SNARE-complex disassembly by NSF follows synaptic-vesicle fusion

Soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNAR E)-mediated fusion of synaptic vesicles with the presynaptic-plasma membran e is essential for communication between neurons. Disassembly of the SNARE complex requires the ATPase N-ethylmaleimide-sensitive fusion protein (NSF) . To determine where in the synaptic-vesicle cycle NSF functions, we have u ndertaken a genetic analysis of comatose (dNSF-1) in Drosophila. Characteri zation of 16 comatose mutations demonstrates that NSF mediates disassembly of SNARE complexes after synaptic-vesicle fusion. Hypomorphic mutations in NSF cause temperature-sensitive paralysis, whereas null mutations result in lethality. Genetic-interaction studies with para demonstrate that blocking evoked fusion delays the accumulation of assembled SNARE complexes and beh avioral paralysis that normally occurs in comatose mutants, indicating NSF activity is not required in the absence of vesicle fusion. In addition, the entire vesicle pool can be depleted in shibire comatose double mutants, de monstrating that NSF activity is not required for the fusion step itself. M ultiple rounds of vesicle fusion in the absence of NSF activity poisons neu rotransmission by trapping SNAREs into cis-complexes. These data indicate t hat NSF normally dissociates and recycles SNARE proteins during the interva l between exocytosis and endocytosis. In the absence of NSF activity, there are sufficient fusion-competent SNAREs to exocy-tose both the readily rele ased and the reserve pool of synaptic vesicles.ceptor (SNARE)-mediated fusi on of synaptic vesicles with the presynaptic-plasma membrane is essential f or communication between neurons. Disassembly of the SNARE complex requires the ATPase N-ethylmaleimide-sensitive fusion protein (NSF). To determine w here in the synaptic-vesicle cycle NSF functions, we have undertaken a gene tic analysis of comatose (dNSF-1) in Drosophila. Characterization of 16 com atose mutations demonstrates that NSF mediates disassembly of SNARE complex es after synaptic-vesicle fusion. Hypomorphic mutations in NSF cause temper ature-sensitive paralysis, whereas null mutations result in lethality. Gene tic-interaction studies with para demonstrate that blocking evoked fusion d elays the accumulation of assembled SNARE complexes and behavioral paralysi s that normally occurs in comatose mutants, indicating NSF activity is not required in the absence of vesicle fusion. In addition, the entire vesicle pool can be depleted in shibire comatose double mutants, demonstrating that NSF activity is not required for the fusion step itself. Multiple rounds o f vesicle fusion in the absence of NSF activity poisons neurotransmission b y trapping SNAREs into cis-complexes. These data indicate that NSF normally dissociates and recycles SNARE proteins during the interval between exocyt osis and endocytosis. In the absence of NSF activity, there are sufficient fusion-competent SNAREs to exocy-tose both the readily released and the res erve pool of synaptic vesicles.