Phosphatidylinositol 3,5-bisphosphate regulates the transition between trans-SNARE complex formation and vacuole membrane fusion

Phosphoinositides (PIs) regulate a myriad of cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P ₂ ) on vacuole fusion remains unknown. PI(3,5)P ₂ is made by the PI3P 5-kinase Fab1 and has been characterized as a regulator of vacuole fission during hyperosmotic shock, where it interacts with the TRP Ca ²⁺ channel Yvc1. Here we demonstrate that exogenously added dioctanoyl (C8) PI(3,5) P ₂ abolishes homotypic vacuole fusion. This effect was not linked to Yvc1, as fusion was equally affected using yvc1Δ vacuoles. Thus, the effects of C8-PI(3,5)P ₂ on fusion and fission operate through distinct mechanisms. Further testing showed that C8-PI(3,5)P ₂ inhibited vacuole fusion after trans-SNARE pairing. Although SNARE complex formation was unaffected, we found that C8-PI(3,5)P ₂ blocked outer leaflet lipid mixing. Overproduction of endogenous PI(3,5)P ₂ by the fab1 ^T2250A hyperactive kinase mutant also inhibited the lipid mixing stage, bolstering the model in which PI(3,5)P ₂ inhibits fusion when present at elevated levels. Taken together, this work identifies a novel function for PI(3,5)P ₂ as a regulator of vacuolar fusion. Moreover, it suggests that this lipid acts as a molecular switch between fission and fusion.