[Determination of intracellular pH by the distribution of benzoic acid in S. cerevisiae. Amino acid transport and proton gradient]. / Determinación del pH intracelular por distribución de ácido benzoico en S. cerevisiae. Transporte de aminoácidos y gradiente de protones.

The internal pH (pHi) of Saccharomyces cerevisiae, wild type strain and its mutant rho- has been measured by the intra-extracellular distribution of 14C-benzoic acid. The values of pHi (external pH 4.5) change with the yeast strain and depend on the cellular metabolic conditions. The values of pHi and proton gradient in the wild type yeast are higher in energized than in starved cells: in energized cells pHi, 6.15 to 6.40, delta pH 1.65 to 1.90 or -97 to -112 mV; starved cells pH 5.90, delta pH 1.40 or -82 mV. In the rho- mutant, the values are lower than in the wild type yeast, in the same metabolic conditions. Energized rho- mutant cells, pH 6.05, delta pH 1.55 or -91 mV; starved cells, pHi 5.70, delta pH 1.20 or -71 mV. The proton conductors, DNP and PCP produce a decrease in pHi and delta pH and inhibition of L-leucine entrance by system S1, high affinity and low velocity and system S2, low affinity and high velocity. The obtained values of delta pH decrease and L-leucine transport inhibition, demonstrate that there is no strict relationship between the proton gradient across the cell membrane and the process of transport of L-leucine in yeast.

Determinación del pH intracelular por distribución de ácido benzoico en S. cerevisiae. Transporte de aminoácidos y gradiente de protones. / [Determination of intracellular pH by the distribution of benzoic acid in S. cerevisiae. Amino acid transport and proton gradient]

The internal pH (pHi) of Saccharomyces cerevisiae, wild type strain and its mutant rho- has been measured by the intra-extracellular distribution of 14C-benzoic acid. The values of pHi (external pH 4.5) change with the yeast strain and depend on the cellular metabolic conditions. The values of pHi and proton gradient in the wild type yeast are higher in energized than in starved cells: in energized cells pHi, 6.15 to 6.40, delta pH 1.65 to 1.90 or -97 to -112 mV; starved cells pH 5.90, delta pH 1.40 or -82 mV. In the rho- mutant, the values are lower than in the wild type yeast, in the same metabolic conditions. Energized rho- mutant cells, pH 6.05, delta pH 1.55 or -91 mV; starved cells, pHi 5.70, delta pH 1.20 or -71 mV. The proton conductors, DNP and PCP produce a decrease in pHi and delta pH and inhibition of L-leucine entrance by system S1, high affinity and low velocity and system S2, low affinity and high velocity. The obtained values of delta pH decrease and L-leucine transport inhibition, demonstrate that there is no strict relationship between the proton gradient across the cell membrane and the process of transport of L-leucine in yeast.

Amino acid uptake by yeasts. IV. Effect of thiol reagents on L-leucine transport in Saccharomyces cerevisiae.

(1) N-Ethylmaleimide (a penetrating SH- reagent) inactivated L-[14C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with N-ethylmaleimide, N-ethylmaleimide concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With D-glucose-energized yeast, N-ethylmaleimide inhibited L-[14C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit L-[14C]leucine binding, except when the cells were preincubated with L-leucine. With the rho- respiratory-deficient mutant (energized cells), N-ethylmaleimide inhibited L-[14C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the N-ethylmaleimide effect as a function of L-[14C]leucine concentration showed a significant decrease of Jmax values of the high- (S1) and low- (S2) affinity amino acid transport systems, but KT values were not significantly modified. (3) When assayed in the presence of D-glucose, N-ethylmaleimide inhibition of D-glucose uptake and respiration contributed significantly to inactivation of L-[14C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of L-[14C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate L-[14C]leucine entrance, while p-chloromercuribenzoate, a slowly penetrating SH-reagent, inactivated it to a limited extent. When compared with the effect of N-ethylmaleimide, these negative results indicate that thiol groups of the L-[14C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.

Kinetics of L-[14C]leucine transport in Saccharomyces cerevisiae: effect of energy coupling inhibitors.

1. L-[14C]Leucine transport into Saccharomyces cerevisiae involves a high-affinity, low-velocity system (system 1) and a low-affinity, high-velocity system (system 2). These systems are characterized by the different values of the kinetic parameters KT and Jmax, and are both capable of concentrative transport. The general amino acid permease is assumed to be a part of the high-affinity system. 2. The kinetics of L-[14C]leucine entrance show and initial rapid phase (the 'very early uptake') before reaching the steady-state rate. The contribution of the very early uptake to total entrance values affects the values of KT and Jmax, especially when the steady-state rate is relatively slow, as with starved yeast, and then negative KT and Jmax values may result. The very early uptake is increased by pretreatment of starved yeast and D-glucose, this latter effect being counteracted by iodoacetate. 3. After energization of starved yeast by pretreatment with D-glucose or propionaldehyde, the apparent KT,2 value greatly decreases whilst the KT,1 value decreases to a much more limited extent, or does not vary. With the energized yeast, KT,2 decreases throughout incubation whilst KT,1 variation is insignificant. Energization increases Jmax,1 and Jmax,2 several-fold and with the energized yeast at the steady-state phase, Jmax,2 greater than or equal to 4Jmax,1. Variation of KT and Jmax values as a function of the metabolic state of yeast cells may be explained in terms of variation of rate constants k-1, k+1 and k+2 for each transport system. 4. Dicyclohexylcarbodiimide, quercetin and diethylstilbestrol inhibit tranport at 0.05 mM L-[14C]leucine, in good agreement with a function of the plasmalemma ATPase for the operation of system 1. Dio-9, propionic and isobutyric acids, pentachlorophenol, carbonylcyanide 3-chlorophenylhydrazone and carbonylcyanide 4-trifluoromethoxyphenylhydrazone, which affect the proton gradient and/or the membrane potential inhibit L-[14C]leucine uptake at all the assayed amino acid concentrations. 5. The polyene antibiotic, nystatin, which forms channels in membranes permeable to K+ and H+, inhibits systems 1 and 2 activity but enniatin (also a K+ ionophore) does not.

Energy requirements for the uptake of L-leucine by Saccharomyces cerevisiae.

(1) Substrates capable of activating mitochondrial electron transfer and oxidative phosphorylation, namely, pyruvate, acetate, propionaldehyde and butanol, stimulated the concentrative uptake (transport and accumulation) of L-[14-C]leucine by Saccharomyces cerevisiae (wild type strain 207, starved cells). Under adequate experimental conditions, the L-[14-C]leucine uptake versus the oxygen uptake ratio was almost the same with either pyruvate, acetate or D-glucose as energy sources. Substrate oxidation also increased L-[14-C]leucine incorporation into the cell protein. (2) With S. cerevisiae D261 and D247-2 and propionaldehyde as an energy source, or with strain 207 and glucose as energy source, 2,4-dinitrophenol (50 muM) inhibited L-[14-C]leucine uptake, the inhibition being accompanied by stimulation of respiration. With S. cerevisiae 207 and propionaldehyde as energy source, 2,4-dinitrophenol inhibited both respiration and L-[14-C]leucine uptake, but with respiration being less affected than uptake. Displacement of accumulated L-[14-C]leucine was also inhibited by 2,4-dinitrophenol. (3) In the presence of glucose, and for relatively brief incubation periods, anaerobically grown cells of S. cerevisiae 207 and of a p-minus "petite" mutant of this strain incorporated L-[14-C]leucine with less efficiency than the original wild type strain 207, grown aerobically. With D-glucose as energy source, 2,4-dinitrophenol and iodoacetate inhibited alike L-[14-C]leucine uptake by the respiration competent cells. (4) It is postulated that in respiration-competent yeasts, the mitochondrion contributes to 6-[14-C]leucine uptake by supplying high-energy compounds required for amino acid transport and accumulation. Conversely, the promitochondrion in the anaerobically grown yeast, or the modified mitochondrion in the respiratory deficient mutant, competes for high energy compounds generated by glycolysis in the cytosol.