Disassembly of membrane-associated NSF 20S complexes is slow relative to vesicle fusion and is Ca(2+)-independent

N-ethylmaleimide-sensitive fusion protein (NSF) and its co-factor soluble NSF attachment protein (alpha)-SNAP) are essential components of the synaptic vesicle fusion machinery and form part of a structurally-conserved 20S protein complex. However, their precise function, relative to fusion itself, is not clear. Using a UV-activated cross-linking approach, we have measured the rate at which a single round of NSF-driven ATP hydrolysis leads to 20S complex disassembly within synaptic membranes. Although this rate is substantially faster than previous estimates of NSF-dependent ATP hydrolysis, it remains much lower than published rates for fusion of synaptic vesicles. Furthermore, the stability of 20S complexes is unaffected by Ca(2+) at concentrations that elicit rapid membrane fusion. We conclude that the ATPase activity of NSF does not contribute directly to vesicle fusion, but more likely plays an earlier role in the synaptic vesicle cycle.

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Alpha-Synuclein and Calpains Disrupt SNARE-Mediated Synaptic Vesicle Fusion During Manganese Exposure in SH-SY5Y Cells

Cells ◽

10.3390/cells7120258 ◽

2018 ◽

Vol 7 (12) ◽

pp. 258 ◽

Cited By ~ 3

Author(s):

Can Wang ◽

Zhuo Ma ◽

Dong-Ying Yan ◽

Chang Liu ◽

Yu Deng ◽

...

Keyword(s):

Synaptic Vesicle ◽

Vesicle Fusion ◽

Alpha Synuclein ◽

Convincing Evidence ◽

Snare Complex ◽

Attachment Protein ◽

Over Expression ◽

Synaptic Vesicle Fusion

Synaptic vesicle fusion is mediated by an assembly of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs), composed of syntaxin 1, soluble NSF-attachment protein (SNAP)-25, and synaptobrevin-2/VAMP-2. Previous studies have suggested that over-exposure to manganese (Mn) could disrupt synaptic vesicle fusion by influencing SNARE complex formation, both in vitro and in vivo. However, the mechanisms underlying this effect remain unclear. Here we employed calpeptin, an inhibitor of calpains, along with a lentivirus vector containing alpha-synuclein (α-Syn) shRNA, to examine whether specific SNAP-25 cleavage and the over-expression of α-Syn disturbed the formation of the SNARE complex in SH-SY5Y cells. After cells were treated with Mn for 24 h, fragments of SNAP-25-N-terminal protein began to appear; however, this effect was reduced in the group of cells which were pre-treated with calpeptin. FM1-43-labeled synaptic vesicle fusion decreased with Mn treatment, which was consistent with the formation of SNARE complexes. The interaction of VAMP-2 and α-Syn increased significantly in normal cells in response to 100 μM Mn treatment, but decreased in LV-α-Syn shRNA cells treated with 100 μM Mn; similar results were observed in terms of the formation of SNARE complexes and FM1-43-labeled synaptic vesicle fusion. Our data suggested that Mn treatment could increase [Ca2+]i, leading to abnormally excessive calpains activity, which disrupted the SNARE complex by cleaving SNAP-25. Our data also provided convincing evidence that Mn could induce the over-expression of α-Syn; when combined with VAMP-2, α-Syn prevented VAMP-2 from joining the SNARE complex cycle.

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