Munc181, a protein essential for regulated
exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like
(SM) proteins. In vitro, Munc181 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and
synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc181 to syntaxin 1 with its biological function. We now show that in intact and
exocytosis-competent lawns of plasma membrane, Munc181 forms a complex with syntaxin that allows formation of SNARE complexes. Munc181 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25deficient mice are used. We conclude that Munc181 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis.