The Interaction of Munc18-1 Helix 11 and 12 with the Central Region of the VAMP2 SNARE Motif Is Essential for SNARE Templating and Synaptic Transmission

André, Timon; Classen, Jessica; Brenner, Philipp; Betts, Matthew J.; Dörr, Bernhard; Kreye, Susanne; Zuidinga, Birte; Meijer, Marieke; Russell, Robert B.; Verhage, Matthijs; Söllner, Thomas H.

doi:10.1523/eneuro.0278-20.2020

Cited by 25 publications

(32 citation statements)

References 56 publications

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“…3J). These results are consistent with our cryo-EM structures and support the notion that the physiological effects of the Q301D mutation arise from impairment of Munc18-1 binding to synaptobrevin (35) rather than to Munc13-1 (34).…”

Section: Alteration Of Munc18-1-snare Interactions By Mutagenesissupporting

confidence: 91%

“…To impair binding of Munc18-1 to syntaxin-1 at a different site, we used an S42Q mutation expected to disrupt interactions with the syntaxin-1 SNARE motif C-terminus. We also included the L307R and L348R mutations that disrupt synaptobrevin binding and/or template complex formation ( 24, 30 ); a E352K mutation that we designed to also disrupt Munc18-1-synaptobrevin binding; and a Q301D mutation that impairs neurotransmitter release and was reported to impair binding of Munc18-1 to synaptobrevin or to Munc13-1 ( 34, 35 ). The locations of the mutated residues are shown in Figs.…”

Section: Main Textmentioning

confidence: 99%

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SNARE assembly enlightened by cryo-EM structures of a synaptobrevin-Munc18-1-syntaxin-1 complex

Stepien

Zhang

et al. 2022

Preprint

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Munc18-1 forms a template to recruit and organize assembly of the SNARE complex that triggers neurotransmitter release, binding first to a closed conformation of syntaxin-1 where its N-terminal region interacts with the SNARE motif, and later binding to synaptobrevin. However, the mechanism of SNARE complex assembly remains unclear. Here we report two cryo-EM structures of Munc18-1 bound to cross-linked syntaxin-1 and synaptobrevin. The structures allow visualization of how syntaxin-1 opens and reveal how part of the syntaxin-1 N-terminal region can help nucleate interactions between the N-termini of the syntaxin-1 and synaptobrevin SNARE motifs while their C-termini bind to distal sites of Munc18-1. Mutagenesis, SNARE complex assembly assays and reconstitution experiments support a model whereby these interactions are critical to initiate SNARE complex assembly.One Sentence SummaryCryo-EM structures reveal key insights into the molecular mechanism of neuronal SNARE complex assembly templated by Munc18-1

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Section: Alteration Of Munc18-1-snare Interactions By Mutagenesissupporting

confidence: 91%

Section: Main Textmentioning

confidence: 99%

SNARE assembly enlightened by cryo-EM structures of a synaptobrevin-Munc18-1-syntaxin-1 complex

Stepien

Zhang

et al. 2022

Preprint

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“…The first four control internal membrane trafficking, while the three MUNC18 proteins control exocytosis (for a review, see Toonen and Verhage, 2003). MUNC18 proteins bind syntaxins (Thurmond et al, 1998;Misura et al, 2000;Kauppi et al, 2002;Dulubova et al, 2007;Burkhardt et al, 2008;Bin et al, 2013), promote docking of secretory vesicles (Voets et al, 2001; and probably serve as a template for SNAREcomplex assembly, which drives exocytosis (Parisotto et al, 2014;Sitarska et al, 2017;Jiao et al, 2018;Meijer et al, 2018;Wang et al, 2019;André et al, 2020). MUNC18-2 acts in blood platelets, cytotoxic T lymphocytes, natural killer cells and mast cells (Côte et al, 2009;Hackmann et al, 2013;Gutierrez et al, 2018;Cardenas et al, 2019); MUNC18-3 in adipocytes (Tamori et al, 1998;Thurmond et al, 2000); and MUNC18-1 in chromaffin cells and the posterior pituitary (Voets et al, 2001;Korteweg et al, 2005) and in synaptic vesicle (SV) exocytosis in neurons (Verhage et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Munc18-1 Is Essential for Neuropeptide Secretion in Neurons

Puntman¹,

Arora

Farina³

et al. 2021

J. Neurosci.

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Neuropeptide secretion from dense-core vesicles (DCVs) controls many brain functions. Several components of the DCV exocytosis machinery have recently been identified, but the participation of a SEC1/MUNC18 (SM) protein has remained elusive. Here, we tested the ability of the three exocytic SM proteins expressed in the mammalian brain, MUNC18-1/2/3, to support neuropeptide secretion. We quantified DCV exocytosis at a single vesicle resolution on action potential (AP) train-stimulation in mouse CNS neurons (of unknown sex) using pHluorin-tagged and/or mCherry-tagged neuropeptide Y (NPY) or brainderived neurotrophic factor (BDNF). Conditional inactivation of Munc18-1 abolished all DCV exocytosis. Expression of MUNC18-1, but not MUNC18-2 or MUNC18-3, supported DCV exocytosis in Munc18-1 null neurons. Heterozygous (HZ) inactivation of Munc18-1, as a model for reduced MUNC18-1 expression, impaired DCV exocytosis, especially during the initial phase of train-stimulation, when the release was maximal. These data show that neurons critically and selectively depend on MUNC18-1 for neuropeptide secretion. Impaired neuropeptide secretion may explain aspects of the behavioral and neurodevelopmental phenotypes that were observed in Munc18-1 HZ mice.

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“…Vps33 only has very weak homology with UNC-18, and yet the engineered Sec1 provided significant rescue. The rescue we observed provides the strongest evidence yet for the physiological importance of the templating functions of the SM proteins, which thus far have only been minimally explored in vivo (André et al, 2020). Second, the recent structure of the yeast Golgi SNARE Tlg2 bound to Vps45 indicates that SM proteins can interact with the Habc domain and the SNARE domain in an open conformation (Eisemann et al, 2020).…”

Section: Discussionmentioning

confidence: 80%

Syntaxin Habc is required to open Unc18 to template SNARE assembly

Parra-Rivas

Palfreyman

et al. 2022

Preprint

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SNARE and Unc18 proteins form the core of the membrane fusion complex at synapses. The fusion machinery is evolutionarily ancient and mediates constitutive fusion in yeast. We demonstrate that the SNARE and Unc18 machinery in the nematode C. elegans can be replaced by yeast proteins and still carry out synaptic transmission. However, substitutions of individual components from yeast disrupts fusion. To understand the functional interactions within the core machinery we adopted an interspecies complementation approach using yeast. Synaptic transmission could be restored in chimeras when two key interfaces were present: a novel Habc-Unc18 contact site and an Unc18-SNARE motif contact site. An open form of Unc18 could bypass the requirement for the Habc-Unc18 interface. Together, these data suggest that the Habc domain of syntaxin is required for Unc18 to adopt an open conformation; open Unc18 then templates SNARE complex formation.

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The Interaction of Munc18-1 Helix 11 and 12 with the Central Region of the VAMP2 SNARE Motif Is Essential for SNARE Templating and Synaptic Transmission

Abstract: The interaction of Munc18-1 helix 11 and 12 with the central region of the VAMP2 SNARE motif is essential for SNARE templating and synaptic transmission

Cited by 25 publications

References 56 publications

SNARE assembly enlightened by cryo-EM structures of a synaptobrevin-Munc18-1-syntaxin-1 complex

SNARE assembly enlightened by cryo-EM structures of a synaptobrevin-Munc18-1-syntaxin-1 complex

Munc18-1 Is Essential for Neuropeptide Secretion in Neurons

Syntaxin Habc is required to open Unc18 to template SNARE assembly

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