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
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