The N-peptide-binding mode is critical to Munc18-1 function in synaptic exocytosis

J Biol Chem. 2018 Nov 23;293(47):18309-18317. doi: 10.1074/jbc.RA118.005254. Epub 2018 Oct 1.

Abstract

Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons. The physiological role of the N-peptide-binding mode, however, remains unclear. In this work, we addressed this key question using a "clogged" Munc18-1 protein, in which an ectopic copy of the syntaxin N-peptide motif was directly fused to Munc18-1. We found that the ectopic N-peptide motif blocks the N-peptide-binding pocket of Munc18-1, preventing the latter from binding to the native N-peptide motif on syntaxin-1. In a reconstituted system, we observed that clogged Munc18-1 is defective in promoting SNARE zippering. When introduced into induced neuronal cells (iN cells) derived from human pluripotent stem cells, clogged Munc18-1 failed to mediate synaptic exocytosis. As a result, both spontaneous and evoked synaptic transmission was abolished. These genetic findings provide direct evidence for the crucial role of the N-peptide-binding mode of Munc18-1 in synaptic exocytosis. We suggest that clogged SM proteins will also be instrumental in defining the physiological roles of the N-peptide-binding mode in other vesicle-fusion pathways.

Keywords: Munc18; N-peptide; SM protein; SNARE proteins; exocytosis; membrane function; membrane fusion; membrane proteins; membrane reconstitution; organelle; synaptic transmission; vesicle transport.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Exocytosis*
  • Humans
  • Munc18 Proteins / chemistry*
  • Munc18 Proteins / genetics
  • Munc18 Proteins / metabolism*
  • Neurons / chemistry
  • Neurons / metabolism
  • Peptides / chemistry
  • Peptides / metabolism*
  • Protein Binding
  • Protein Transport
  • Signal Transduction
  • Synapses / chemistry
  • Synapses / genetics
  • Synapses / metabolism*
  • Synaptic Transmission
  • Syntaxin 1 / chemistry
  • Syntaxin 1 / genetics
  • Syntaxin 1 / metabolism

Substances

  • Munc18 Proteins
  • Peptides
  • Syntaxin 1

Associated data

  • PDB/3PUJ