Regulated membrane fusion is essential for cell compartmentation and growth, neurotransmission, and hormone release. Fusion occurs by a highly complex, and conserved, pathway of SNAREs, Ypt/Rabs, their effectors and chaperones (NSF/SNAP and SM proteins), phosphoinositides, and actin. In addition to regulating organelle movement, actin remodeling is essential for the assembly and function of docking and fusion membrane microdomains, as shown with the yeast vacuole. Vacuole fusion is catalyzed by a """"""""vertex"""""""" ring of proteins and lipids, including actin, which surrounds the closely apposed membrane surfaces. Jasplakinolide sensitive actin remodeling is required for SNARE and Ypt/Rab proteins and 3- phosphoinositides to assemble into this ring microdomain, and they in turn govern actin enrichment at the ring. After docking, fusion requires further latrunculin-sensitive actin remodeling. To test our working model, that actin remodeling from F to G regulates lipid and protein assembly into the vertex ring and then contributes to fusion by being further remodeled from G to F, we will: 1. Establish the abundance and microdomain localization of F and G actin at each step of vacuole fusion, exploiting their distinct physical properties and ligand binding, 2. Perform genetic screens to identify both actin mutants with specific effects on vacuole fusion and actin regulatory protein cascades which remodel vacuolar actin, using the extensive and available collections of mutants, 3. Identify actin's vacuolar receptor and the proteins which regulate its remodeling, using actin-affinity isolation methods, 4. Establish the mutual relationships between actin and the fusion regulatory lipids (sterol, phosphoinositides, and diacylglycerol) by which they regulate each other's assembly into the ring domain, using ratiometric fluorescence microscopy assays of vertex enrichment and actin and lipid ligands and modifying enzymes, 5. Establish the order, or interdependence, of trans-SNARE pairing, actin polymerization, and lipid rearrangements for vacuole fusion, using reversible inhibitory ligands and our colorimetric assay of in vitro vacuole fusion, and 6. Reconstitute the functional dependence of each step of vacuole fusion on purified actin and actin regulatory proteins through systematic purification of actin and actin regulatory factors and reconstitution into fusion subreactions, on the vacuole at first and then in liposome model systems. ? ? ?
