Phosphatidylinositol-3,5-bisphosphate mediated vacuolar morphology modulation is integral to ethanol stress response

Abstract

Vacuoles enlarge in response to ethanol. We used quantitative imaging to show that even brief exposure to ethanol causes a rapid transition from multilobed vacuoles to a single compartment. Vacuole unlobing is essential as mutants restricted for vacuole fusion exhibit greater ethanol sensitivity. This response involves the inhibition of vacuole fission via downregulation of the activity of Fab1, a lipid kinase generating the signaling lipid phosphatidylinositol-3,5-bisphosphate. Ethanol exposure results in the redistribution of a phosphatidylinositol-3,5-bisphosphate sensor from the perivauolar dots to the cytoplasm and the dissociation of the effector of this lipid, Atg18, from the vacuolar membrane to the cytoplasm. Impaired Atg18 membrane recruitment also entails a loss of interaction between a scaffold of the Fab1 complex, Vac14, and Atg18. A hyperactive Fab1 mutant, with elevated pools of phosphatidylinositol-3,5-bisphosphate, shows delayed vacuole enlargement; the partial relocation of the phosphatidylinositol-3,5-bisphosphate probe to the perivacuolar dots and of Atg18 to the vacuole membrane. Since Atg18 is responsible for vacuole fragmentation, our results show that, under ethanol stress, cells actively regulate vacuole morphology by modulating phosphatidylinositol-3,5-bisphosphate levels.