Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

AbstractSec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. Next, SNAP-25 binds the templated SNAREs to form a partially-zippered SNARE complex. Finally, full zippering displaces Munc18-1. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

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Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

eLife ◽

10.7554/elife.41771 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 34

Author(s):

Junyi Jiao ◽

Mengze He ◽

Sarah A Port ◽

Richard W Baker ◽

Yonggang Xu ◽

...

Keyword(s):

Membrane Fusion ◽

Single Molecule ◽

Force Spectroscopy ◽

Sm Proteins ◽

Single Molecule Force Spectroscopy ◽

Helix Bundle ◽

Four Helix Bundle ◽

The Stability ◽

Sm Protein ◽

Template Complex

Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

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The Sec1/Munc18 protein Vps45 holds the Qa-SNARE Tlg2 in an open conformation

eLife ◽

10.7554/elife.60724 ◽

2020 ◽

Vol 9 ◽

Author(s):

Travis J Eisemann ◽

Frederick Allen ◽

Kelly Lau ◽

Gregory R Shimamura ◽

Philip D Jeffrey ◽

...

Keyword(s):

Snare Complex ◽

Snare Proteins ◽

Snare Protein ◽

Sm Proteins ◽

Closed Conformation ◽

Open Conformation ◽

Helical Hairpin ◽

Snare Motif ◽

Sm Protein ◽

Template Complex

Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

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Mso1p Regulates Membrane Fusion through Interactions with the Putative N-Peptide–binding Area in Sec1p Domain 1

Molecular Biology of the Cell ◽

10.1091/mbc.e09-07-0546 ◽

2010 ◽

Vol 21 (8) ◽

pp. 1362-1374 ◽

Cited By ~ 11

Author(s):

Marion Weber ◽

Konstantin Chernov ◽

Hilkka Turakainen ◽

Gerd Wohlfahrt ◽

Maria Pajunen ◽

...

Keyword(s):

Membrane Fusion ◽

Protein Interactions ◽

Peptide Binding ◽

Complex Function ◽

Snare Complex ◽

Targeted Mutagenesis ◽

Rab Gtpase ◽

Binding Modes ◽

Sm Proteins ◽

Sm Protein

Sec1p/Munc18 (SM) family proteins regulate SNARE complex function in membrane fusion through their interactions with syntaxins. In addition to syntaxins, only a few SM protein interacting proteins are known and typically, their binding modes with SM proteins are poorly characterized. We previously identified Mso1p as a Sec1p-binding protein and showed that it is involved in membrane fusion regulation. Here we demonstrate that Mso1p and Sec1p interact at sites of exocytosis and that the Mso1p–Sec1p interaction site depends on a functional Rab GTPase Sec4p and its GEF Sec2p. Random and targeted mutagenesis of Sec1p, followed by analysis of protein interactions, indicates that Mso1p interacts with Sec1p domain 1 and that this interaction is important for membrane fusion. In many SM family proteins, domain 1 binds to a N-terminal peptide of a syntaxin family protein. The Sec1p-interacting syntaxins Sso1p and Sso2p lack the N-terminal peptide. We show that the putative N-peptide binding area in Sec1p domain 1 is important for Mso1p binding, and that Mso1p can interact with Sso1p and Sso2p. Our results suggest that Mso1p mimics N-peptide binding to facilitate membrane fusion.

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SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion

The Journal of Cell Biology ◽

10.1083/jcb.201003148 ◽

2010 ◽

Vol 190 (1) ◽

pp. 55-63 ◽

Cited By ~ 68

Author(s):

Jingshi Shen ◽

Shailendra S. Rathore ◽

Lavan Khandan ◽

James E. Rothman

Keyword(s):

Membrane Fusion ◽

Complete Removal ◽

Snare Complex ◽

Sm Proteins ◽

Membrane Bilayer ◽

Core Domain ◽

Target Membrane ◽

Sm Protein ◽

Typical Configuration ◽

Intracellular Membrane Fusion

Sec1/Munc18 (SM) proteins activate intracellular membrane fusion through binding to cognate SNAP receptor (SNARE) complexes. The synaptic target membrane SNARE syntaxin 1 contains a highly conserved Habc domain, which connects an N-peptide motif to the SNARE core domain and is thought to participate in the binding of Munc18-1 (the neuronal SM protein) to the SNARE complex. Unexpectedly, we found that mutation or complete removal of the Habc domain had no effect on Munc18-1 stimulation of fusion. The central cavity region of Munc18-1 is required to stimulate fusion but not through its binding to the syntaxin Habc domain. SNAP-25, another synaptic SNARE subunit, contains a flexible linker and exhibits an atypical conjoined Qbc configuration. We found that neither the linker nor the Qbc configuration is necessary for Munc18-1 promotion of fusion. As a result, Munc18-1 activates a SNARE complex with the typical configuration, in which each of the SNARE core domains is individually rooted in the membrane bilayer. Thus, the SNARE four-helix bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of fusion.

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Autoinhibition of SNARE complex assembly by a conformational switch represents a conserved feature of syntaxins

Biochemical Society Transactions ◽

10.1042/bst0380209 ◽

2010 ◽

Vol 38 (1) ◽

pp. 209-212 ◽

Cited By ~ 9

Author(s):

Chris MacDonald ◽

Mary Munson ◽

Nia J. Bryant

Keyword(s):

Membrane Fusion ◽

Membrane Trafficking ◽

Snare Complex ◽

Cellular Transport ◽

Protein Families ◽

Sm Proteins ◽

Complex Assembly ◽

Receptor Complexes ◽

Snare Motif ◽

Sm Protein

Regulation and specificity of membrane trafficking are required to maintain organelle integrity while performing essential cellular transport. Membrane fusion events in all eukaryotic cells are facilitated by the formation of specific SNARE (soluble N-ethylmaleimide-sensitive fusion proteinattachment protein receptor) complexes between proteins on opposing lipid bilayers. Although regulation of SNARE complex assembly is not well understood, it is clear that two conserved protein families, the Sx (syntaxin) and the SM (Sec1p/Munc18) proteins, are central to this process. Sxs are a subfamily of SNARE proteins; in addition to the coiled-coil SNARE motif, Sxs possess an N-terminal, autonomously folded, triple-helical (Habc) domain. For some Sxs, it has been demonstrated that this Habc domain exerts an autoinhibitory effect on SNARE complex assembly by making intramolecular contacts with the SNARE motif. SM proteins regulate membrane fusion through interactions with their cognate Sxs. One hypothesis for SM protein function is that they facilitate a switch of the Sx from a closed to an open conformation, thus lifting the inhibitory action of the Habc domain and freeing the SNARE motif to participate in SNARE complexes. However, whether these regulatory mechanisms are conserved throughout the Sx/SM protein families remains contentious as it is not clear whether the closed conformation represents a universal feature of Sxs.

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