Figure 1.

Inhibition of vacuole fusion by ligands of phosphatidylinositol phosphates. (A) Vacuoles (6 μg) were preincubated for 5 min on ice without inhibitor or with the indicated amounts of monoclonal antibodies to phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol 4,5-diphosphate (PIP₂) in PS buffer with 50 mM KCl. The samples were then supplemented with salts and an ATP-regenerating system to yield standard fusion reactions. Cytosol was added as indicated. After 70 min at 27°C, fusion was assayed via alkaline phosphatase activity. (B) As in (A) but with neomycin as a phosphoinositide ligand. (C) The inhibitory effect is specific for PI(4)P and PI(4,5)P₂ antibodies. Fusion reactions with cytosol were performed as in (A), in the absence of cytosol. Vacuoles were preincubated with monoclonal antibodies to PI(4)P, PI(4,5)P₂, phosphatidylserine (PS), or phosphatidylcholine (PC; 35 μM each), or the respective control buffers.