Regulated exocytosis requires tight coupling of the membrane fusion machinery to a triggering signal and a fast response time. Complexins are part of this regulation and, together with synaptotagmins, control calcium-dependent exocytosis. Stimulatory and inhibitory functions have been reported for complexins. To test if complexins directly affect membrane fusion, we analyzed the 4 known mammalian complexin isoforms in a reconstituted fusion assay. In contrast to complexin III (CpxIII) and CpxIV, CpxI and CpxII stimulated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-pin assembly and membrane fusion. This stimulatory effect required a preincubation at low temperature and was specific for neuronal t-SNAREs. Stimulation of membrane fusion was lost when the carboxy-terminal domain of CpxI was deleted or serine 115, a putative phosphorylation site, was mutated. Transfer of the carboxy-terminal domain of CpxI to CpxIII resulted in a stimulatory CpxIII-I chimera. Thus, the carboxyterminal domains of CpxI and CpxII promote the fusion of highcurvature liposomes.exocytosis ͉ SNARE

Sec1p/Munc18 proteins and SNAP receptors (SNAREs) are key components of the intracellular membrane fusion machinery. Compartment-specific v-SNAREs on a transport vesicle pair with their cognate t-SNAREs on the target membrane and drive lipid bilayer fusion. In a reconstituted assay that dissects the sequential assembly of t-SNARE (syntaxin 1⅐SNAP-25) and v-/t-SNARE (VAMP2⅐syntaxin 1⅐SNAP-25) complexes, and finally measures lipid bilayer merger, we resolved the inhibitory and stimulatory functions of the Sec1p/Munc18 protein Munc18-1 at the molecular level. Inhibition of membrane fusion by Munc18-1 requires a closed conformation of syntaxin 1. Remarkably, the concurrent preincubation of Munc18-1-inhibited syntaxin 1 liposomes with both VAMP2 liposomes and SNAP-25 at low temperature releases the inhibition and effectively stimulates membrane fusion. VAMP8 liposomes can neither release the inhibition nor exert the stimulatory effect, demonstrating the need for a specific Munc18-1/VAMP2 interaction. In addition, Munc18-1 binds to the N-terminal peptide of syntaxin 1, which is obligatory for a robust stimulation of membrane fusion. In contrast, this interaction is neither required for the inhibitory function of Munc18-1 nor for the release of this block. These results indicate that Munc18-1 and the neuronal SNAREs already have the inherent capability to function as a basic stage-specific off/on switch to control membrane fusion.Membrane fusion in eukaryotic cells is mediated by a conserved machinery consisting of compartment-specific v-SNAREs 3 on transport vesicles and t-SNAREs on the target membrane (1-4). SNAREs are characterized by SNARE motifs, stretches of 60 -70 amino acids, which contain heptad repeats with a central "0" layer and assemble into specific four-helix bundles (5). The formation of SNAREpins, trans v-/t-SNARE complexes bridging two membranes, occurs in a zipper-like manner that starts at the membrane distal (N-terminal) end of the SNAREpins and proceeds toward the (C-terminal) membrane-spanning anchors of the SNAREs (6, 7). Zippering brings the two lipid bilayers in close apposition, finally resulting in membrane merger (2,8). Thus, the energy required for membrane fusion is provided by the exergonic folding of the largely unstructured v-and t-SNARE proteins into stable four-helix bundles (2, 5, 9). Although SNAREs can be considered to be the minimal membrane fusion machinery, in the physiological cellular environment, an array of accessory proteins and lipids controls the spatial and temporal activity of SNARE proteins (10).One class of accessory proteins, the SM (Sec1p/Munc18) proteins, directly bind to SNAREs, control their activity, and are required for membrane fusion in vivo (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). SM proteins contain about 600 amino acids, which are folded into an archshaped structure. At least two SNARE-binding modes have been described. In the first mode, the SM protein binds the t-SNARE component syntaxin in a "closed" conformation, in which the N-terminal three...