Effects of ethanol and other alkanols on transport of acetic acid in Saccharomyces cerevisiae

Abstract

In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of Vmax versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S. cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.

FIG. 1

Initial rates of uptake (V) of undissociated acetic acid (as a function of concentration) by glucose-grown cells of S. cerevisiae IGC 4072 at pH 6.0 in the absence of ethanol (•) or in the presence of ethanol at a concentration of 0.52 M (○), 1.19 M (▪), or 1.72 M (□). (Insert) Dependence on ethanol concentration of the diffusion constants (μ_dif) calculated from the slopes, which increased exponentially in accordance with the following equation: μ_dif^x = μ_dif⁰e^k_dif^enh[x], where μ_dif^x and μ_dif⁰ are the values of the diffusion constants at ethanol concentrations of x and zero, respectively, and k_dif^enh is an exponential constant that expresses the exponential enhancement of acid influx by the alcohol.

FIG. 2

Intracellular concentration of total labelled acetic acid in glucose-grown cells of S. cerevisiae IGC 4072 measured in the absence and presence of ethanol at pH 3.0. The numbers next to the lines indicate the extracellular ethanol concentrations (expressed as percentages, weight/volume). The initial extracellular concentration of total acetic acid was 1 mM.

FIG. 3

Effects of alcohols on the acid transport. Semilog plots of the relative maximum rates of uptake of labelled acetic acid (12 mM) at pH 3.0 by acetic acid-grown cells of S. cerevisiae IGC 4072 as a function of the concentration of ethanol (•) and butanol (▪). The V_max values decreased exponentially in accordance with the following equation (16): V_max^x = V_max⁰e^−k_i[x], where V_max^x and V_max⁰ are the maximum initial uptake rates at external alcohol concentrations of x and zero, respectively. The dependence on ethanol concentration of the intracellular concentration of total labelled acetic acid calculated at the maximum level of accumulation by glucose-grown cells of S. cerevisiae IGC 4072 was also determined (○); the values were extrapolated from data shown in Fig. 2. In addition, the dependence on ethanol concentration of the intracellular concentration of total labelled propionic acid calculated at the maximum level of accumulation by acetic acid-grown cells of S. cerevisiae IGC 4072 was determined (□); the values were extrapolated from data shown in Fig. 4.

FIG. 4

Intracellular concentration of total labelled propionic acid in acetic acid-grown cells of S. cerevisiae IGC 4072, measured in the absence and presence of ethanol, at pH 3.0. The numbers next to the lines indicate the extracellular ethanol concentrations (expressed as percentages, weight/volume). The initial extracellular concentration of total propionic acid was 1.0 mM.