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: Having established that Munc18a binds to the cytoplasmic (non-membrane anchored) t-SNARE complex, we monitored cytoplasmic ternary SNARE complex formation in the absence or presence of Munc18a. The kinetics of ternary SNARE complex formation was measured by fluorescence anisotropy, by adding fluorescently labeled synaptobrevin-2 cytoplasmic domain (synaptobrevin-2(1–96)-Alexa488) to a preformed cytoplasmic t-SNARE complex consisting of Stx1a(1–267) and SNAP-25. An increase in anisotropy, which is positively correlated with an increase of molecular mass, indicates the formation of SNARE complex. Using a 1:1 molar ratio of Stx1a(1–267) and SNAP-25 present at 6–100-fold molar excess over synaptobrevin-2(1–96)-Alexa488, we monitored the rate of ternary SNARE complex formation under three conditions (Figs. 5 and 6): 1) no Munc18a (Fig. 5, red line); 2) incubating Munc18a with the t-SNARE complex for 0.5–2 h prior to initiating the complex formation reaction (Fig. 5, blue line); and 3) starting from a gel filtration-purified 1:1:1 complex of Munc18a·Stx1a(1–267)·SNAP-25 (Fig. 5, green line). As a control, we confirmed that adding Munc18a to Stx1a(1–267) before adding SNAP-25, using a 1:1:1 molar ratio of Munc18a·Stx1a(1–267)·SNAP-25 over a range of concentrations, inhibits neuronal SNARE complex formation (31).
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.