The Longin SNARE VAMP7/TI-VAMP Adopts a Closed Conformation
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Abstract:
SNARE protein complexes are key mediators of exocytosis by juxtaposing opposing membranes, leading to membrane fusion. SNAREs generally consist of one or two core domains that can form a four-helix bundle with other SNARE core domains. Some SNAREs, such as syntaxin target-SNAREs and longin vesicular-SNAREs, have independent, folded N-terminal domains that can interact with their respective SNARE core domains and thereby affect the kinetics of SNARE complex formation. This autoinhibition mechanism is believed to regulate the role of the longin VAMP7/TI-VAMP in neuronal morphogenesis. Here we use nuclear magnetic resonance spectroscopy to study the longin-SNARE core domain interaction for VAMP7. Using complete backbone resonance assignments, chemical shift perturbations analysis, and hydrogen/deuterium exchange experiments, we conclusively show that VAMP7 adopts a preferentially closed conformation in solution. Taken together, the closed conformation of longins is conserved, in contrast to the syntaxin family of SNAREs for which mixtures of open and closed states have been observed. This may indicate different regulatory mechanisms for SNARE complexes containing syntaxins and longins, respectively.Keywords:
SNARE complex
STX1A
Munc-18
Neuroexocytosis requires the formation of a protein complex called the SNARE complex, formed when the v-SNARE VAMP2 located on vesicles containing the neurotransmitter, bind to the t-SNAREs syntaxin-1 and SNAP25 on the target plasma membrane. Munc18-1 contributes to exocytosis not only through its interaction with syntaxin-1 during its transport to the cell surface, but also by promoting SNARE complex formation. The focus of this thesis is to characterise these two functions at the molecular level by investigating how Munc18-1 interaction with syntaxin-1 is affected in early infantile epileptic encephalopathy (EIEE), and to elucidate the trafficking pathway underpinning Munc18-1-mediated syntaxin-1 transport to the cell surface in neurosecretory cells. Munc18-1 binding to the syntaxin-1 N-terminus has been proposed to promote SNARE complex formation leading to fusion, suggesting that Munc18-1 might play a role in priming of exocytosis. In Chapter 2, the significance of the Munc18-1:Syntaxin-1 N-terminus interaction in exocytosis and syntaxin-1 transport to the cell surface was investigated. Munc18-1 recombinant protein harbouring specific mutations in the hydrophobic pocket (F115E and E132A) designed to selectively abrogate the N-terminus interaction slightly reduced the binding affinity of Munc18-1 to syntaxin-1 but fully blocked binding to the SNARE complex. Overexpression of the Munc18-1- F115E/E132A double mutant in Munc18-1 knock down PC12 cells (KD43) not only rescued the transport of syntaxin-1 to the plasma membrane but also stimulated secretion of hGH and NPY. Total internal reflection fluorescence microscopy analysis show that the secretory vesicles had a reduced rate of vesicle fusion at the onset of stimulation. The Munc18-1 hydrophobic pocket is therefore essential for SNARE complex binding but removal of the Munc18-1:Syntaxin-1 Nterminus binding produced a limited effect on Ca2+-dependent exocytosis in PC12 cells. This suggests that alternative mechanisms are in place to support Munc18-1-mediated SNARE complex formation and priming. Munc18-1 binds syntaxin-1 in a closed conformation, where the N-terminal Habc domain of syntaxin-1 folds back onto its C-terminal H3 domain (syntaxin-1 SNARE motif) with high affinity. This binding prevents syntaxin-1 from participating in SNARE complex formation, and thus inhibits exocytosis. In Chapter 3, we dissected the contribution of the closed conformation binding and the N-terminus using Munc18-1/-2 double knockdown PC12 (DKD PC12) cells. In these cells, endogenous syntaxin-1 is mislocalised and regulated secretion inhibited. The expressions levels of syntaxin-1, -2 and -3 were also greatly reduced in the DKD PC12 cells. The Munc18-1- K46E/E59K mutant, specifically designed to inhibit syntaxin-1 closed conformation binding, was unable to restore syntaxin-1 expression levels, syntaxin-1 transport to cells surface and regulated viii secretion in these PC12 cells. Munc18-1-F115E/E132A mutant, previously shown to have impaired binding to syntaxin-1 N-terminus, was able to rescue syntaxin-1 transport to the cell surface and exocytosis in DKD PC12 cells. These results suggest that Munc18-1/-2 regulate syntaxin expression levels, its transport to the cell surface and exocytosis in PC12 cells. The binding of Munc18-1 to the syntaxin-1 closed conformation is therefore necessary for Munc18-1 stimulatory action, whereas the binding to syntaxin-1 N-terminus plays a limited role in neurosecretion. The roles of Munc18-1 in neuroexocytosis was further dissected in Chapter 4 by analysing the importance of Munc18-1 domain 1 in mediating binding to syntaxin-1 closed conformation and its contribution in chaperoning syntaxin-1 during its transport to the cell surface. The binding affinity of Munc18-1 domain 1 mutants (D34N, M38V, K46E, E59K, K63E, E66A) was tested and their effect on syntaxin-1 transport to the plasma membrane and secretion in DKD PC12 cells investigated. We found that Munc18-1 domain 1 mutant’s ability to rescue syntaxin-1 transport to the plasma membrane and exocytosis in DKD PC12 cells was correlated to the mutant’s binding affinity to syntaxin-1. These results demonstrated that Munc18-1 domain 1 is critical for syntaxin-1 closed conformation binding and that this binary interaction underpins Munc18-1 chaperoning activity. The significance of Munc18-1:Syntaxin-1 interaction during syntaxin-1 transport/traffic to the cell surface was characterised in Chapter 5. Using Munc18-1-C180Y, a structurally unstable mutant linked to early infantile epileptic encephalopathy (EIEE), we investigated the trafficking pathway underlying syntaxin-1 transport to the plasma membrane in DKD PC12 cells. Overexpression of the Munc18-1-C180Y-EmGFP in DKD PC12 resulted in the formation of fluorescent aggregates and failure to restore syntaxin-1 plasma membrane localisation. Regulated exocytosis was also affected due to the lack of the t-SNARE synatxin-1 on the plasma membrane. Importantly, Munc18-1-C180Y binding affinity to syntaxin-1 was similar to that of wild type Munc18-1. Our results demonstrate that structural instability of Munc18-1-C180Y interferes with the Munc18-1-mediated syntaxin-1 transport to the plasma membrane, which may be an underlying factor in EIEE. Using immunoisolation we purified Munc18-1 trafficking cargoes and identified Rab33b as a critical player in this trafficking pathway. This thesis elucidates the molecular basis of Munc18-1-mediated syntaxin-1 transport/traffic to the plasma membrane, a necessary step for functional regulated exocytosis, and highlights a novel role for Rab33b in promoting Munc18-1-dependent syntaxin-1 traffic. Abolishing Munc18- 1:Syntaxin-1 binding impairs Munc18-1-mediated syntaxin-1 trafficking and contributes to the establishment of diseases such as EIEE.
Munc-18
SNARE complex
Syntaxin 3
Vesicle fusion
STX1A
Synaptobrevin
SNAP25
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SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins are supposed to mediate the docking and/or fusion of the vesicle with the plasma membrane. However, it is not clearly understood how this process is regulated. In a search for potential SNARE regulators, we recently identified septin 5 (Sept5) as a novel SNARE interacting protein. Septins were first identified as filamentous proteins required for cytokinesis in yeast. Several septins have now been identified in mammals but little is known about their functions. We have previously shown that Sept5 is predominantly expressed in the brain, where it associates with vesicles and membranes through its interaction with the SNARE domain of syntaxin 1A. Furthermore, Sept5 appears to inhibit exocytosis, possibly by regulating vesicle targeting and/or fusion events. To gain insight into the role of Sept5, we have mapped the Sept5 domains important for syntaxin binding. We also investigated the ability of Sept5 to bind to syntaxin when in various protein complexes. Although Sept5 cannot bind an nSec1–syntaxin complex, it can bind syntaxin in a SNARE complex. This interaction is occluded by the binding of α-SNAP, suggesting that Sept5 may regulate the availability of SNARE proteins through its interaction with syntaxin and the 7 S complex.
Septin
Syntaxin 3
Munc-18
SNARE complex
STX1A
Vesicle fusion
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The fusion of the secretory vesicle with the plasma membrane requires the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes formed by synaptobrevin, syntaxin-1, and SNAP-25. Within the pathway leading to exocytosis, the transitions between the “open” and “closed” conformations of syntaxin-1 function as a switch for the fusion of vesicles with the plasma membranes; rapid assembly and disassembly of syntaxin-1 clusters on the plasma membrane provide docking and fusion sites for secretory vesicles in neuroendocrine cells; and the fully zippered trans-SNARE complex, which requires the orderly, rapid and accurate binding of syntaxin-1 to other SNARE proteins, play key roles in triggering fusion. All of these reactions that affect exocytosis under physiological conditions are tightly regulated by multiple factors. Here, we review the current evidence for the involvement of syntaxin-1 in the mechanism of neuroendocrine cell exocytosis, discuss the roles of multiple factors such as proteins, lipids, protein kinases, drugs, and toxins in SNARE complex-mediated membrane fusion, and present an overview of syntaxin-1 mutation-associated diseases with a view to developing novel mechanistic therapeutic targets for the treatment of neuroendocrine disorders.
Munc-18
Synaptobrevin
Syntaxin 3
STX1A
Vesicle fusion
SNAP25
SNARE complex
SNAP23
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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 H(abc) 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. PMID: 17301226 Funding information This work was supported by: NINDS NIH HHS, United States Grant ID: NS 37200 NINDS NIH HHS, United States Grant ID: R01 NS037200
SNARE complex
Munc-18
STX1A
Vesicular Transport Proteins
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SNARE complex
Munc-18
SNAP23
SNAP25
Synaptotagmin 1
Synaptobrevin
STX1A
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The regulation of SNARE complex assembly likely plays an important role in governing the specificity as well as the timing of membrane fusion. Here we identify a novel brain-enriched protein, amisyn, with a tomosyn- and VAMP-like coiled-coil-forming domain that binds specifically to syntaxin 1a and syntaxin 4 both in vitro and in vivo, as assessed by co-immunoprecipitation from rat brain. Amisyn is mostly cytosolic, but a fraction co-sediments with membranes. The amisyn coil domain can form SNARE complexes of greater thermostability than can VAMP2 with syntaxin 1a and SNAP-25 in vitro, but it lacks a transmembrane anchor and so cannot act as a v-SNARE in this complex. The amisyn coil domain prevents the SNAP-25 C-terminally mediated rescue of botulinum neurotoxin E inhibition of norepinephrine exocytosis in permeabilized PC12 cells to a greater extent than it prevents the regular exocytosis of these vesicles. We propose that amisyn forms nonfusogenic complexes with syntaxin 1a and SNAP-25, holding them in a conformation ready for VAMP2 to replace it to mediate the membrane fusion event, thereby contributing to the regulation of SNARE complex formation. PMID: 12145319
SNARE complex
Munc-18
STX1A
Syntaxin 3
Synaptobrevin
Immunoprecipitation
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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.
Synaptobrevin
STX1A
Munc-18
Syntaxin 3
SNARE complex
SNAP23
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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 H(abc) 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.
Munc-18
SNARE complex
STX1A
Vesicular Transport Proteins
Vesicle fusion
Synaptobrevin
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Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of alpha-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of alpha-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of alpha-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of alpha-SNAP potently inhibits Ca(2+)-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an alpha-SNAP mutant defective in NSF activation is used. We conclude that alpha-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for alpha-SNAP in the SNARE cycle that drives exocytotic membrane fusion.
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Synaptobrevin
Munc-18
STX1A
Vesicle fusion
SNAP25
Syntaxin 3
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Previously, we have demonstrated physical and functional interactions of the voltage-gated potassium channel Kv2.1 with the plasma membrane protein components of the exocytotic SNARE complex, syntaxin 1A, and the t-SNARE, syntaxin 1A/SNAP-25, complex. Importantly, the physical interaction of Kv2.1 with syntaxin was shown to be involved in the facilitation of secretion from PC12 cells, which was independent of potassium currents. Recently, we showed that also VAMP2, the vesicular SNARE, interacts physically and functionally with Kv2.1. Here, we first set out to test the interaction of the full SNARE, syntaxin/SNAP-25/VAMP2, complex with the channel. Using the interaction of VAMP2 with Kv2.1 in Xenopus oocytes as a probe, we showed that coexpression of the t-SNARE complex with VAMP2 abolished the VAMP2 effect on channel inactivation and reduced the amount of VAMP2 that coprecipitated with Kv2.1. Further, in vitro pull down assays showed that the full SNARE complex failed to interact with Kv2.1 N- and C-termini in tandem, in contrast to the individual SNARE components. This suggests that the interactions of the SNARE components with Kv2.1 are abolished upon their recruitment into a full SNARE complex, which does not interact with the channel. Other important findings arising from the in vitro study are that the t-SNARE complex, in addition to syntaxin, interacts with a specific C-terminal channel domain, C1a, shown to mediate the facilitation of release by Kv2.1 and that the presence of Kv2.1 N-terminus has crucial contribution to these interactions. These findings provide important insights into the understanding of the complex molecular events involved in the novel phenomenon of secretion facilitation in neuroendocrine cells by Kv2.1.
SNARE complex
Munc-18
Syntaxin 3
STX1A
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