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-[ 14C]leucine by starved Saccharomyces ellipsoideus. Under adequate experimental conditions the ratio of l-[ 14C]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-[ 14C]leucine entrance and accumulation but the inhibition was less than that of respiration. With propionaldehyde as the energy source, cycloheximide did not affect l-[ 14C]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-[ 14C]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-[ 14C]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-[ 14C]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-[ 14C]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-[ 14C]leucine transport either by d-glucose or by propionaldehyde. 5. (5) The kinetics of l-[ 14C]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-[ 14C]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-[ 14C]leucine entrance irrespective of the time addition. 7. (7) Preincubation of yeast with cyclic AMP caused an increased rate of l-[ 14C]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-[ 14C]leucine. This difference indicates that l-[ 14C]leucine transport System B was the more responsive to cyclic AMP.