title: Dual modes of Munc18-1/SNARE interactions are coupled by functionally critical binding to syntaxin-1 N terminus.
abstract: 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.
authors.affiliation: Department of Neuroscience, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. mkhvoc@mednet.swmed.edu
authors.forename: Mikhail
authors.initials: M
authors.lastname: Khvotchev
authors.forename: Irina
authors.initials: I
authors.lastname: Dulubova
authors.forename: Jianyuan
authors.initials: J
authors.lastname: Sun
authors.forename: Han
authors.initials: H
authors.lastname: Dai
authors.forename: Josep
authors.initials: J
authors.lastname: Rizo
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Südhof
mesh_terms: D000818:Animals; D005089:Exocytosis / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D051938:Munc18 Proteins / Q000737:chemistry / Q000378:metabolism*; D016716:PC12 Cells; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D050600:SNARE Proteins / Q000737:chemistry / Q000378:metabolism*; D050827:Syntaxin 1 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D051938:Munc18 Proteins; D010446:Peptide Fragments; D050600:SNARE Proteins; D050827:Syntaxin 1