Munc18-1: sequential interactions with the fusion machinery stimulate vesicle docking and priming

TY - JOUR

T1 - Munc18-1: sequential interactions with the fusion machinery stimulate vesicle docking and priming

AU - Gulyás-Kovács, Attila

AU - de Wit, Heidi

AU - Milosevic, Ira

AU - Kochubey, Olexiy

AU - Toonen, Ruud

AU - Klingauf, Jürgen

AU - Verhage, Matthijs

AU - Sørensen, Jakob B

N1 - Keywords: Amino Acid Sequence; Animals; Cattle; Cell Line; Cells, Cultured; Chromaffin Cells; Exocytosis; Humans; Membrane Fusion; Mice; Molecular Sequence Data; Munc18 Proteins; Protein Binding; Protein Structure, Secondary; Synaptic Vesicles

PY - 2007

Y1 - 2007

N2 - Exocytosis of secretory or synaptic vesicles is executed by a mechanism including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Munc18-1 is a part of this fusion machinery, 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 stimulatory 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. In Munc18-1 null chromaffin cells, vesicle docking is abolished and syntaxin levels are reduced. A mutation that diminished Munc18 binding to syntaxin1 in vitro attenuated the vesicle-docking step but rescued vesicle priming in excess of docking. Conversely, expressing the Munc18-2 isoform, which also displays binding to closed syntaxin1, rescued vesicle docking identical with Munc18-1 but impaired more 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.

AB - Exocytosis of secretory or synaptic vesicles is executed by a mechanism including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Munc18-1 is a part of this fusion machinery, 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 stimulatory 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. In Munc18-1 null chromaffin cells, vesicle docking is abolished and syntaxin levels are reduced. A mutation that diminished Munc18 binding to syntaxin1 in vitro attenuated the vesicle-docking step but rescued vesicle priming in excess of docking. Conversely, expressing the Munc18-2 isoform, which also displays binding to closed syntaxin1, rescued vesicle docking identical with Munc18-1 but impaired more 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.

U2 - 10.1523/JNEUROSCI.0658-07.2007

DO - 10.1523/JNEUROSCI.0658-07.2007

M3 - Journal article

C2 - 17687045

SN - 0270-6474

VL - 27

SP - 8676

EP - 8686

JO - The Journal of neuroscience : the official journal of the Society for Neuroscience

JF - The Journal of neuroscience : the official journal of the Society for Neuroscience

IS - 32

ER -