Extensive research has yielded crucial insights into the mechanism of neurotransmitter release, and working models for the functions of key proteins involved in release. The SNAREs Syntaxin-1, Synaptobrevin, and SNAP-25 play a central role in membrane fusion, forming SNARE complexes that bridge the vesicle and plasma membranes and that are disassembled by NSF-SNAPs. Exocytosis likely starts with Syntaxin-1 folded into a self-inhibited closed conformation that binds to Munc18-1. Munc13s open Syntaxin-1, orchestrating SNARE complex assembly in an NSF-SNAP-resistant manner together with Munc18-1. In the resulting primed state, with partially assembled SNARE complexes, fusion is inhibited by Synaptotagmin-1 and Complexins, which also perform active functions in release. Upon influx of Ca(2+), Synaptotagmin-1 activates fast release, likely by relieving the inhibition caused by Complexins and cooperating with the SNAREs in bringing the membranes together. Although alternative models exist and fundamental questions remain unanswered, a definitive description of the basic release mechanism may be available soon.

Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca2+-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a ‘primed’ state that does not fuse but is ready for fast fusion upon Ca2+ influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion.DOI: http://dx.doi.org/10.7554/eLife.13696.001

SUMMARY Rapid neurotransmitter release depends on the Ca2+-sensor Synaptotagmin-1 and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Synaptotagmin-1 triggers release remains unclear, in part because elucidating high-resolution structures of Synaptotagmin-1-SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode where basic residues in the concave side of the Synaptotagmin-1 C2B domain β-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Synaptotagmin-1 that markedly impair SNARE-complex binding in vitro and Synaptotagmin-1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Synaptotagmin-1 function. Our results support a model whereby their dynamic interaction facilitates cooperation between synaptotagmin-1 and the SNAREs in inducing membrane fusion.

Interferon-inducible guanylate-binding proteins (GBPs) mediate cell-autonomous antimicrobial defences. Shigella flexneri, a Gram-negative cytoplasmic free-living bacterium that causes bacillary dysentery, encodes a repertoire of highly similar type III secretion system effectors called invasion plasmid antigen Hs (IpaHs). IpaHs represent a large family of bacterial ubiquitin-ligases, but their function is poorly understood. Here we show that S. flexneri infection induces rapid proteasomal degradation of human guanylate binding protein-1 (hGBP1). We performed a transposon screen to identify a mutation in the S. flexneri gene ipaH9.8 that prevented hGBP1 degradation. IpaH9.8 targets hGBP1 for degradation via Lys48-linked ubiquitination. IpaH9.8 targets multiple GBPs in the cytoplasm independently of their nucleotide-bound states and their differential function in antibacterial defence, promoting S. flexneri replication and resulting in the death of infected mice. In the absence of IpaH9.8, or when binding of GBPs to IpaH9.8 was disrupted, GBPs such as hGBP1 and mouse GBP2 (mGBP2) translocated to intracellular S. flexneri and inhibited bacterial replication. Like wild-type mice, mutant mice deficient in GBP1-3, 5 and 7 succumbed to S. flexneri infection, but unlike wild-type mice, mice deficient in these GBPs were also susceptible to S. flexneri lacking ipaH9.8. The mode of IpaH9.8 action highlights the functional importance of GBPs in antibacterial defences. IpaH9.8 and S. flexneri provide a unique system for dissecting GBP-mediated immunity.

Complexins facilitate and inhibit neurotransmitter release through distinct domains, and their function was proposed to be coupled to the Ca2+ sensor Synaptotagmin-1. However, the mechanisms underlying Complexin function remain unclear. We now uncover an interaction between the Complexin N terminus and the SNARE complex C terminus, and show that disrupting this interaction abolishes the facilitatory function of Complexins in mouse neurons. Analyses of hypertonically induced exocytosis demonstrate that Complexins enhance synaptic vesicle fusogenicity. Genetic experiments crossing Complexin and Synaptotagmin-1 null mice indicate a functional interaction between these proteins, but also show that Complexins can promote Ca2+-triggered release in the absence of Synaptotagmin-1. We propose that the Complexin N terminus stabilizes the SNARE complex C terminus and/or helps release the inhibitory function of Complexins, thereby activating the fusion machinery in a manner that may cooperate with Synaptotagmin-1, but does not require Synaptotagmin-1.

Munc13–1 acts as a master regulator of neurotransmitter release, mediating docking-priming of synaptic vesicles and diverse presynaptic plasticity processes. It is unclear how the functions of the multiple domains of Munc13–1 are coordinated. The crystal structure of a Munc13–1 fragment including its C1, C2B and MUN domains (C1C2BMUN) reveals a 19.5 nm-long multi-helical structure with the C1 and C2B domains packed at one end. The similar orientations of the respective diacyglycerol- and Ca2+-binding sites of the C1 and C2B domains suggest that the two domains cooperate in plasma-membrane binding and that activation of Munc13–1 by Ca2+ and diacylglycerol during short-term presynaptic plasticity are closely interrelated. Electrophysiological experiments in mouse neurons support the functional importance of the domain interfaces observed in C1C2BMUN. The structure imposes key constraints for models of neurotransmitter release and suggests that Munc13–1 bridges the vesicle and plasma membranes from the periphery of the membrane-membrane interface.DOI: http://dx.doi.org/10.7554/eLife.22567.001

Munc13-1 plays a crucial role in neurotransmitter release. We recently proposed that the C-terminal region encompassing the C1, C2B, MUN and C2C domains of Munc13-1 (C1C2BMUNC2C) bridges the synaptic vesicle and plasma membranes through interactions involving the C2C domain and the C1-C2B region. However, the physiological relevance of this model has not been demonstrated. Here we show that C1C2BMUNC2C bridges membranes through opposite ends of its elongated structure. Mutations in putative membrane-binding sites of the C2C domain disrupt the ability of C1C2BMUNC2C to bridge liposomes and to mediate liposome fusion in vitro. These mutations lead to corresponding disruptive effects on synaptic vesicle docking, priming, and Ca2+-triggered neurotransmitter release in mouse neurons. Remarkably, these effects include an almost complete abrogation of release by a single residue substitution in this 200 kDa protein. These results show that bridging the synaptic vesicle and plasma membranes is a central function of Munc13-1.

Munc18-1 orchestrates SNARE complex assembly together with Munc13-1 to mediate neurotransmitter release. Munc18-1 binds to synaptobrevin, but the relevance of this interaction and its relation to Munc13 function are unclear. NMR experiments now show that Munc18-1 binds specifically and non-specifically to synaptobrevin. Specific binding is inhibited by a L348R mutation in Munc18-1 and enhanced by a D326K mutation designed to disrupt the ‘furled conformation’ of a Munc18-1 loop. Correspondingly, the activity of Munc18-1 in reconstitution assays that require Munc18-1 and Munc13-1 for membrane fusion is stimulated by the D326K mutation and inhibited by the L348R mutation. Moreover, the D326K mutation allows Munc13-1-independent fusion and leads to a gain-of-function in rescue experiments in Caenorhabditis elegans unc-18 nulls. Together with previous studies, our data support a model whereby Munc18-1 acts as a template for SNARE complex assembly, and autoinhibition of synaptobrevin binding contributes to enabling regulation of neurotransmitter release by Munc13-1.DOI: http://dx.doi.org/10.7554/eLife.24278.001