Inhibition of vacuolar proton pumps by ethanol impairs grape berry compartmentation

Tonoplast vesicles were extracted from mature grape berries in order to study the effect of ethanol on their vacuolar proton pumps, vacuolar ATPase (V-ATPase) and inorganic pyrophosphatase (PPase). Ethanol significantly inhibited their functioning; PPase was more sensitive than V-ATPase. The effectiveness of alcohols with different carbon-chain length indicated that a localised modification of the lipidic environment of the pump may be involved in this phenomenon. However, ethanol did not seem to significantly increase passive proton permeability of tonoplast vesicles. The inhibition of the hydrolytic activity of the inorganic pyrophosphatase PPase led to a decrease in the proton pumping rate into the vacuole and consequently in the generated pH gradient. PPase inhibition by ethanol was not compensated by an activation of the less sensitive V-ATPase when both pumps operated simultaneously. The response of proton pumps to ethanol would be expected to impair cellular compartmentation during anaerobic metabolism. This phenomenon would explain the cell death in grape berries during 'carbonic maceration' or 'vinification beaujolaise'.