Munc18a Does Not Alter Fusion Rates Mediated by Neuronal SNAREs, Synaptotagmin, and Complexin
Autor: | Jiajie Diao, Sandro Vivona, Axel T. Brunger, Ucheor B. Choi, Richard A. Pfuetzner, Ying Lai, William I. Weis, Niket Shah, Yunxiang Zhang, Karen N. Colbert, Mark S. Padolina, Susanne Ressl, Daniel J. Cipriano |
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Rok vydání: | 2015 |
Předmět: |
Vesicle-Associated Membrane Protein 2
Fusion Machinery Gene Expression Membrane Fusion Munc18 Synaptic Transmission Biochemistry 0302 clinical medicine Neurobiology Single Vesicle Assay Phosphorylation Neurons 0303 health sciences Vesicle SNAP25 Recombinant Proteins Cell biology Synaptotagmin I Thermodynamics Synaptic Vesicles biological phenomena cell phenomena and immunity SNARE Proteins Protein Binding endocrine system Vesicle fusion Synaptic Vesicle Synaptosomal-Associated Protein 25 Nerve Tissue Proteins Complexin Biology Synaptic vesicle Exocytosis Synaptotagmin 1 03 medical and health sciences Munc18 Proteins Escherichia coli Animals Molecular Biology 030304 developmental biology Lipid bilayer fusion Cell Biology Synaptotagmin Rats Adaptor Proteins Vesicular Transport Kinetics nervous system Calcium Neurotransmitter Release 030217 neurology & neurosurgery |
Zdroj: | The Journal of Biological Chemistry |
ISSN: | 0021-9258 |
DOI: | 10.1074/jbc.m114.630772 |
Popis: | Background: Munc18-1 is an important factor for synaptic transmitter release, but its molecular mechanism remains an enigma. Results: Munc18a does not affect fusion kinetics. It can sequester syntaxin-1A molecules from the syntaxin-1A·SNAP-25 t-SNARE complex. Conclusion: Munc18a has no effect on complete fusion in conjunction with synaptotagmin-1, complexin-1, and neuronal SNAREs. Significance: This work provides new insights into Munc18-1 and its interactions with other synaptic proteins. Sec1/Munc18 (SM) proteins are essential for membrane trafficking, but their molecular mechanism remains unclear. Using a single vesicle-vesicle content-mixing assay with reconstituted neuronal SNAREs, synaptotagmin-1, and complexin-1, we show that the neuronal SM protein Munc18a/nSec1 has no effect on the intrinsic kinetics of both spontaneous fusion and Ca2+-triggered fusion between vesicles that mimic synaptic vesicles and the plasma membrane. However, wild type Munc18a reduced vesicle association ∼50% when the vesicles bearing the t-SNAREs syntaxin-1A and SNAP-25 were preincubated with Munc18 for 30 min. Single molecule experiments with labeled SNAP-25 indicate that the reduction of vesicle association is a consequence of sequestration of syntaxin-1A by Munc18a and subsequent release of SNAP-25 (i.e. Munc18a captures syntaxin-1A via its high affinity interaction). Moreover, a phosphorylation mimic mutant of Munc18a with reduced affinity to syntaxin-1A results in less reduction of vesicle association. In summary, Munc18a does not directly affect fusion, although it has an effect on the t-SNARE complex, depending on the presence of other factors and experimental conditions. Our results suggest that Munc18a primarily acts at the prefusion stage. |
Databáze: | OpenAIRE |
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