title: Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.
abstract: 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 Ca(2+)-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.
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and the Howard Hughes Medical Institute, Stanford University, Stanford, California 94305.
authors.forename: Jiajie
authors.initials: J
authors.lastname: Diao
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Structural Biology, and.
authors.forename: Karen N
authors.initials: KN
authors.lastname: Colbert
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.
authors.forename: Ying
authors.initials: Y
authors.lastname: Lai
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and the Howard Hughes Medical Institute, Stanford University, Stanford, California 94305.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Pfuetzner
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and the Howard Hughes Medical Institute, Stanford University, Stanford, California 94305.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Padolina
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.
authors.forename: Sandro
authors.initials: S
authors.lastname: Vivona
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.
authors.forename: Susanne
authors.initials: S
authors.lastname: Ressl
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and the Howard Hughes Medical Institute, Stanford University, Stanford, California 94305.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Cipriano
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and the Howard Hughes Medical Institute, Stanford University, Stanford, California 94305.
authors.forename: Ucheor B
authors.initials: UB
authors.lastname: Choi
authors.affiliation: Structural Biology, and.
authors.forename: Niket
authors.initials: N
authors.lastname: Shah
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Structural Biology, and Photon Science and bill.weis@stanford.edu.
authors.forename: William I
authors.initials: WI
authors.lastname: Weis
authors.affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and the Howard Hughes Medical Institute, Stanford University, Stanford, California 94305 axel.brunger@stanford.edu.
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008561:Membrane Fusion; D051938:Munc18 Proteins / Q000235:genetics* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D009435:Synaptic Transmission; D013572:Synaptic Vesicles / Q000737:chemistry / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism; D050863:Synaptotagmin I / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; C095992:complexin I; D002118:Calcium
keywords: Complexin; Exocytosis; Fusion Machinery; Membrane Fusion; Munc18; Neurotransmitter Release; SNARE Proteins; Single Vesicle Assay; Synaptic Vesicle; Synaptotagmin