Structure of the Munc18c/Syntaxin4 N-peptide complex defines universal features of the N-peptide binding mode of Sec1/Munc18 proteins

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...

Section: Discussioncontrasting

confidence: 48%

“…The hydrophobic residues insert into a peripheral pocket on the cognate SM protein ( Fig. 1 A and B) (33,34). First shown in the Golgi and endocytic SNAREs (35,36), the N-peptide binding mode was later found to be widespread among SM-syntaxin pairs (37)(38)(39)(40).…”

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

116

142

“…The structures of SNARE complexes are conserved across vesicle fusion pathways (72)(73)(74). Similarly, all SM proteins adopt a compact, arch-like configuration (34,35,58,75,76). However, the architecture of the SNARE/ SM complex appears to differ significantly across vesicle fusion pathways.…”

Section: Munc18mentioning

confidence: 87%

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-...

“…Since the finding of the interaction between the Munc18 hydrophobic pocket and the N-peptide of syntaxin (25,26), which has been shown to support the binding of Munc18-1 to the SNARE complex and facilitates the SNARE-mediated liposome fusion (20), many efforts have been made to identify the crucial role of this interaction in physiological membrane fusion. Several studies suggest the important role of syntaxin-1 N-peptide (27,28,30).…”

Section: Discussionmentioning

confidence: 99%

“…The priming function of Munc18 is believed to be mediated in part through the interaction between the Munc18 hydrophobic pocket region and the syntaxin Npeptide. X-ray crystallography determined the structure of the hydrophobic pocket region of Munc18-1 and Munc18-3 bound with the N-peptide of their cognate syntaxins: syntaxin-1 and syntaxin-4, respectively (25,26).…”

mentioning

confidence: 99%

Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation

Bin

Jung

Piggott

et al. 2013

The function of the Munc18-1 protein hydrophobic pocket, which interacts with the syntaxin-1 N-terminal peptide, has been highly controversial in neurosecretion. Recent analysis of patients with familial hemophagocytic lymphohistiocytosis type 5 has identified the E132A mutation in the hydrophobic pocket of Munc18-2, prompting us to examine the role of this region in the context of immune cell secretion. Double knockdown of Munc18-1 and Munc18-2 in RBL-2H3 mast cells eliminates both IgE-dependent and ionomycin-induced degranulation and causes a significant reduction in syntaxin-11 without altering expressions of the other syntaxin isoforms examined. These phenotypes were effectively rescued on reexpression of wild-type Munc18-1 or Munc18-2 but not the mutants (F115E, E132A, and F115E/E132A) in the hydrophobic pocket of Munc18. In addition, these mutants show that they are unable to directly interact with syntaxin-11, as tested through protein interaction experiments. Our results demonstrate the crucial roles of the hydrophobic pocket of Munc18 in mast cell degranulation, which include the regulation of syntaxin-11. We also suggest that the functional importance of this region is significantly different between neuronal and immune cell exocytosis.membrane fusion | FHL5