Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin [Molecular Biophysics]

February 25th, 2015 by Zhang, Y., Diao, J., Colbert, K. N., Lai, Y., Pfuetzner, R. A., Padolina, M. S., Vivona, S., Ressl, S., Cipriano, D. J., Choi, U. B., Shah, N., Weis, W. I., Brunger, A. T.

Sec1/Munc18 (SM) proteins are essential 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, wildtype Munc18a reduced vesicle association approximately 50% when the vesicles bearing the t-SNAREs syntaxin-1A and SNAP-25 were pre-incubated 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 pre-fusion stage.