Cellular/Molecular Munc18-1: Sequential Interactions with the Fusion Machinery Stimulate Vesicle Docking and Priming

Exocytosis of secretory or synaptic vesicles is executed by amechanism including the SNARE (solubleN-ethylmaleimide-sensitive factor attachmentprotein receptor) proteins.Munc18-1 is apart of this fusionmachinery, but its role is controversial because it is indispensable for fusion but also inhibits the assembly of purified SNAREs in vitro. This inhibition reflects the binding of Munc18-1 to a closed conformation of the target-SNARE syntaxin1. The controversy would be solved if binding to closed syntaxin1 were shown to be stimula-tory for vesicle fusion and/or additional essential interactions were identified between Munc18-1 and the fusion machinery. Here, we provide evidence for both notions by dissecting sequential steps of the exocytotic cascade while expressing Munc18 variants in the Munc18-1 null background. InMunc18-1 null chromaffin cells, vesicle docking is abolished and syntaxin levels are reduced. Amutation that diminished Munc18 binding to syntaxin1 in vitro attenuated the vesicle-docking step but rescued vesicle priming in excess of docking.Conversely, expressing theMunc18-2 isoform,whichalsodisplaysbinding to closed syntaxin1, rescuedvesicle docking identical withMunc18-1 but impairedmore downstream vesicle priming steps. All Munc18 variants restored syntaxin1 levels at least to wild-type levels, showing that the docking phenotype is not caused by syntaxin1 reduction. None of the Munc18 variants affected vesicle fusion kinetics or fusion pore duration. In conclusion, binding of Munc18-1 to closed syntaxin1 stimulates vesicle docking and a distinct interaction mode regulates the consecutive priming step. Key words:Munc18-1; chromaffin cells; amperometry; capacitance measurements; SNARE proteins; exocytosi