Munc18a clusters SNARE-bearing liposomes prior to trans-SNARE zippering

Abstract

Sec1-Munc18 (SM) proteins co-operate with SNAREs {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] receptors} to mediate membrane fusion in eukaryotic cells. Studies of Munc18a/Munc18-1/Stxbp1 in neurotransmission suggest that SM proteins accelerate fusion kinetics primarily by activating the partially zippered trans-SNARE complex. However, accumulating evidence has argued for additional roles for SM proteins in earlier steps in the fusion cascade. Here, we investigate the function of Munc18a in reconstituted exocytic reactions mediated by neuronal and non-neuronal SNAREs. We show that Munc18a plays a direct role in promoting proteoliposome clustering, underlying vesicle docking during exocytosis. In the three different fusion reactions examined, Munc18a-dependent clustering requires an intact N-terminal peptide (N-peptide) motif in syntaxin that mediates the binary interaction between syntaxin and Munc18a. Importantly, clustering is preserved under inhibitory conditions that abolish both trans-SNARE complex formation and lipid mixing, indicating that Munc18a promotes membrane clustering in a step that is independent of trans-SNARE zippering and activation.

Keywords: Munc18; SNARE proteins; docking; exocytosis; membrane fusion; reconstitution.

Figure 1. Munc18a selectively promotes the clustering of SNARE-bearing liposomes

Donor and acceptor proteoliposomes as specified were mixed in a 1:8 molar ratio, with either Munc18a or Munc18a buffer. Following overnight incubation on ice, diluted samples were subject to confocal fluorescence microscopy. Cluster/particle sizes in four randomly collected images were measured and their cumulative distribution is presented on the vertical axis. The Wilcoxon test indicates that the particle size distributions for the “– Munc18a” condition and the “+ Munc18” condition in C), E), and G) are significantly different (P < 0.001). This experiment is a representative of 3 repeats.

Figure 2. N-peptide motifs are required in Munc18a-dependent clustering and lipid-mixing acceleration

VAMP2-bearing donor liposomes were incubated 3 h on ice with acceptor proteoliposomes as specified, with or without Munc18a. Each sample was then aliquoted for clustering and lipid-mixing assays. A) Cumulative distribution plots show the results of the clustering assay. B) Following the lipid-mixing assay, the mean values of the maximal lipid-mixing rates are presented and error bars indicate SD (n=4). Student’s t-test was used to assess the significance of the difference between the “– Munc18a” results and the “+ Munc18” results. * indicates P < 0.05 whereas ** indicates P < 0.01. Note that the extent of Munc18a-dependent acceleration of lipid mixing does not necessarily correlate linearly with the extent of change in clustering, which implies either the insufficient resolution of the clustering assay or possibly different modes of action exploited by Munc18a in clustering and lipid mixing respectively. C) To determine the biochemical interaction between Munc18a and Q-SNAREs, His₆-Munc18a was incubated with specified proteoliposomes at 4°C overnight before Ni-NTA agarose was added to bring down His₆-Munc18a and associating molecules. Eluates were subject to SDS-PAGE followed by Coomassie Brilliant Blue staining. The top and bottom panels are from two separate gels. D) Densitometry was performed and the intensity of the syntaxin bands in the pull-down was normalized for the input. The values of the wild-type syntaxin3 and syntaxin4 were set as 100% respectively and then used to calculate the relative levels of mutant syntaxins in the pull-down. Student’s t-test was used to assess the significance of the difference between the “WT” results and the “∆N” results. ** indicates P < 0.01. Error bars represent SD (n=3).

Figure 3. The impact of synthetic N-peptides on Munc18a’s function

Donor and acceptor proteoliposomes as indicated were incubated 3 h on ice alone or with Munc18a (2μM) that had been preincubated overnight with specified amounts of Stx4-N^wt, Stx4-N^L8A, or control buffer. The reaction mixtures were then divided for lipid-mixing assay and clustering assay respectively. A) To assess the potential of N-peptide to stimulate Munc18a function in reactions containing VAMP2-bearing donor liposomes and syntaxin4∆N/SNAP23-bearing acceptor liposomes, the ratio of the maximal lipid-mixing rate of each Munc18a-containing reaction over that of the SNARE-only reaction was calculated and shown as fold of stimulation. B) To assess the potential of N-peptide to inhibit Munc18a activity in reactions containing VAMP2-bearing donor liposomes and syntaxin4/SNAP23-bearing acceptor liposomes, the maximal lipid-mixing rate of the peptide-free reaction (but with Munc18a) was set at 100% and then used to calculate the relative values of the other reactions. Student’s t-test was used in statistical analysis. ** indicates P < 0.01. Error bars indicate SD (n=4). Cumulative distribution plots in A) and B) highlight the impact of the wild-type and mutant N-peptides on membrane clustering.

Figure 4. Requirement for Qbc-SNAREs in Munc18a-dependent clustering

VAMP2-bearing donor liposomes and various acceptor liposomes as specified were incubated on ice for 3 h, with or without Munc18a. The samples were examined with confocal microscopy and cluster/particle sizes were determined by ImageJ. The cumulative distribution plot of a typical experiment (out of 7) is presented.

Figure 5. Munc18a-dependent trans-SNARE assembly on ice

A) Illustration of the experimental procedure. B) In Triton X-100 lysates, His₆-tagged Q-SNAREs were precipitated by Ni-NTA resin and subjected to SDS-PAGE and immunoblotting, using antibodies specific to SNAP23 (lanes 1 to 8; top panel) or SNAP25 (lanes 9–12, top panel; 50% input is shown to gauge the relative efficiency of each pull-down experiment. The empty space between lanes 3 and 4, 7 and 8, 11 and 12 specifies two regions of the same blot from one gel. VAMP2 co-precipitated with His₆-tagged Q-SNAREs was probed by monoclonal antibody (lanes 1 to 12; bottom panel; 5% of the Input is shown). To control for Munc18a-dependent SNARE complex formation in detergent lysates, acceptor and donor liposomes received Munc18a immediately after detergent addition in lanes 1, 5, and 9. This experiment is a representative of 3 independent repeats.

Figure 6. Munc18a promotes proteoliposome clustering in a step prior to trans-SNARE zippering

A) Incubation procedure. Syntaxin4/His₆-SNAP23-bearing acceptor liposomes were incubated with either the soluble, inhibitory VAMPs (10-fold molar excess in comparison to the membrane-bound VAMP2 added next) or buffer for 1 h on ice before receiving the VAMP2-bearing donor liposomes and Munc18a. Following another 15 h incubation on ice, samples were aliquoted and subject to B) lipid-mixing assay, C) pull-down assays using Ni-NTA resin, D) co-immunoprecipitation using immobilized anti-Stx4 antibody, and E) clustering assay. Student’s t-test was used to assess the statistical difference between the results in lane 3 and the rest in B). * indicates P < 0.05. Error bars indicate SD (n=3).