G.W.F.H. Borst-Pauwels

Cotransport of phosphate and sodium by yeast

Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a Km of 30 micronM and is independent of sodium, and a sodium-dependent mechanism with a Km of 0.6 micronM, both Km values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na+, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Ribidium has no effect on the stimulation of phosphate uptake by sodium. Phosphate and arsenate enhance sodium uptake at pH 7.2. The Km of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake. The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed.

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH, cations and phosphate.

The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.

Uptake of the lipophilic cation dibenzyldimethylammonium into Saccharomyces cerevisiae. Interaction with the thiamine transport system

The distribution ratio of the lipophilic cation dibenzyldimethylammonium between the cells of Saccharomyces cerevisiae and the medium appears to reflect changes in the membrane potential in a way that is qualitatively correct: the addition of a proton conductor or of an agent which blocks metabolism causes an apparent depolarization of the cell membrane; monovalent cations cause also a lowering of the equilibrium distribution, whereas the addition of divalent cations results in an increase of the partition ratio. However, uptake of dibenzyldimethylammonium and probably also of other liophilic cations proceeds via the thiamine transport system of the yeast. Dibenzyldimethylammonium transport is inducible, like thiamine transport. A kinetic analysis of the mutual interaction between thiamine and dibenzyldimethylammonium uptake shows that these compounds share a common transport system; moreover, dibenzyldimethylammonium uptake is inhibited complete by thiamine disulfide, a competitive inhibitor of thiamine transport and dibenzyldimethylammonium uptake in a thiamine-transport mutant is reduced considerably. It is concluded that one should be cautious when using lipophilic cations to measure the membrane potential of cells of S. cerevisiae.

Kinetics of sulfate uptake by yeast

Uptake of sulfate by yeast requires the presence of a metabolic substrate and is dependent on the time during which the cells have been metabolizing in the absence of sulfate. At low concentrations of sulfate, uptake can be described by simple saturation kinetics. Uptake of sulfate is accompanied by a net proton influx of 3 H+ and an efflux of 1 K+ for each sulfate ion taken up. Divalent cations stimulate sulfate uptake at low concentrations of sulfate; the maximal rate of uptake is not significantly affected but Km is lowered. Stimulation by divalent cations shows an optimum at a cation concentration of about 4 mM. Monovalent cations are less effective, trivalent cations are more effective in stimulating sulfate uptake. The results are qualitatively in accordance with the notion, that the effect of cations is due to an effect via the surface potential.

Kinetics of ion translocation across charged membranes mediated by a two-site transport mechanism: Effects of polyvalent cations upon rubidium uptake into yeast cells

(1) The effect of surface charge upon the kinetics of monovalent cation translocation via a two-site mechanism is investigated theroretically. (2) According to the model dealt with, typical relations are expected for the dependence of the kinetic parameters of the translocation process upon the concentration of a polyvalent cation, differing essentially from those derived for the case in which the membrane carries no excess charge. (3) Even when a polyvalent cation does not compete with the substrate cation for binding to the translocation sites, apparently competitive inhibition may occur when the membrane is negatively charged. (4) The model is tested experimentally by studying the effects of the polyvalent cations Mg2+, Sr2+, Ca2+, Ba2+ and Al3+ upon Rb+ uptake into yeast cells at pH 4.5 A good applicability is found. (5) Equimolar concentrations of polyvalent cations reduce the rate of the Rb+ uptake into yeast cells in the order Mg2+ less than Sr2+ less than Ca2+ less than Ba2+ less than Al3+. (6) The conclusion is reached that the reduction in the rate of Rb+ uptake caused by the polyvalent cations applied results mainly from screening of the negative fixed charges on the membrane surface and binding to these negative sites rather than competition with Rb+ for the transport sites. (7) The results of our investigation indicate the affinity of the alkaline-earth cations for the negative fixed charges on the surface to the yeast cell membrane increases in the orther Mg2+ less than Sr2 less than Ca2+ less than Ba2+. (8) Probably mainly phosphoryl groups determine the net charge on the membrane of the yeast cell at a medium pH of 4.5.

The interaction of 2,4-dinitrophenol with anaerobic Rb+ transport across the yeast cell membrane

Abstract 1. 1. The Rb+ uptake mechanism of yeast is activated by small amounts of Rb- or K+, giving rise to a deviation from normal Michaelis-Menten kinetics at concentrations below 0.6 mM Rb+. 2. 2. The affinity of Rb+ and K+ for the activation sites appeared to be much higher than for the substrate sites. 3. 3. 2,4-Dinitrophenol inhibits the Rb+ uptake by lowering the affinity for both the activation sites and the substrate sites. 4. 4. 2,4-Dinitrophenol causes a sudden increase in the Rb+ permeability of the cells. This increase is only transient except at relatively high dinitrophenol concentrations.

Activation of Rb+ and Na+ uptake into yeast by monovalent cations

Abstract 86 Rb + uptake by yeast was not only stimulated by Rb + or K + but also by Na + . The uptake of 22 Na + was enhanced by both Rb + and K + , but not by Na + , which was inhibitory at all concentrations applied. Inhibition of 22 Na + uptake by inactive Na + occurred in two phases: one phase refers to inhibition at low Na + concentrations and the other to inhibition at high Na + concentrations. Our results can be qualitatively described by a two-site transport mechanism, having two cation binding sites, which must be occupied with monovalent cations before transport can occur.

Some characteristics of tetraphenylphosphonium uptake into Saccharomyces cerevisiae

The characteristics of the uptake of the lipophilic cation tetraphenylphosphonium (TPP+) into Saccharomyces cerevisiae have been investigated in order to establish whether this compound can be used to monitor the membrane potential of his organism. Unlike dibenzyldimethylammonium, TPP+ is not translocated via the thiamine transport system, nor via another inducible translocation mechanism. On changing the experimental conditions the equilibrium potential of TPP+ varies according to expected changes of the membrane potential. TPP+ accumulation is higher in metabolizing cells than in non-metabolizing cells. In addition, decreasing the medium pH, addition of the proton conductor 2,4-dinitrophenol and addition of K+ all cause an apparent depolarization, whereas Ca2+ apparently hyperpolarizes the cell membrane. It is concluded that TPP+, if applied at low concentrations, can be used to measure the membrane potential of S. cerevisiae.

Inhibition of phosphate and arsenate uptake in yeast by monoiodoacetate, fluoride, 2,4-dinitrophenol and acetate

Abstract 1. Arsenate and phosphate uptake by the yeast Saccharomyces cerevisiae Delft II is inhibited completely by 3 mM monoiodoacetate, 20 mM fluoride, 0.1 mM 2,4-dinitrophenol and 60 mM acetate under anaerobic conditions at pH 4.5. 2. Monoiodoacetate, 2,4-dinitrophenol and acetate decrease not only the maximum rate of uptake but also the K m for this process. 3. The percent decrease in the rates of glycolysis and phosphate uptake caused by monoiodoacetate is about the same. The three other inhibitors inhibit glycolysis only at concentrations much higher than those which decrease phosphate uptake. 4. The cellular ATP concentration is strongly decreased by 3 mM monoiodoacetate and 20 mM fluoride (90%), but much less by 0.1 mM 2,4-dinitrophenol (60%) and by 60 mM acetate (25%). 5. Possible ways in which the inhibitors may affect P i and As i uptake are discussed.

Possible energization of K+ accumulation into metabolizing yeast by the protonmotive force binding correction to be applied in the calculation of the yeast membrane potential from tetraphenylphosphonium distribution

Membrane potentials of yeast cells, Saccharomyces cerevisiae, calculated from the equilibrium distribution of tetraphenylphosphonium (TPP) between cell-water and medium should be corrected for a contribution due to binding of TPP to intracellular constituents. The magnitude of this correction depends upon the way in which it is determined. In cells permeabilized by boiling, cell-binding is much higher than in cells permeabilized by repeated freezing and thawing. The binding corrections are 75 +/- 1 mV and 49 +/- 7 mV, respectively. The binding correction obtained from TPP distribution between deenergized cells and medium is much lower and amounts to 19 +/- 9 mV. The latter value is probably more reliable. It is supposed that permeabilization of the cells by boiling or repeated freezing and thawing unmasks potential TPP binding groups in the cell. The K+ accumulation into anaerobically metabolizing yeast cells can be accounted for almost quantitatively by a cotransport of protons and K+ ions if the lower binding correction is applied. This means that K+ accumulation into the yeast cell may be driven by the sum of the protonmotive force and the membrane potential.