Abstract
Intracellular membrane fusion is mediated by dynamic assembly and disassembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs). α-SNAP guides NSF to disassemble SNARE complexes after membrane fusion. Recent experiments showed that α-SNAP also dramatically enhances SNARE assembly and membrane fusion. How α-SNAP is involved in these opposing activities is not known. Here, we examine the effect of α-SNAP on the stepwise assembly of the synaptic SNARE complex using optical tweezers. We found that α-SNAP destabilized the linker domain (LD) of the SNARE complex but stabilized its C-terminal domain (CTD) through a conformational selection mechanism. In contrast, α-SNAP minimally affected assembly of the SNARE N-terminal domain (NTD), indicating that α-SNAP barely bound the partially assembled trans-SNARE complex. Thus, α-SNAP recognizes the folded CTD for SNARE disassembly with NSF and subtly modulates membrane fusion by altering the stabilities of the SNARE CTD and LD.
Highlights
; Weber et al, 2000), an essential intermediate for calciumtriggered synaptic exocytosis (Su€dhof and Rothman, 2009)
We find that a-sensitive factor (NSF) attachment protein (SNAP) attenuates linker domain (LD) zippering but enhances C-terminal domain (CTD) zippering, via conformational selection
A-SNAP Destabilizes the LD Synaptic SNARE complexes consist of syntaxin 1A and SNAP25B, which are located on plasma membranes (t-SNAREs), and VAMP2, which is anchored in synaptic vesicles (v-SNARE) (Sollner et al, 1993) (Figure 1A)
Summary
Using a single-molecule approach, Ma et al find that a-SNAP plays distinct roles in stepwise SNARE assembly: it destabilizes the SNARE linker domain, stabilizes the C-terminal domain, but hardly affects the N-terminal domain.
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