SNAREpin/Munc18 promotes adhesion and fusion of large vesicles to giant membranes

In neurons, soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1-24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Here we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a-Syx1a complex. membrane trafficking | SM proteins | protein crystallography N eurons communicate across specialized intercellular junctions called synapses. Arrival of an action potential in a presynaptic neuron triggers a calcium (Ca 2+ ) influx, which leads to the exocytosis of presynaptic vesicles and release of neurotransmitters for uptake via receptors in the postsynaptic neuron. Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of neurotransmitter-containing vesicles to the presynaptic plasma membrane, a critical step in Ca 2+ -triggered exocytosis. Three neuronal SNARE proteinssyntaxin 1a (Syx1a) and synaptosomal-associated protein of 25 kDa (SNAP25), located on the synaptic membrane, and vesicleassociated membrane protein 2 (VAMP2, also called synaptobrevin) combine to form a four-helix bundle, a process that drives membrane fusion. Syx1a contains an N-terminal regulatory region in addition to its C-terminal SNARE domain and adopts two conformations: a closed conformation, in which a three-helix bundle (designated Habc) in the N-terminal region binds intramolecularly to the SNARE domain, and an open conformation in which the SNARE domain binds SNAP25 and VAMP2 to form the ternary SNARE complex.The neuronal Sec1/Munc18a (SM)-like protein Munc18a is essential for synaptic vesicle fusion and associates with the neuronal SNARE proteins in at least two distinct modes. Munc18a

Section: *Derived From Crysol (30)mentioning

confidence: 90%

Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation

Colbert

Hattendorf

Weiß

et al. 2013

“…Munc18-1 has been shown to play dual roles in synaptic vesicle fusion. First, Munc18-1 positively regulates the SNARE-dependent fusion reaction by interacting with the trans-SNARE complex and accelerating the fusion kinetics (12,(25)(26)(27)(28)(29)(30)(31)(32)(33). Second, Munc18-1 binds to syntaxin-1 monomer and locks the latter into a "closed" configuration that prevents SNARE complex formation (34-36).…”

mentioning

confidence: 99%

Comparative studies of Munc18c and Munc18-1 reveal conserved and divergent mechanisms of Sec1/Munc18 proteins

Rathore

Lopez

et al. 2013

Self Cite

154

Sec1/Munc18 (SM) family proteins are essential for every vesicle fusion pathway. The best-characterized SM protein is the synaptic factor Munc18-1, but it remains unclear whether its functions represent conserved mechanisms of SM proteins or specialized activities in neurotransmitter release. To address this question, we dissected Munc18c, a functionally distinct SM protein involved in nonsynaptic exocytic pathways. We discovered that Munc18c binds to the trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex and strongly accelerates the fusion rate. Further analysis suggests that Munc18c recognizes both vesicle-rooted SNARE and target membrane-associated SNAREs, and promotes trans-SNARE zippering at the postdocking stage of the fusion reaction. The stimulation of fusion by Munc18c is specific to its cognate SNARE isoforms. Because Munc18-1 regulates fusion in a similar manner, we conclude that one conserved function of SM proteins is to bind their cognate trans-SNARE complexes and accelerate fusion kinetics. Munc18c also binds syntaxin-4 monomer but does not block target membrane-associated SNARE assembly, in agreement with our observation that six-to eightfold increases in Munc18c expression do not inhibit insulinstimulated glucose uptake in adipocytes. Thus, the inhibitory "closed" syntaxin binding mode demonstrated for Munc18-1 is not conserved in Munc18c. Unexpectedly, we found that Munc18c recognizes the N-terminal region of the vesicle-rooted SNARE, whereas Munc18-1 requires the C-terminal sequences, suggesting that the architecture of the SNARE/SM complex likely differs across fusion pathways. Together, these comparative studies of two distinct SM proteins reveal conserved as well as divergent mechanisms of SM family proteins in intracellular vesicle fusion. membrane fusion | vesicle transport | exocytosis T he fusion of intracellular vesicles with target membranes requires two classes of conserved proteins: SNAREs and SM (Sec1/Munc18) proteins (1, 2). SNAREs are membrane-associated proteins that contain characteristic stretches of 60-70 amino acids known as core domains or SNARE motifs. Fusion is initiated when the core domains of the vesicle-rooted SNARE (v-SNARE) and the target membrane-associated SNAREs (t-SNAREs) zipper into a four-helix trans-SNARE complex between two apposed bilayers (2-5). N-to C-terminal zippering of the trans-SNARE complex brings the two membranes into close apposition to fuse (6-8).First isolated in genetic screens in yeast and nematodes (9, 10), SM proteins are hydrophilic factors of 60-70 kDa that regulate membrane fusion through binding to their cognate SNAREs (11-13). SM proteins exhibit a similar loss-of-function phenotype as that of SNAREs (i.e., abrogation of fusion) and are essential for every pathway of intracellular vesicle fusion (14-16). Mutations of SM proteins give rise to a number of human diseases, including epilepsy and inflammatory disorders, as well as arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome (17-...

“…The most intensely studied form of intracellular membrane fusion is calcium-triggered neurotransmitter release at the chemical synapse, which serves as the brain's major form of cell-cell communication (15)(16)(17)(18)(19). Neurotransmitter secretion is mediated by the fusion of exocytic vesicles with the plasma membrane and requires the v-SNARE vesicle-associated membrane protein 2 (VAMP2; also known as synaptobrevin-2), the t-SNAREs syntaxin-1 and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-25, and the SM protein Munc18-1/ nSec1 (UNC-18 in nematodes and ROP in flies) (20)(21)(22)(23)(24)(25)(26)(27)(28).…”

mentioning

confidence: 99%

Syntaxin N-terminal peptide motif is an initiation factor for the assembly of the SNARE–Sec1/Munc18 membrane fusion complex

Rathore

Bend

et al. 2010

Self Cite

116

142

Intracellular membrane fusion is mediated by the concerted action of N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. During fusion, SM proteins bind the N-terminal peptide (N-peptide) motif of the SNARE subunit syntaxin, but the function of this interaction is unknown. Here, using FRET-based biochemical reconstitution and Caenorhabditis elegans genetics, we show that the N-peptide of syntaxin-1 recruits the SM protein Munc18-1/nSec1 to the SNARE bundle, facilitating their assembly into a fusion-competent complex. The recruitment is achieved through physical tethering rather than allosteric activation of Munc18-1. Consistent with the recruitment role, the Npeptide is not spatially constrained along syntaxin-1, and it is functional when translocated to another SNARE subunit SNAP-25 or even when simply anchored in the target membrane. The N-peptide function is restricted to an early initiation stage of the fusion reaction. After association, Munc18-1 and the SNARE bundle together drive membrane merging without further involving the N-peptide. Thus, the syntaxin N-peptide is an initiation factor for the assembly of the SNARE-SM membrane fusion complex.I ntracellular membrane fusion is the basis of a wide range of fundamental biological processes, including organelle maintenance, hormone secretion, and inside-outside distribution of receptors and transporters. The merging of intracellular membrane bilayers is mediated by a fusion complex comprised of SNAREs and Sec1/Munc18 (SM) proteins (1). The core of the fusion machinery is the trans-SNARE complex (SNAREpin) formed by the pairing of the vesicle-rooted SNARE (v-SNARE) with the target membrane-associated SNAREs (t-SNAREs) (2-5). N-to C-terminal zippering of the trans-SNARE complex brings two membranes into close apposition and helps to overcome the energy barrier for fusion (6-10). SM proteins are soluble factors of 60-70 kDa that directly interact with their cognate trans-SNARE complexes to promote the speed and specificity of a fusion reaction (11)(12)(13)(14).Each fusion pathway in the cell requires a specific subset of SNAREs and SM proteins (15). The most intensely studied form of intracellular membrane fusion is calcium-triggered neurotransmitter release at the chemical synapse, which serves as the brain's major form of cell-cell communication (15)(16)(17)(18)(19). Neurotransmitter secretion is mediated by the fusion of exocytic vesicles with the plasma membrane and requires the v-SNARE vesicle-associated membrane protein 2 (VAMP2; also known as synaptobrevin-2), the t-SNAREs syntaxin-1 and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-25, and the SM protein Munc18-1/ nSec1 (UNC-18 in nematodes and ROP in flies) (20-28).The interaction between SNAREs and SM proteins involves multiple binding modes. The primary target of SM proteins is believed to be the four-helix SNARE bundle (29-31). Assembled from the SNARE motifs and the transmembrane domains of t-and vSNAREs (4, 5), the SNARE bun...