Background: Munc18-1 has multiple roles in neuronal exocytosis by regulating SNARE proteins. Results: Mutations within domain-3a of Munc18-1 perturb syntaxin-1 chaperoning function and exocytosis. Conclusion: Domain-3a plays a crucial role in syntaxin-1 chaperoning in addition to the priming function, and Pro-335 is pivotal in regulating the balance between these two functions. Significance: This work provides mechanistic insights about how Munc18-1 controls exocytosis.

Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of syntaxin isoforms. However, the key syntaxin that, crucially, participates in the degranulation -whose levels and intracellular localization are regulated by Munc18-2 -remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' syntaxin-3. Furthermore, knockdown of syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed syntaxin-3, which is essential for degranulation by chaperoning syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion.

It is proposed that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which depends on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane.