Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins

PLoS One. 2017 Oct 31;12(10):e0187302. doi: 10.1371/journal.pone.0187302. eCollection 2017.

Abstract

The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone.

MeSH terms

  • Adipocytes / metabolism*
  • Membrane Fusion
  • Munc18 Proteins / metabolism*
  • Neurons / metabolism*
  • Protein Binding
  • Qa-SNARE Proteins / metabolism*
  • SNARE Proteins / metabolism*

Substances

  • Munc18 Proteins
  • Qa-SNARE Proteins
  • SNARE Proteins

Grants and funding

This work was supported by the Australian National Health and Medical Research Council (NHMRC, www.nhmrc.gov.au) through program grant 535921 (to JLM) and then project grants 1066069 and 1080995 (to AEW and JLM). AEW was supported by an NHMRC Peter Doherty Fellowship (569864); BMC by an NHMRC Career Development Fellowship (APP1061574); JLM by an Australian Research Council (www.arc.gov.au) Laureate Fellowship (FL0992138) and then NHMRC Fellowship (455829). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.