Abstract
Assembly of SNARE complexes that mediate neurotransmitter release requires opening of a ‘closed’ conformation of UNC-64/syntaxin. Rescue of
unc-13/Munc13
mutant phenotypes by overexpressed ...open UNC-64/syntaxin suggested a specific function of UNC-13/Munc13 in opening UNC-64/ syntaxin. Here, we revisit the effects of open
unc-64
/syntaxin by generating knockin (KI) worms. The KI animals exhibit enhanced spontaneous and evoked exocytosis compared to WT animals. Unexpectedly, the open syntaxin KI partially suppresses exocytosis defects of various mutants, including
snt-1
/synaptotagmin,
unc-2/
P/Q/N-type Ca
2+
channel alpha-subunit and
unc-31/
CAPS, in addition to
unc-13/
Munc13 and
unc-10/
RIM, and enhanced exocytosis in
tom-1/
Tomosyn mutants. However, open syntaxin aggravates the defects of
unc-18/
Munc18 mutants. Correspondingly, open syntaxin partially bypasses the requirement of Munc13 but not Munc18 for liposome fusion. Our results show that facilitating opening of syntaxin enhances exocytosis in a wide range of genetic backgrounds, and may provide a general means to enhance synaptic transmission in normal and disease states.
Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting ...hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.
Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are ...bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate, but the mechanism by which they kill bacteria is unknown. Here we elucidate the mechanistic basis for RegIII bactericidal activity. We show that human RegIIIα (also known as HIP/PAP) binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore. We derive a three-dimensional model of the RegIIIα pore by docking the RegIIIα crystal structure into a cryo-electron microscopic map of the pore complex, and show that the model accords with experimentally determined properties of the pore. Lipopolysaccharide inhibits RegIIIα pore-forming activity, explaining why RegIIIα is bactericidal for Gram-positive but not Gram-negative bacteria. Our findings identify C-type lectins as mediators of membrane attack in the mucosal immune system, and provide detailed insight into an antibacterial mechanism that promotes mutualism with the resident microbiota.
SNARE proteins and synaptotagmin are key components of the complex machinery that controls Ca
2+-triggered neurotransmitter release but their mechanisms of action are under debate. Recent research ...has shed light on which biochemical and/or biophysical properties underlie SNARE and synaptotagmin function. SNARE proteins most likely have a role in membrane fusion owing to their ability to bring the synaptic vesicle and plasma membranes together and to perturb lipid bilayers through their transmembrane regions. Synaptotagmin acts as a Ca
2+ sensor and might cooperate with the SNAREs in accelerating fusion by binding simultaneously to the two membranes. However, recent research has strongly challenged the validity of models proposing that the SNAREs (with or without synaptotagmin) constitute ‘minimal membrane fusion machineries’ and has emphasized the essential nature of other proteins for exocytosis. Understanding the functions of these proteins will be crucial to reach a faithful description of the mechanisms of membrane fusion and neurotransmitter release.
Abstract
The presynaptic active zone protein Munc13 is essential for neurotransmitter release, playing key roles in vesicle docking and priming. Mechanistically, it is thought that the C
2
A domain ...of Munc13 inhibits the priming function by homodimerization, and that RIM disrupts the autoinhibitory homodimerization forming monomeric priming-competent Munc13. However, it is unclear whether the C
2
A domain mediates other Munc13 functions in addition to this inactivation–activation switch. Here, we utilize mutations that modulate the homodimerization and heterodimerization states to define additional roles of the Munc13 C
2
A domain. Using electron microscopy and electrophysiology in hippocampal cultures, we show that the C
2
A domain is critical for additional steps of vesicular release, including vesicle docking. Optimal vesicle docking and priming is only possible when Munc13 heterodimerizes with RIM via its C
2
A domain. Beyond being a switching module, our data suggest that the Munc13-RIM heterodimer is an active component of the vesicle docking, priming and release complex.
Reconstitution assays with proteoliposomes provide a powerful tool to elucidate the mechanism of neurotransmitter release, but it is important to understand how these assays report on membrane ...fusion, and recent studies with yeast vacuolar SNAREs uncovered asymmetry in the results of lipid mixing assays. We have investigated whether such asymmetry also occurs in reconstitution assays with the neuronal SNAREs, using syntaxin-1-SNAP-25-containing liposomes and liposomes containing synaptobrevin (T and V liposomes, respectively), and fluorescent probes to monitor lipid and content mixing simultaneously. Switching the fluorescent probes placed on the T and V liposomes, we observed a striking asymmetry in both lipid and content mixing stimulated by a fragment spanning the two C
domains of synaptotagmin-1, or by a peptide that spans the C-terminal half of the synaptobrevin SNARE motif. However, no such asymmetry was observed in assays performed in the presence of Munc18-1, Munc13-1, NSF and αSNAP, which coordinate the assembly-disassembly cycle of neuronal SNARE complexes. Our results show that switching fluorescent probes between the two types of liposomes provides a useful approach to better understand the reactions that occur between liposomes and detect heterogenous behavior in these reactions.
Ca
2+ binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca
2+-triggered exocytosis ...requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca
2+ binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca
2+-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca
2+-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca
2+-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca
2+-triggered neurotransmitter release.
Neurotransmitter release depends on the SNARE complex formed by syntaxin-1, synaptobrevin and SNAP-25, as well as on complexins, which bind to the SNARE complex and play active and inhibitory roles. ...A crystal structure of a Complexin-I fragment bearing a so-called 'superclamp' mutation bound to a truncated SNARE complex lacking the C-terminus of the synaptobrevin SNARE motif (SNAREΔ60) suggested that an 'accessory' α-helix of Complexin-I inhibits release by inserting into the C-terminus of the SNARE complex. Previously, isothermal titration calorimetry (ITC) experiments performed in different laboratories yielded apparently discrepant results in support or against the existence of such binding mode in solution (Trimbuch et al., 2014; Krishnakumar et al., 2015). Here, ITC experiments performed to solve these discrepancies now show that the region containing the Complexin-I accessory helix and preceding N-terminal sequences does interact with SNAREΔ60, but the interaction requires the polybasic juxtamembrane region of syntaxin-1 and is not affected by the superclamp mutation within the experimental error of these experiments.
Both SM proteins (for Sec1/Munc18-like proteins) and SNARE proteins (for soluble NSF-attachment protein receptors) are essential for intracellular membrane fusion, but the general mechanism of ...coupling between their functions is unclear, in part because diverse SM protein/SNARE binding modes have been described. During synaptic vesicle exocytosis, the SM protein Munc18-1 is known to bind tightly to the SNARE protein syntaxin-1, but only when syntaxin-1 is in a closed conformation that is incompatible with SNARE complex formation. We now show that Munc18-1 also binds tightly to assembled SNARE complexes containing syntaxin-1. The newly discovered Munc18-1/SNARE complex interaction involves contacts of Munc18-1 with the N-terminal Habc domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. Together with earlier studies, our results suggest that binding of Munc18-1 to closed syntaxin-1 is a specialization that evolved to meet the strict regulatory requirements of neuronal exocytosis, whereas binding of Munc18-1 to assembled SNARE complexes reflects a general function of SM proteins involved in executing membrane fusion.
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