nitrogen source. Under these conditions, the specific activity of the leucine-binding component also increases over threefold. Regulation of leucine uptake was studied by using wild-type strain 60615 and a mutant 60615/fl(2) with a constitutively elevated leucine uptake system. Leucine pool formation in the mutant was accompanied by an overshoot, leading to a loss of leucine from the pool. The phenomenon could be observed in the wild type under certain conditions. The mechanism of this process was examined. The leucine uptake system was found to be stable in the absence of protein synthesis. The rate of leucine uptake increased on reduction of the pool of amino acids, and in strain 60615/fl(2) the ability to overshoot was rapidly recovered on depletion of the leucine pool. The results suggest a control of leucine uptake by feedback inhibition, in which leucine or other amino acids, e.g., isoleucine, inhibit leucine uptake. The results do not exclude control by a rapidly activated-inactivated system.     

Short polyhistidine peptides penetrate effectively into Nicotiana tabacum-cultured cells and Saccharomyces cerevisiae cells

The polyhistidine peptides (PHPs) have been previously reported as novel cell-penetrating peptides and are efficiently internalized into mammal cells; however, penetration of PHPs into other cell types is unknown. In this study, the cellular uptake of PHPs in plant and yeast cells was found to be dependent on the number of histidines, and short PHPs (H6–H10 peptides) showed effective internalization. The H8 peptide showed the highest cell-penetrating capacity and localized to vacuoles in plant and yeast cells. Low-temperature conditions inhibited significantly the cellular uptake of short PHPs by both cells. However, net charge neutralization of PHPs also completely inhibited cellular uptake by plant cells, but not by yeast cells. These results indicate that short PHPs penetrate effectively into plant and yeast cells by similar mechanism with the exception of net charge dependency. The findings show the short PHPs are promising candidates for new delivery tools into plant and yeast cells.     

Uptake of chromium(III) and chromium(VI) compounds in the yeast cell structure

The study presented in this article investigated the influence of different Cr(III) and Cr(VI) compounds in the cultivation medium on the uptake and localization of chromium in the cell structure of the yeast Candida intermedia. The morphology of the yeast cell surface was observed by the scanning electron microscopy. Results demonstrated that the growth inhibitory concentration of Cr(III) in the cultivation medium induced changes in the yeast cell shape and affected the budding pattern, while inhibitory concentration of Cr(VI) did not cause any visible effects on morphological properties of the yeast cells. The amount of total accumulated chromium in yeast cells and the distribution of chromium between the yeast cell walls and spheroplasts were determined by atomic absorption spectroscopy. No significant differences were found neither in total chromium accumulation nor in the distribution of chromium in yeast cell walls and spheroplasts between the two of Cr(VI) compounds. Conversely, substantial differences between Cr(III) compounds were demonstrated in the total uptake as well as the localization of chromium in yeast cells.     

Growth phase-dependent active transport of pyridoxine in a fission yeast, Schizosaccharomyces pombe

Schizosaccharomyces pombe showed maximum pyridoxine uptake activity around 10 h after starting cultivation. High concentrations of thiamine and pyridoxine in the medium did not affect the activity or the time but changed intracellular levels of vitamin B₆ compounds. Pyridoxine was taken up by a saturable mechanism with two kinds of affinity (K_m 22.4 μM and 118 μM). The uptake depended on the energy produced anaerobically with an optimum pH of 4.5. The uptake was completely inhibited by amiloride, sodium azide or 2,4-dinitrophenol. The uptake system of the fission yeast was different in various respects from that of a budding yeast.     

The further preparation of inorganic cationic yeasts and some of their chief properties

1. A method is described for replacing the intracellular K(+) of the yeast cell by Rb(+), Cs(+), Li(+) or Ca(2+) ions. In the formation of a calcium yeast it is necessary to proceed first through a sodium yeast (Conway & Moore, 1954) as in the formation of a magnesium yeast (Conway & Beary, 1962). This concludes the series of such yeasts in which almost all the usual K(+) is replaced by another cation, and for which the effect on the properties of fermentation, oxygen uptake and of growth are described. 2. Previous work has shown that all these inorganic cations that can be accumulated in quantity at pH7.0 are taken up by the same carrier, that the uptake of Mg(2+) is almost completely inhibited by anoxia and cyanide (0.2mm) and that in the uptake of Mg(2+) ions a practically equivalent amount of H(+) ions is excreted. It is suggested that these facts amount to a definitive demonstration that the carrier is a cytochrome.     

Vanadate-resistant mutants of Candida albicans show alterations in phosphate uptake

Phosphate uptake studies in different strains of the dimorphic pathogenic yeast Candida albicans were undertaken to show that this yeast actively transported phosphate with an apparent Km in the range of 90-170 microM. The uptake was pH dependent and derepressible under phosphate starvation. Vanadate-resistant (van) mutants of C. albicans showed a 20-70% reduction in the rate of phosphate uptake in high phosphate medium and was associated with an increased Km and reduced Vmax. The magnitude of derepression under phosphate starvation was different between van mutants. These results demonstrate that van mutants may have developed resistance by modifying the rate of entry of vanadate.     

Transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine in Saccharomyces cerevisiae

The transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) was studied in resting cells of Saccharomyces cerevisiae. Hydroxymethylpyrimidine uptake was an energy- and temperature-dependent process which has an optimal pH at 4.5. The apparent Km for hydroxymethylpyrimidine uptake was 0.37 microM, and the uptake was inhibited by 2-methyl-4-amino-5-aminomethylpyrimidine, thiamin and pyrithiamin. Furthermore, hydroxymethylpyrimidine uptake was inhibited by 4-azido-2-nitrobenzoylthiamin, a specific and irreversible inhibitor of the yeast thiamin transport system and it was greatly impaired in the thiamin transport mutant of S. cerevisiae. Thus, hydroxymethylpyrimidine is taken up by a common transport system with thiamin in S. cerevisiae, but in contrast to thiamin transport, accumulated hydroxymethylpyrimidine is released from yeast cells showing an overshoot phenomenon.     

Cystine reductase in the dimorphic fungus Histoplasma capsulatum.

Organo-sulfur compounds favor the transition of mycelia of Histoplasma capsulatum to the yeast form (6, 8). Investigation of the role of cystine in the transition revealed that the two phases concentrated this amino acid at comparable rates and that mutants defective in the uptake of cystine were still able to undergo the transition normally. Uptake of cystine is therefore probably not a requirement for transition to or maintenance of the yeast phase. Both phases contained a reduced nicotinamide adenine dinucleotide phosphate-dependent glutathione reductase; but a reduced nicotinamide adenine dinucleotide-dependent cystine reductase was detectable only in the yeast phase. The cystine reductase appeared early in the transition of mycelium to yeast. Treatment of mycelia with p-chloromercuriphenylsulfonic acid, which prevented the transition to yeast, had no effect on cystine uptake but strongly inhibited the cystine reductase. These results suggest that cystine reductase may provide reduced sulfhydryl groups involved in the transition of mycelium to yeast.     

An investigation into the feasibility of using yeast protoplasts to study the ion transport properties of the plasma membrane

A method for studying ion uptake in enzymatically isolated protoplasts from the yeast, Saccharomyces cerevisiae, is described. The kinetics of K⁺ and Rb⁺ uptake, metabolic proton extrusion and cell electrophoretic mobility bave been determined. Enzymic removal of the cell wall does not significantly alter the above-mentioned properties of the yeast cells. It is concluded that studies of these properties can be performed equally well with intact yeast cells or protoplasts. However, in studies aimed at determining effects of complex organic substances, e.g., antibiotics, on plasma membrane function the use of protoplasts is recommended. The effectiveness of the antibiotic, Dio-9, for example, in reversing the metabolic proton extrusion into a net proton influx is at least 50 times higher after enzymic removal of the yeast cell wall.     

Saccharomyces cerevisiae: the effect of different forms and concentrations of iodine on uptake and yeast growth

The essentiality of iodine for humans, especially in the early stages of life, is well recognized. The chemical forms of iodine in food supplements, infant formulae and iodated salt are either iodide (KI) or iodate (KIO₃). Because there are no or rare data about iodine uptake by yeasts, we investigated the influence of different sources of iodine, as KI, KIO₃ and periodate (KIO₄), on its uptake in and growth of the model yeast Saccharomyces cerevisiae . KIO₃ inhibited the growth of the yeast the most and already at a 400 μM initial concentration in the growth medium; the OD was reduced by 23% in comparison with the control, where no KIO₃ was added. The uptake of different iodine sources by the yeast S. cerevisiae was minimal, in total <1%. Tracer experiments with radioactive ¹³¹I added as KI showed that the yeast S. cerevisiae does not have the ability to transform KI into volatile species. We investigated the specificity of iodine uptake added as KIO₃ in the presence of Na₂SeO₄ or ZnCl₂ or K₂CrO₄ in the growth medium, and it was found that chromate had the most influence on reduction of KIO₃ uptake.