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Mass Spectrometry Uncovers Protein Interactions and Stoichiometry of Intermediates and Sub-complexes for SNARE Complex Assembly

ABSTRACT: The neuronal SNARE complex assembles from vesicular Synaptobrevin-2 as well as Syntaxin-1 and SNAP25 anchored to the presynaptic membrane. It mediates fusion of synaptic vesicles with the presynaptic plasma membrane resulting in exocytosis of neurotransmitters into the synaptic cleft. While the general sequence of SNARE complex formation is well-established, our knowledge on possible intermediates is still incomplete. We, therefore, follow the step-wise 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 preferred sub-complexes and monitor oligomerisation of various assemblies. Importantly, we find that interactions with Complexin-1 reduce self-association of the ternary SNARE complex. Chemical cross-linking provides detailed insights into these interactions suggesting a role during membrane fusion. Taken together, we unravel possible intermediates and preferred sub-complexes and compile a road map of SNARE complex assembly including regulation by Complexin-1.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Marie Alfes

LAB HEAD: Carla Schmidt

PROVIDER: PXD030619 | Pride | 2023-01-25

REPOSITORIES: Pride

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