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 -