Effect of ethanol on fluxes of water and protons across the plasma membrane of Saccharomyces cerevisiae

Abstract

Plasma membrane integrity, ability to transport substrates and maintenance of homeostasis represent obligatory requirements for efficient ethanol production by Saccharomyces cerevisiae. The effect of ethanol on water diffusion through the bilayer and on mediated water movements was evaluated by stopped flow spectroscopy. Ethanol stimulated water diffusion and inhibited mediated water transport. In a strain overexpressing AQY1, the activation energy for water transport increased progressively (from 5.9 to 12.7 kcal mol(-1)) for increasing ethanol concentrations (up to 12% v/v), indicating that mediated water transport lost importance as compared with water diffusion through the bilayer. The effect of ethanol on proton movements (inward by passive diffusion and outward through the PMA1 H(+)-ATPase) was evaluated by measuring the rate of extracellular alcalinization and acidification of unbuffered cell suspensions at different temperatures. Above 10% ethanol, H(+) diffusion was strongly increased at 30 degrees C, but no effect was observed at 20 degrees C up to 12%, indicating the existence of a threshold above which ethanol has a marked effect. On H(+) extrusion, ethanol had no effect at 20 degrees C, but induced a monotonous decrease at higher temperatures. Our results support the view that above a threshold of ethanol concentration, the membrane structure is disrupted, becoming very leaky to H(+).

Peculiar H + Homeostasis of Saccharomyces cerevisiae during the Late Stages of Wine Fermentation

“…In yeast, under acidic pH, proton homeostasis is essentially maintained by a balance between the flux of H ϩ pumped out by the PMA1 ATPase and the influx of H ϩ by passive diffusion through the lipid bilayer and by secondary transporters (20). This balance is also supported by the cell buffering capacity (22) and vacuolar transport (34).…”

“…Fermentations were performed at 15, 25, and 30°C with low magnetic stirring (150 rpm). For comparison with standard lab conditions, cells were also grown up to an optical density at 640 nm (OD 640 ) of Ϸ1 (mid-exponential phase) in mineral medium (20) with 2% glucose at 25°C with low magnetic stirring (150 rpm).…”

“…This balance is also supported by the cell buffering capacity (22) and vacuolar transport (34). Ethanol interferes with this delicate equilibrium by inhibiting mediated transport and enhancing passive diffusion (20,35), disturbing both intracellular pH (pH in ) and proton motive force across the plasma membrane (18). In the past, the ability of yeasts to acidify the environment has been suggested to be an indicator of good metabolic performance and ethanol tolerance (11,23).…”

“…While transforming must into wine, the physiological state of the yeast suffers important changes; resting cells are physiologically very different from the cells at the first stages of the fermentation process (31). Several authors reported that ethanol affects key features of yeast physiology, impairing the major membrane transport systems assayed under lab conditions (14,20), reducing metabolic activity (16,21), decreasing cell growth (4), and inducing apoptotic responses and cell death (12). Although ethanol toxicity in yeasts is a complex mechanism, the main target of such stress seems to be cell membranes (1,2).…”

“…Although ethanol toxicity in yeasts is a complex mechanism, the main target of such stress seems to be cell membranes (1,2). Ethanol causes an increase in membrane fluidity and a loss in its structural integrity (7), with an accompanying increase of leakiness (13,20) and changes in lipid composition (10), generating deleterious nonmediated diffusion processes (30,33). Some of the above biochemical changes induced in yeast by ethanol stress are similar to those caused by thermal stress (27), and a synergistic effect between ethanol and temperature is well documented (17).…”