SNARE-complex disassembly by NSF follows synaptic-vesicle fusion

Abstract

Soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-mediated fusion of synaptic vesicles with the presynaptic-plasma membrane 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 undertaken a genetic analysis of comatose (dNSF-1) in Drosophila. Characterization 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. Geneticinteraction studies with para demonstrate that blocking evoked fusion delays the accumulation of assembled SNARE complexes and behavioral 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, demonstrating that NSF activity is not required for the fusion step itself. Multiple rounds of vesicle fusion in the absence of NSF activity poisons neurotransmission by trapping SNAREs into cis-complexes. These data indicate that NSF normally dissociates and recycles SNARE proteins during the interval between exocytosis and endocytosis. In the absence of NSF activity, there are sufficient fusion-competent SNAREs to exocytose both the readily released and the reserve pool of synaptic vesicles.neurotransmitter release ͉ exocytosis ͉ synapse ͉ Drosophila T he core components of the vesicle-fusion apparatus consist of the plasma-membrane soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptors (SNAREs) syntaxin and soluble NSF attachment protein of 25 kDa (SNAP-25) and the vesicle SNARE synaptobrevin͞vesicle-associated membrane protein. Neuronal SNAREs assemble into highly stable SDS-resistant 7S SNARE complexes (1). The ATPase NSF has been implicated in numerous membrane-trafficking steps, including regulated exocytosis (2-8). NSF is recruited to the 7S complex by means of the adapter proteins ␣͞␤-and ␥-SNAP to form a larger 20S complex that is subsequently disassembled by the ATPase activity of NSF (1). Both assembly and disassembly of the SNARE complex are essential for synaptic-vesicle trafficking in vivo (6-10). NSF belongs to the AAA family of ATPases (ATPases with diverse cellular activities) and forms a hollow cylindrical hexamer when viewed with high-resolution electron microscopy (11, 12). The structure of the NSF hexamer depends on the presence or absence of bound ATP or ADP and undergoes substantial conformational changes upon ATP hydrolysis (11). Each monomer consists of three domains (13): an N-terminal domain essential for interaction with the SNAP͞SNARE complex, and two tandem ATPase domains termed D1 and D2. The D2 domain is required for hexamerization of NSF (11,(14)(15)(16). The D1 domain contains a high-affinity ATP-binding and hydrolysis sit...

Transcellular spreading of huntingtin aggregates in theDrosophilabrain

“…To test the requirement of SNARE-mediated fusion events in the spread of Htt aggregates throughout the Drosophila brain, we knocked down expression of NSF1, encoded by comatose (comt). NSF1 is required for the disassembly and recycling of SNARE complexes involved in synaptic transmission (32)(33)(34) and is also required for fusion events involving lysosomal trafficking and autophagy (35). To inhibit NSF1 function in neurons expressing UAS-mRFP.…”

Characterization of AtCDC48. Evidence for Multiple Membrane Fusion Mechanisms at the Plane of Cell Division in Plants

“…vesicle (v)-SNAREs pair with target membrane (t)-SNAREs) to yield SNARE complexes (Jahn and Sü dhof, 1999;Brunger, 2001). Sec18p/NSF functions as a molecular chaperone to disassemble, at the expense of ATP, these SNARE complexes to facilitate another round of secretory membrane targeting and fusion (Littleton et al, 2001;May et al, 2001). …”

Protein-tyrosine Phosphatase 1B Modulates Early Endosome Fusion and Trafficking of Met and Epidermal Growth Factor Receptors

“…8) in which NSF is recruited to and potentially phosphorylated by the Met receptor followed by the fusion of endosomes mediated by the actions of v-SNAREs and t-SNAREs. Because the first round of endosome fusion does not require NSF (55), small vesicles containing Met are thus formed. We propose that PTP1B then dephosphorylates NSF, allowing the removal of ␣-SNAP and permitting the next round of endosome fusion to occur.…”