Mass spectrometry uncovers intermediates and off-pathway complexes for SNARE complex assembly.

The SNARE complex assembles from vesicular Synaptobrevin-2 as well as Syntaxin-1 and SNAP25 both anchored to the presynaptic membrane. It mediates fusion of synaptic vesicles with the presynaptic plasma membrane resulting in exocytosis of neurotransmitters. While the general sequence of SNARE complex formation is well-established, our knowledge on possible intermediates and stable off-pathway complexes is incomplete. We, therefore, follow the stepwise assembly of the SNARE complex and target individual SNAREs, binary sub-complexes, the ternary SNARE complex as well as interactions with Complexin-1. Using native mass spectrometry, we identify the stoichiometry of sub-complexes and monitor oligomerisation of various assemblies. Importantly, we find that interactions with Complexin-1 reduce multimerisation of the ternary SNARE complex. Chemical cross-linking provides detailed insights into these interactions suggesting a role for membrane fusion. In summary, we unravel the stoichiometry of intermediates and off-pathway complexes and compile a road map of SNARE complex assembly including regulation by Complexin-1.