Abstract
Mammalian-regulated secretion is absolutely dependent on four evolutionarily conserved proteins: three SNARE proteins and munc18. Dissecting the functional outcomes of the spatially organized protein interactions between these factors has been difficult because of the close interrelationship between different binding modes. Here, we investigated the spatial distribution of single munc18 molecules at the plasma membrane of cells and the underlying interactions between syntaxin and munc18. Disruption of munc18 binding to the N-terminal peptide motif of syntaxin did not alter munc18 localization on the plasma membrane but had a pronounced influence on the behavior of secretory vesicles and their likelihood to undergo fusion. We therefore conclude that interaction with the syntaxin N-peptide can confer differential release probabilities to secretory vesicles and may contribute to the delineation of secretory vesicle pools.
Highlights
In all specialized secretory cells, regulated exocytosis is mediated by three central players; the vesicular membrane protein synaptobrevin (v-SNARE) and plasma membrane proteins SNAP-25 and syntaxin (t-SNAREs) [1, 2]
It was suggested that munc18 interacts with the v-SNARE synaptobrevin in vitro, albeit at a far lower affinity than that observed for syntaxin [28]
Mode 1 binding is important in facilitating the trafficking of syntaxin to the plasma membrane [9] whereas modes 2 and 3 are involved in vesicle mobilization [21], SNARE complex binding [24], regulating the rate of membrane fusion in vitro [25], and in synaptic vesicle “priming” [29]
Summary
In all specialized secretory cells, regulated exocytosis is mediated by three central players; the vesicular membrane protein synaptobrevin (v-SNARE) and plasma membrane proteins SNAP-25 (synaptosome-associated protein 25 kDa) and syntaxin (t-SNAREs) [1, 2]. We investigated the spatial distribution of single munc18 molecules at the plasma membrane of cells and the underlying interactions between syntaxin and munc18. Fluorescence intensity covariance analysis in these cells confirmed that the three mutants that disrupted mode 2/3 interaction in vitro had a significant effect on syntaxin intracellular localization, with munc18[I127A] having the largest influence (Fig. 1D and supplemental Fig. 2).
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