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

Abstract

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.