Sterols modulate vacuolar proton transport and autophagy in yeast

Abstract

Inhibitors of the sterol-D8, D7-isomerase (ERG2 gene in yeast) are widely used as fungicides and comprise a family known as amine fungicides. Recently, it has been proposed that V-ATPase inhibition is behind the fungicidal effect of the closely related azole fungicides. However, the cellular mode of action of amine fungicides is still debatable. Here we show that abnormal D8-unsaturated sterols inhibit proton transport by negatively affecting vacuolar H+-ATPase functionality in yeast. Accordingly, cells defective in ERG2 show V-ATPase-like phenotypes such as sensitivity to alkaline pH or zinc. Plants typically show a double set of electrogenic proton pumps at their vacuoles and are resistant to these fungicides. Expression of an alternative proton pump, a chimaera of the plant vacuolar H+-pyrophosphatase AVP1, efficiently complements V-ATPase-like defects in erg2- mutants. Tridemorph, an Erg2p-specific fungicide, induces V-ATPase defects even at low concentrations. However, V-ATPase inhibition is not the cause of cell death since erg2- mutants can grow under standard conditions, while fungicide-treated wild-type cells die by apoptosis. A consequence of V-ATPase inhibition are defects in the process of autophagy akin to those found in V-ATPase mutants. Being this a paramount process to maintain chronological lifespan, we have done a series of experiments to assess the importance of proton transport in erg2- mutants.