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

Abstract

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.

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

“…The E.coli expression constructs for N-terminally MBP-tagged rat VAMP2, VAMP8, VAMP2cd, VAMP8cd, syntaxin3 (Stx3), syntaxin4 (Stx4), SNAP23, Munc18a, and for TEV were described previously (46). The cDNAs for full-length or N-terminally truncated rat syntaxin3 and syntaxin4 were inserted individually into the LIC site of pET MBP His 6 LIC cloning vector (gift from Scott Gradia; Addgene plasmid # 37237) to generate pET-Stx3-TCS(Tev Cleavable Site)-MBP-His 6 , pET-Stx3ΔN-TCS-MBP-His 6 , pET-Stx4-TCS-MBP-His 6 , and pET-Stx4ΔN-TCS-MBP-His 6 .…”

“…Recombinant proteins including MBP-TCS-VAMP2, MBP-TCS-VAMP8, MBP-TCS-SNAP23, His 6- SNAP23, Stx3-TCS-MBP-His 6 , Stx3∆N-TCS-MBP-His 6 , Stx4-TCS-MBP-His 6 , Stx4∆N-TCS-MBP-His 6 , Stx1-His 6 /His 6 -SNAP25, Stx1∆N-His 6 /His 6 -SNAP25, Stx4/His 6 -SNAP23 were purified according to previously reported procedures (28, 36, 46). His 6 -Munc18a was isolated from Rosetta2(DE3)/pPROEX-HTb-rMunc18a as described below.…”

“…Wherever MBP-Munc18a was used, the MBP tagged was removed by premixing MBP-Munc18a with His 6 -TEV at a molar ratio of 2:1. We routinely used Munc18a at 3.4 μM (unless otherwise specified), a concentration at which lipid-mixing rate reaches saturation (46). Following either 3 h or overnight incubation on ice, reaction mixtures were transferred to a 396 well plate and the NBD fluorescent signal, initially quenched by Rhodamine on the same donor membrane, was measured (λex = 460 nm, λem = 538 nm, emission cutoff = 515 nm) in a SpectraMAX Gemini XPS plate reader (Molecular Devices) at 37°C.…”

“…The trimeric syntaxin4/SNAP23/VAMP2 complex is best known for catalyzing Glut4 translocation along with Munc18c in muscle cells and adipocytes (44, 45). However, Munc18a has also been detected in these cells (41) (Gene expression database Bgee) and was recently shown to promote syntaxin4/SNAP23/VAMP2-mediated lipid mixing in reconstitution (46). Considering that syntaxin4 is required for both the initial and 2 nd phase in insulin secretion (47), whereas its “cognate” SM protein Munc18c is required only in the 2 nd phase (48), it is conceivable that a Munc18a/syntaxin4/SNAP-23/VAMP2 complex might play an under-appreciated role in the first phase of insulin release.…”

“…Considering that syntaxin4 is required for both the initial and 2 nd phase in insulin secretion (47), whereas its “cognate” SM protein Munc18c is required only in the 2 nd phase (48), it is conceivable that a Munc18a/syntaxin4/SNAP-23/VAMP2 complex might play an under-appreciated role in the first phase of insulin release. The physiological importance of the Munc18a/syntaxin3/SNAP23/VAMP2 complex is less clear, but Munc18a was shown to promote the lipid-mixing reaction mediated by syntaxin3, SNAP23, and VAMP2 in reconstitution (46). Interestingly, all these four components are expressed in mast cells and pancreatic islet β cells (49, 50), although it remains to be determined whether this particular SM/SNARE combination is required in any form of exocytosis in these cells.…”