Munc18b regulates core SNARE complex assembly and constitutive exocytosis by interacting with the N-peptide and the closed-conformation C-terminus of syntaxin 3.
The interaction between SM (Sec1/Munc18) and SNARE (soluble N-ethylmaleimide-sensitive factor-attachment receptor) proteins constitutes the core eukaryotic membrane fusion machinery which manages exocytosis by mediating fusion of constitutively exocytic vesicles with the plasma membrane. However, mechanistic details on the nature and the physiological impact of SM-SNARE interactions remain largely elusive. Detailed characterization of the interaction profiles between Munc18b and its cognate SNAREs, Stx3 (syntaxin 3), SNAP-23 (soluble N-ethylmaleimide-attachment protein 23) and VAMP8 (vesicle-associated membrane protein 8), revealed that Munc18b binds Stx3, VAMP8 and the assembled core SNARE complex consisting of Stx3, SNAP-23 and VAMP8. Dissection of the Munc18b-Stx3 heterodimer suggested that Munc18b interacts with Stx3's conserved N-peptide as well as with its closed-conformation C-terminus encompassing the Habc domain, a linker and the SNARE (H3) motif. Deletion of the Habc domain or mutations interrupting the intramolecular binding of the Habc and H3 domains abrogated the Munc18b-Stx3 interaction. Although only the N-peptide deletion mutant, but not the soluble wild-type Stx3, is assembled into the core SNARE complex in the presence of Munc18b in vitro, ectopic expression of this SM protein increases constitutive exocytosis in mammalian cells. Our results suggest that Munc18b is functionally coupled to the assembly of exocytic SNARE complexes and increases exocytosis by interacting with the N-peptide and closed-conformation C-terminus of Stx3, thereby neutralizing the secretion-inhibitory effect of this SNARE.
Peng
Ren-Wang
RW
Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
Guetg
Claudio
C
Abellan
Eric
E
Fussenegger
Martin
M
D005089:Exocytosis; D006367:HeLa Cells; D006801:Humans; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*
D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins