Phosphatidylinositol 4,5-bisphosphate regulates two steps of homotypic vacuole fusion

Yeast vacuoles undergo cycles of fragmentation and fusion as part of their transmission to the daughter cell and in response to changes of nutrients a nd the environment. Vacuole fusion can be reconstituted in a cell free syst em. We now show that the vacuoles synthesize phosphoinositides during in vi tro fusion. Of these phosphoinositides, phosphatidylinositol 4-phosphate an d phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) are important for fusi on. Monoclonal antibodies to PI(4,5)P-2, neomycin (a phosphoinositide ligan d), and phosphatidylinositol-specific phospholipase C interfere with the re action. Readdition of PI(4,5)P-2 restores fusion in each case. Phosphatidyl inositol 3-phosphate and PI(3,5)P-2 synthesis are not required. PI(4,5)P-2 is necessary for priming, i.e., for the Sec18p (NSF)-driven release of Sec1 7p (alpha-SNAP), which activates the vacuoles for subsequent tethering and docking. Therefore, it represents the kinetically earliest requirement iden tified for vacuole fusion so far. Furthermore, PI(4,5)P-2 is required at a step that can only occur after docking but before the BAPTA sensitive step in the latest stage of the reaction. We hence propose that PI(4,5)P-2 contr ols two steps of vacuole fusion.