Functional pairing of SNAREs examined under enhanced tethering conditions.

<p>Donor RPLs bearing VAMP2 (A), VAMP3 (B), VAMP7 (C) or VAMP8 (D) were incubated with acceptor RPLs in standard fusion reactions (lanes 1 to 3), 4 x reactions (lanes 4 to 6), where the concentrations of the donor and acceptor RPLs were increased 4 fold to 200mM and 1600mM respectively. Fusion reactions including 4% PEG6000 (v/v) are presented in lanes 7 to 9. The maximal early rates of dequenching/lipid mixing were calculated as described in Materials and Methods. The mean values are presented and error bars represent standard deviations from at least three independent experiments. Where appropriate, p values calculated using Student’s t test are presented.</p

αSNAP/NSF-dependent inhibition of SNARE-mediated lipid mixing.

<p>(A and B) VAMP2-bearing donor RPLs were incubated with specified acceptor RPLs in fusion reactions containing 0.5mM ATP, 0.5mM MgCl₂, and 4% PEG6000. Also included in the reactions are specified amounts of NSF along with 0.5μM αSNAP (A) or 0.15μM αSNAP (B). (C and D) VAMP3-bearing donor RPLs were incubated with specified acceptor RPLs in fusion reactions containing 1mM ATP, 1mM MgCl₂, and 4% PEG6000. Also included in the reactions are specified amounts of NSF along with 0.15μM αSNAP. (E and F) VAMP8-bearing donor RPLs were incubated with specified acceptor RPLs in standard reactions containing 0.5mM ATP and 0.5mM MgCl₂. Also included in the reactions are specified amounts of NSF along with 0.1μM αSNAP (E) or 0.02μM αSNAP (F). All samples contained the same amounts of αSNAP buffer and NSF buffer. The maximal early rates of lipid mixing for the SNARE-only reactions were used to generate the “standard” value (the lipid-mixing rate from SNARE-free RPLs was treated as a background and subtracted) and set as 100%. The values for other conditions were adjusted relative to the “standard” value. Error bars represent standard deviations from three independent experiments.</p

Munc18a selectively regulates different trans-SNARE complexes.

<p>Various combinations of donor and acceptor RPLs as specified were incubated overnight at 4°C with Munc18a (2μM) or control buffer, before transferring to 37°C. Error bars represent standard deviations from three independent experiments. p values were calculated using Student’s t test. ** indicates p < 0.01.</p

Differential Effects of Munc18s on Multiple Degranulation-Relevant Trans-SNARE Complexes

<div><p>Mast cell exocytosis, which includes compound degranulation and vesicle-associated piecemeal degranulation, requires multiple Q- and R- SNAREs. It is not clear how these SNAREs pair to form functional trans-SNARE complexes and how these trans-SNARE complexes are selectively regulated for fusion. Here we undertake a comprehensive examination of the capacity of two Q-SNARE subcomplexes (syntaxin3/SNAP-23 and syntaxin4/SNAP-23) to form fusogenic trans-SNARE complexes with each of the four granule-borne R-SNAREs (VAMP2, 3, 7, 8). We report the identification of at least six distinct trans-SNARE complexes under enhanced tethering conditions: i) VAMP2/syntaxin3/SNAP-23, ii) VAMP2/syntaxin4/SNAP-23, iii) VAMP3/syntaxin3/SNAP-23, iv) VAMP3/syntaxin4/SNAP-23, v) VAMP8/syntaxin3/SNAP-23, and vi) VAMP8/syntaxin4/SNAP-23. We show for the first time that Munc18a operates synergistically with SNAP-23-based non-neuronal SNARE complexes (i to iv) in lipid mixing, in contrast to Munc18b and c, which exhibit no positive effect on any SNARE combination tested. Pre-incubation with Munc18a renders the SNARE-dependent fusion reactions insensitive to the otherwise inhibitory R-SNARE cytoplasmic domains, suggesting a protective role of Munc18a for its cognate SNAREs. Our findings substantiate the recently discovered but unexpected requirement for Munc18a in mast cell exocytosis, and implicate post-translational modifications in Munc18b/c activation.</p></div

Reconstituted proteoliposomes (RPLs) bearing R- and Q- SNAREs involved in mast cell exocytosis.

<p>(A) Coomassie blue-stained SDS-PAG of reconstituted proteoliposomes. A total of 20 nmol (based on total lipids) of donor RPLs (lanes 1 to 4) and acceptor RPLs (lanes 5 and 6) were used in each lane. Small amounts of His₆-Tev used in the reconstitution get incorporated as well (specified by the arrowhead). The positions of protein markers are indicated on the left. (B to E) Standard fusion reactions. The fluorescence of NBD-DHPE reconstituted in the donor RPLs was measured every min and the dequenching of NBD-DHPE fluorescence (due to lipid mixing) is presented as Ft/F₀, with Ft being the NBD-DHPE fluorescence at any time point and F₀ being the fluorescence at the first minute. Represented by x are controls (not readily visible in C and D), in which donor RPLs were incubated with the SNARE-free acceptor RPLs. A representative result from more than three biological replicates is shown.</p

Munc18a-dependent stimulation is sensitive to inhibitory proteins at an early stage of the fusion reaction.

<p>(A). Acceptor RPLs bearing untagged syntaxin4/His₆-SNAP-23 and VAMP2-bearing donor RPLs were incubated with inhibitory proteins VAMP2cd (2μM) or VAMP8cd (2μM) or buffer on ice overnight and then received 5μM Munc18a or MBP (control). The incubation was continued on ice for another 90 min before shifting to 37°C. Fold increases in the initial lipid-mixing rates of the fusion reactions are shown. In (B), the same RPLs were incubated first with 5μM Munc18a on ice for 90 min before the addition of VAMP2cd (2μM) or VAMP8cd (2μM). Following overnight incubation on ice, samples were transferred to 37°C to monitor NBD fluorescence. The maximal early rates of lipid mixing for the Munc18a-only reactions were used to generate the “standard” value and set as 100%. The values for other conditions were adjusted relative to the “standard” value. Error bars represent standard deviations from three independent experiments.</p