The function of synaptotagmin as a Ca(2+) sensor in neurotransmitter release involves Ca(2+)-dependent phospholipid binding to its two C(2) domains, but this activity alone does not explain why ...Ca(2+) binding to the C(2)B domain is more critical for release than Ca(2+) binding to the C(2)A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca(2+) induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca(2+) binding to the C(2)B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C(2)B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.
Unc13/Munc13s play a crucial function in neurotransmitter release through their MUN domain, which mediates the transition from the Syntaxin-1/Munc18-1 complex to the SNARE complex. The MUN domain was ...suggested to be related to tethering factors, but no MUN-domain structure is available to experimentally validate this notion and address key unresolved questions about the interactions and minimal structural unit required for Unc13/Munc13 function. Here we identify an autonomously folded module within the MUN domain (MUN-CD) and show that its crystal structure is remarkably similar to several tethering factors. We also show that the activity in promoting the Syntaxin-1/Munc18-1 to SNARE complex transition is strongly impaired in MUN-CD. These results show that MUN domains and tethering factors indeed belong to the same family and may have a common role in membrane trafficking. We propose a model whereby the MUN-CD module is central for Munc13 function but full activity requires adjacent sequences.
► The crystal structure of a C-terminal module of Munc13-1 (MUN-CD) is described ► MUN-CD is at the heart of Munc13 function, but full activity requires adjacent modules ► The structure of MUN-CD is very similar to those of diverse tethering factors ► MUN-domain containing proteins and tethering factors form a protein family
Munc18-1 and soluble NSF attachment protein receptors (SNAREs) are critical for synaptic vesicle fusion. Munc18-1 binds to the SNARE syntaxin-1 folded into a closed conformation and to SNARE ...complexes containing open syntaxin-1. Understanding which steps in fusion depend on the latter interaction and whether Munc18-1 competes with other factors such as complexins for SNARE complex binding is critical to elucidate the mechanisms involved. In this study, we show that lentiviral expression of Munc18-1 rescues abrogation of release in Munc18-1 knockout mice. We describe point mutations in Munc18-1 that preserve tight binding to closed syntaxin-1 but markedly disrupt Munc18-1 binding to SNARE complexes containing open syntaxin-1. Lentiviral rescue experiments reveal that such disruption selectively impairs synaptic vesicle priming but not Ca²⁺-triggered fusion of primed vesicles. We also find that Munc18-1 and complexin-1 bind simultaneously to SNARE complexes. These results suggest that Munc18-1 binding to SNARE complexes mediates synaptic vesicle priming and that the resulting primed state involves a Munc18-1-SNARE-complexin macromolecular assembly that is poised for Ca²⁺ triggering of fusion.
SNARE proteins play a critical role in intracellular membrane fusion by forming tight complexes that bring two membranes together and involve sequences called SNARE motifs. These motifs have a high ...tendency to form amphipathic coiled-coils that assemble into four-helix bundles, and often precede transmembrane regions. NMR studies in dodecylphosphocholine (DPC) micelles suggested that the N-terminal half of the SNARE motif from the neuronal SNARE synaptobrevin binds to membranes, which appeared to contradict previous biophysical studies of synaptobrevin in liposomes. NMR analyses of synaptobrevin reconstituted into nanodiscs and into liposomes now show that most of its SNARE motif, except for the basic C terminus, is highly flexible, exhibiting cross-peak patterns and transverse relaxation rates that are very similar to those observed in solution. Considering the proximity to the bilayer imposed by membrane anchoring, our data show that most of the synaptobrevin SNARE motif has a remarkable reluctance to bind membranes. This conclusion is further supported by NMR experiments showing that the soluble synaptobrevin SNARE motif does not bind to liposomes, even though it does bind to DPC micelles. These results show that nanodiscs provide a much better membrane model than DPC micelles in this system, and that most of the SNARE motif of membrane-anchored synaptobrevin is accessible for SNARE complex formation. We propose that the charge and hydrophobicity of SNARE motifs is optimized to enable formation of highly stable SNARE complexes while at the same time avoiding membrane binding, which could hinder SNARE complex assembly.
The SM (Sec1/Munc18-like) protein Munc18-1 and the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin/VAMP ...(vesicle-associated membrane protein) constitute the core fusion machinery for synaptic vesicle exocytosis. Strikingly, Munc18-1 interacts with neuronal SNARE proteins in two distinct modes (i.e., with isolated syntaxin-1 alone in a "closed" conformation and with assembled SNARE complexes containing syntaxin-1 in an "open" conformation). However, it is unclear whether the two modes of Munc18/SNARE interactions are linked. We now show that both Munc18/SNARE interaction modes involve the same low-affinity binding of the extreme syntaxin-1 N terminus to Munc18-1, suggesting that this binding connects the two Munc18/SNARE interaction modes to each other. Using transfected cells as an in vitro assay system, we demonstrate that truncated syntaxins lacking a transmembrane region universally block exocytosis, but only if they contain a free intact N terminus. This block is enhanced by coexpression of either Munc18-1 or SNAP-25, suggesting that truncated syntaxins block exocytosis by forming an untethered inhibitory SNARE complex/Munc18-1 assembly in which the N-terminal syntaxin/Munc18 interaction is essential. Introduction of an N-terminal syntaxin peptide that disrupts this assembly blocks neurotransmitter release in the calyx of Held synapse, whereas a mutant peptide that does not disrupt the SNARE complex/Munc18 assembly has no effect. Viewed together, our data indicate that binding of Munc18 to the syntaxin N terminus unites different modes of Munc18/SNARE interactions and is essential for exocytic membrane fusion.
Most core components of the neurotransmitter release machinery have homologues in other types of intracellular membrane traffic, likely underlying a universal mechanism of intracellular membrane ...fusion. However, no clear similarity between Munc13s and protein families generally involved in membrane traffic has been reported, despite the essential nature of Munc13s for neurotransmitter release. This crucial function was ascribed to a minimal Munc13 region called the MUN domain, which likely participates in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex (SNARE) assembly and is also found in Ca2+-dependent activator protein for secretion. We have now used comparative sequence and structural analyses to study the structure and evolutionary origin of the MUN domain. We found weak yet significant sequence similarities between the MUN domain and a set of protein subunits from several related vesicle tethering complexes, such as Sec6 from the exocyst complex and Vps53 from the Golgi-associated retrograde protein complex. Such an evolutionary relationship allows structure prediction of the MUN domain and suggests functional similarities between MUN domain-containing proteins and multisubunit tethering complexes such as exocyst, conserved oligomeric Golgi complex, Golgi-associated retrograde protein complex, and Dsl1p. These findings further unify the mechanism of neurotransmitter release with those of other types of intracellular membrane traffic and, in turn, support a role for tethering complexes in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex assembly.
The spindle checkpoint delays chromosome segregation in response to misaligned sister chromatids during mitosis, thus ensuring the fidelity of chromosome inheritance. Through binding to Cdc20, the ...Mad2 spindle checkpoint protein inhibits the target of this checkpoint, the ubiquitin protein ligase APC/C(Cdc20). We now show that without cofactor binding or covalent modification Mad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). The structure of N2-Mad2 has been determined by NMR spectroscopy. N2-Mad2 is much more potent in APC/C inhibition. Overexpression of a Mad2 mutant that specifically sequesters N2-Mad2 partially blocks checkpoint signaling in living cells. The two Mad2 conformers interconvert slowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. Our results suggest that the unusual two-state behavior of Mad2 is critical for spindle checkpoint signaling.
Synaptotagmin 2 resembles synaptotagmin 1, the Ca2+ sensor for fast neurotransmitter release in forebrain synapses, but little is known about synaptotagmin 2 function. Here, we describe a severely ...ataxic mouse strain that harbors a single, destabilizing amino‐acid substitution (I377N) in synaptotagmin 2. In Calyx of Held synapses, this mutation causes a delay and a decrease in Ca2+‐induced but not in hypertonic sucrose‐induced release, suggesting that synaptotagmin 2 mediates Ca2+ triggering of evoked release in brainstem synapses. Unexpectedly, we additionally observed in synaptotagmin 2 mutant synapses a dramatic increase in spontaneous release. Synaptotagmin 1‐deficient excitatory and inhibitory cortical synapses also displayed a large increase in spontaneous release, demonstrating that this effect was shared among synaptotagmins 1 and 2. Our data suggest that synaptotagmin 1 and 2 perform equivalent functions in the Ca2+ triggering of action potential‐induced release and in the restriction of spontaneous release, consistent with a general role of synaptotagmins in controlling ‘release slots’ for synaptic vesicles at the active zone.
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