Diacylglycerol and its formation by phospholipase C regulate Rab- and SNARE-dependent yeast vacuole fusion

Although diacylglycerol (DAG) can trigger liposome fusion, biological membrane fusion requires Rab and SNARE proteins. We have investigated whether DAG and phosphoinositide-specific phospholipase C (PLC) have a role in the Rab- and SNARE-dependent homotypic vacuole fusion in Saccharomyces cerevisiae. Vacuole fusion was blocked when DAG was sequestered by a recombinant C1b domain. DAG underwent ATP-dependent turnover during vacuole fusion, but was replenished by the hydrolysis of phosphatidylinositol 4,5-bisphosphate to DAG by PLC. The PLC inhibitors 3-nitrocoumarin and U73122 blocked vacuole fusion in vitro, whereas their inactive homologues did not. Plclp is the only known PLC in yeast. Yeast cells lacking the PLC1 gene have many small vacuoles, indicating defects in protein trafficking to the vacuole or vacuole fusion, and purified Plc1p stimulates vacuole fusion. Docking-dependent Ca²⁺ efflux is absent in plc1Δ vacuoles and was restored only upon the addition of both Plc1p and the Vam7p SNARE. However, vacuoles purified from plc1Δ strains still retain PLC activity and significant 3-nitrocoumarin- and 1173122-sensitive fusion, suggesting that there is another PLC in S. cerevisiae with an important role in vacuole fusion.