Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.
Bottom Line: 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.
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.Show MeSH
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Mentions: To test this hypothesis, we labeled SNAP-25 at residue 76 and reconstituted a mixture of labeled and unlabeled SNAP-25 molecules into PM vesicles together with unlabeled syntaxin-1A. The same reconstitution and immobilization protocols were performed as for all other single vesicle experiments in this work. We adjusted the ratio of labeled to unlabeled SNAP-25 molecules in order to obtain at most one labeled SNAP-25 molecule per PM vesicle. Note that we used a soluble SNAP-25 construct (i.e. without palmitoylation), so once a SNAP-25 molecule was dislodged from a t-SNARE complex it was expected to diffuse away from the surface. We monitored fluorescence from single SNAP-25 molecules before and 120 min after the Munc18a addition (Fig. 8). Consistent with our hypothesis of syntaxin-1A sequestration, we observed that incubation of Munc18a results in reduction of about 50% of surface-localized SNAP-25 molecules.
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.