Resolving the function of distinct Munc18-1/SNARE protein interaction modes in a reconstituted membrane fusion assay

Abstract

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 1·SNAP-25) and v-/t-SNARE (VAMP2·syntaxin 1·SNAP-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.

FIGURE 1.

Fusion of VAMP2 and syntaxin 1 liposomes requires SNAP-25. A, 30 μl of syntaxin 1 liposomes were preincubated with increasing amounts of soluble SNAP-25 (S25) and 5 μl of v-SNARE liposomes containing rhodamine- and NBD-labeled lipids for 1 h on ice, in a total volume of 70 μl (see also “Experimental Procedures”). Samples were transferred into a prewarmed microwell plate, and lipid mixing was monitored at 37 °C. After 2 h, n-dodecyl-β-d-maltoside was added, and the fusion signals were normalized to the maximum NBD fluorescence as described under “Experimental Procedure.” B, 20% of the fusion reactions shown in A were separated by SDS-PAGE, and the proteins were stained by Coomassie Blue.

FIGURE 2.

Munc18-1 inhibits syntaxin 1 liposome fusion. A, 30 μl of syntaxin 1 liposomes were preincubated with the indicated amounts of Munc18-1 in a final volume of 72 μl at room temperature for 30 min. Reaction vials were transferred into an ice bath and incubated with SNAP-25 (4-fold molar excess over syntaxin 1) for 1 h, and subsequently 5 μl of v-SNARE liposomes were added (final volume 80 μl). The control incubation lacks SNAP-25. Samples were transferred into a prewarmed microwell plate, and fusion was measured at 37 °C. Samples were analyzed as described in the legend to Fig. 1 and “Experimental Procedures.” B, 20% of the fusion reactions shown in A were separated by SDS-PAGE, and the proteins were stained by Coomassie Blue.

FIGURE 3.

Munc18-1 inhibition requires a closed conformation of syntaxin 1 but occurs independently of the syntaxin 1 N-peptide. 30 μl of liposomes containing either wild type syntaxin (wt) or mutant syntaxin (L8A) (A) or syntaxin (B) (open conformation (oc)) were preincubated in the absence or presence of Munc18-1 (2-fold molar excess over syntaxin 1) in a final volume of 60 μl at room temperature for 30 min. Reactions were transferred into an ice-bath and incubated with 5 μl of SNAP-25 (4-fold molar excess over syntaxin 1) for 1 h. Control samples (wt and L8A) lacked SNAP-25 but contained buffer. Reactions were transferred into a preheated microwell plate, and fusion was started by adding 5 μl of v-SNARE liposomes to all samples (total volume of 70 μl). C, 20% of the fusion reactions shown in A were separated by SDS-PAGE, and the proteins were stained by Coomassie Blue. Lanes from the same gel were cropped as indicated. D, bar graph showing the effect of Munc18-1 on the initial rates of fusion reactions containing various syntaxin 1 constructs. For comparison, the initial fusion rate of the reaction containing wild type syntaxin 1 and VAMP2 liposomes was set to 1 and used to normalize the other reactions. Error bars represent the mean ± S.E. The various syntaxin liposomes were preincubated with Munc18-1; subsequently SNAP-25 was added, and finally fusion was started by the addition of VAMP2 liposomes as described in A and B.

FIGURE 4.

Preincubation of Munc18-1-inhibited syntaxin 1 liposomes with VAMP2 liposomes in the presence of SNAP-25 at low temperature stimulates membrane fusion in a v-SNARE-dependent manner. A, scheme illustrates the distinct incubation steps and the order of addition of the reaction partners. Note, compared with previous experiments (upper panel), the v-SNARE liposomes were now added together with SNAP-25 to the Munc18-1 inhibited syntaxin 1 liposomes and incubated for 1 h at 4 °C (lower panel). B, 30 μl of syntaxin 1 (wt) liposomes were incubated according to the scheme shown in the lower panel of A with the indicated components and either with 5 μl of VAMP2 liposomes (v2) (lipid to protein ratio 350:1) or 5 μl of VAMP8 liposomes (v8) (lipid to protein ratio 275:1) in a total volume of 70 μl. Control samples lacked SNAP-25. Fusion was monitored and analyzed as described before. C, 20% of the fusion reactions shown in B were separated by SDS-PAGE and the proteins were stained by Coomassie Blue. D, comparison of the initial fusion rates of reactions containing wild type syntaxin 1 and either VAMP2 or VAMP8 liposomes in the absence or the presence of Munc18-1. The initial fusion rate of the reaction containing wild type syntaxin 1 liposomes and VAMP2 liposomes was set to 1 and used to normalize the other reactions. Error bars represent the means ± S.E.

FIGURE 5.

Syntaxin 1 N-peptide contributes to Munc18-1 mediated stimulation of membrane fusion. A, 30 μl of syntaxin (wt) liposomes or syntaxin (L8A) liposomes were incubated in the absence or the presence of Munc18-1 (2-fold molar excess over syntaxin 1) at room temperature for 30 min in a total volume of 50 μl. Reactions were transferred onto ice and a 3-fold molar excess of SNAP-25 and 5 μl of v-SNARE liposomes were added and incubated for an additional hour in a final volume of 70 μl. Samples were transferred into a preheated microwell plate, and fusion was monitored as described before. B, 20% of the fusion reactions shown in A were separated by SDS-PAGE, and the proteins were stained by Coomassie Blue. Lanes from the same gel were cropped as indicated. C, comparison of the initial rates of fusion reactions containing either wt, or L8A, or d24 syntaxin 1 liposomes in the absence or presence of Munc18-1. The initial fusion rate of the reaction containing wild type syntaxin 1 liposomes and VAMP2 liposomes was set to 1 and used to normalize the other reactions. All reactions contained SNAP-25. Error bars represent the means ± S.E.