Eugenia H. Ramos

Some properties of l-14C]leucine transport in Saccharomyces ellipsoideus☆

Abstract 1. (1.) Propionaldehyde, a substrate capable of activating electron transfer and oxidative phosphorylation, stimulated the concentrative uptake (entrance and accumulation) of l -[ 14 C]leucine by starved Saccharomyces ellipsoideus . Under adequate experimental conditions the ratio of l -[ 14 C]leucine uptake to oxygen uptake was higher with propionaldehyde than with d -glucose or endogenous substrates as energy sources. 2. (2) With d -glucose as energy source, antimycin significantly inhibited l -[ 14 C]leucine entrance and accumulation but the inhibition was less than that of respiration. With propionaldehyde as the energy source, cycloheximide did not affect l -[ 14 C]leucine uptake despite the almost complete inhibition of amino acid incorporation into the cell protein. 3. (3) Preincubation of starved yeast with l -glucose or propionaldehyde before l -[ 14 C]leucine addition significantly increased the amino acid entrance values. The effect of energization on amino acid entrance increased with the time the yeast was preincubated with the oxidizable substrates. Energization of a S. ellipsoideus rho − mutant with d -glucose, caused less l -[ 14 C]leucine entrance than with the wild type yeast. 4. (4) Addition of 2,4-dinitrophenol to the d -glucose or propionaldehyde-energized S. ellipsoideus brought about a significant inhibition of l -[ 14 C]leucine entrance, but the d -glucose-energized yeast was much less sensitive to the uncoupler than its propionaldehyde-energized counterpart. 2,4-Dinitrophenol also inhibited l -[ 14 C]leucine entrance in the d -glucose-energized rho − mutant, to the same extent as with the d -glucose-energized wild type yeast. Preincubation of yeast with 2,4-dinitrophenol prevented to about the same extent the energization of l -[ 14 C]leucine transport either by d -glucose or by propionaldehyde. 5. (5) The kinetics of l -[ 14 C]leucine transport as a function of amino acid concentration indicate the existence of two apparently distinct transport systems, namely, one with high affinity and low transport activity (System A) and the other with low affinity and high transport activity (System B). The kinetic parameters K T and V characterizing these systems were dependent on the energization state of the yeast cells since K TA (starved) K TA (energized); K TB (starved) > K TB (energized), while V (starved) V (energized), (for both systems). Systems A and B could be further differentiated by the effect of pH and temperature. 6. (6) Ammonium ions significantly inhibited l -[ 14 C]leucine entrance in d -glucose energized yeast when added simultaneously with d -glucose, but stimulated the very early entrance when added after energization had occurred. With the glucose-energized rho − mutant, ammonium ions increased l -[ 14 C]leucine entrance irrespective of the time addition. 7. (7) Preincubation of yeast with cyclic AMP caused an increased rate of l -[ 14 C]leucine entrance. The effect (a) was related to the nucleotide concentration, and (b) was higher with 1.0 mM than with 0.1 mM l -[ 14 C]leucine. This difference indicates that l -[ 14 C]leucine transport System B was the more responsive to cyclic AMP.

Study on fatty acid binding by proteins in yeast. Dissimilar results in Saccharomyces cerevisiae and Yarrowia lipolytica

1. The presence of soluble proteins with fatty acid binding activity was investigated in cell-free extracts from Saccharomyces cerevisiae and Yarrowia lipolytica cultures. 2. No significant fatty acid binding by proteins was detected in S. cerevisiae, even when grown on a fatty acid-rich medium, thus indicating that such proteins are not essential to fatty acid metabolism. 3. An inducible fatty acid binding protein (K0.5 = 3-4 microM) was found in Y. lipolytica which had grown on a minimal medium with palmitate as the sole source of carbon and energy. 4. The relative molecular mass of this protein was 100,000 as inferred from Sephacryl S-200 gel filtration.

RAS2/PKA pathway activity is involved in the nitrogen regulation of L-leucine uptake in Saccharomyces cerevisiae.

The aim of the present work is to study the participation of RAS2/PKA signal pathway in the nitrogen regulation of L-leucine transport in yeast cells. The study was performed on Saccharomyces cerevisiae isogenic strains with the normal RAS2 gene, the RAS2val19 mutant and the disrupted ras2::LEU2. These strains bring about different activities of the RAS2/PKA signal pathway, L-(14C)-Amino acid uptake measurements were determined in cells grown in a rich YPD medium with a mixed nitrogen source or in minimal media containing NH4+ or L-proline as the sole nitrogen source. We report herein that in all strains used, even in those grown in a minimal proline medium, the activity of the general amino acid permease (GAP1) was not detected. L-Leucine uptake in these strains is mediated by two kinetically characterized transport systems. Their KT values are of the same order as those of S1 and S2 L-leucine permeases. Mutation in the RAS2 gene alters initial velocities and Jmax values in both high and low affinity L-leucine transport systems. Activation of the RAS2/PKA signalling pathway by the RAS2val19 mutation, blocks the response to a poor nitrogen source whereas inactivation of RAS2 by gene disruption, results in an increase of the same response.

A Simple Chemical Method for Rendering Wild-Type Yeast Permeable to Brefeldin A That Does Not Require the Presence of an erg6 Mutation

The present work aims to develop a growth medium to render a wild-type strain of Saccharomyces cerevisiae permeable to the antifungal drug Brefeldin A. In the current study, a synthetic medium containing 0.1% L-proline and supplemented with 3.0 × 10-3% SDS is employed. When Brefeldin A is added to this medium, a wild-type strain shows increased growth sensitivity and a diminished transport of the amino acid L-leucine. Since Brefeldin A exerts its effect on the endoplasmic reticulum and the Golgi apparatus, the medium permits the study of the drug effect on the intracellular traffic of L-leucine permeases.

Inhibitory action of palmitic acid on the growth ofSaccharomyces cerevisiae

High concentrations of long-chain fatty acids have been found to be harmful to mammalian cells and prokaryotic organisms. This effect was investigated in Saccharomyces cerevisiae. Addition of 3 mmol/L palmitate to a yeast extract-peptone medium caused a significant inhibition of cell growth during the first 2 d of incubation, followed by renewed growth and palmitate utilization. Inhibition was also observed with palmitate concentrations down to 0.1 mmol/L. As inferred from catalase activity determinations, this effect was found to correlate with the absence of peroxisome proliferation. Finally, no inhibition was observed in exponential-phase cultures or in the presence of 0.1 g/L glucose, this suggesting that the physiological state of the cell may determine whether its growth will be inhibited by fatty acids.

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.

Control of leucine transport in yeast by periplasmic binding proteins

The concentrative inward transport of leucine in Saccharomyces carlsbergensis involves two transport systems (S1 and S2); S1 is a system of high affinity and low translocation velocity, and S2 is a system of low affinity and high translocation velocity. The inward transport process of the amino acid is discriminated into two kinetically defined steps: first, binding to periplasmic proteins and second, translocation across the plasmalemma. When cells were incubated with glucose to increase the metabolic energy charge, we observed that JTmax (maximum flux that each system can exhibit for the translocation step) increased for both systems. This increase in JTmax is due to variations in the parameters defining the initial step (Ks (apparent dissociation constant) and N (concentration of binding sites)): for S1, N1 increases and for S2, KS2 diminishes. Dissipation of the electrochemical proton gradient produced an increase of KS1 and a decrease of N2, resulting in a decrease of JTmax in both systems. Instead, osmotic shock decreases N1 and N2, which suggests that periplasmic components were removed, resulting also in a decrease of JTmax in both systems. These results are consistent with the proposition that the total unidirectional flux of the amino acid proceeds by means of a system of multiple components, with the simultaneous operation of two independent transport processes. We propose that the initial interaction of leucine with components of the cellular envelope might be the essential step for the subsequent translocation of the amino acid across the permeability barrier.