Munc18-1 binds to syntaxin-1A via two distinct sites referred to as the "closed" conformation and N terminus binding. The latter has been shown to stimulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis, whereas the former is believed to be inhibitory or dispensable. To precisely define the contributions of each binding mode, we have engineered Munc18-1/-2 double knockdown neurosecretory cells and show that not only syntaxin-1A and -1B but also syntaxin-2 and -3 are significantly reduced as a result of Munc18-1 and -2 knockdown. Syntaxin-1 was mislocalized and the regulated secretion was abolished. We next examined the abilities of Munc18-1 mutants to rescue the defective phenotypes. Mutation (K46E/E59K) of Munc18-1 that selectively prevents binding to closed syntaxin-1 was unable to restore syntaxin-1 expression, localization, or secretion. In contrast, mutations (F115E/ E132A) of Munc18-1 that selectively impair binding to the syntaxin-1 N terminus could still rescue the defective phenotypes. Our results indicate that Munc18-1 and -2 act in concert to support the expression of a broad range of syntaxins and to deliver syntaxin-1 to the plasma membrane. Our studies also indicate that the binding to the closed conformation of syntaxin is essential for Munc18-1 stimulatory action, whereas the binding to syntaxin N terminus plays a more limited role in neurosecretory cells.
Striking correlations exist between the abilities of domain-1 cleft mutants of Munc18-1 to bind and chaperone syntaxin-1 and their ability to restore vesicle docking and secretion.
J. Neurochem. (2010) 115, 1–10.
Abstract
Munc18‐1 plays essential roles in neurosecretion by interacting with syntaxin‐1 and controlling the formation of the soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNARE) complex. At least three important functions of Munc18‐1 have been proposed: (i) molecular chaperone of syntaxin‐1 for appropriate localization and expression of syntaxin‐1, (ii) priming/stimulation of the SNARE‐mediated membrane fusion, and (iii) docking of large dense‐core vesicles to the plasma membrane. Similarly, at least two different binding modes have been proposed for the interaction between Munc18‐1 and syntaxin‐1: (i) binary binding to a ‘closed’ conformation of syntaxin‐1, and (ii) binding to the N‐terminal peptide of syntaxin‐1, which is thought to enable an interaction with the quaternary SNARE complex and/or further stabilize the binary interaction between Munc18‐1 and closed syntaxin‐1. Recent structural analyses have identified critical Munc18‐1 residues implicated in these different modes of binding. These have recently been tested functionally in rescue experiments using Munc18‐1 null neurons, chromaffin cells and Munc18‐1/‐2 knockdown PC12 cells, allowing remarkable progress to be made in the structural/functional understanding of Munc18‐1. In this review, we summarize these recent advances and attempt to propose an updated model of the pleiotropic functions of Munc18‐1 in neuroexocytosis.
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.
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