Munc18-Bound Syntaxin Readily Forms SNARE Complexes with Synaptobrevin in Native Plasma Membranes

Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis

Determinants of liposome fusion mediated by synaptic SNARE proteins

Synaptic exocytosis requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 1, SNAP-25, and synaptobrevin (VAMP). Assembly of the SNAREs into a stable core complex is supposed to catalyze membrane fusion, and proteoliposomes reconstituted with synaptic SNARE proteins spontaneously fuse with each other. We now show that liposome fusion mediated by synaptic SNAREs is inhibited by botulinum neurotoxin E (BoNT/E) but can be rescued by supplementing the C-terminal portion of SNAP-25. Furthermore, fusion is prevented by a SNAP-25-specific antibody known to block exocytosis in chromaffin cells, and it is competed for by soluble fragments of the R-SNAREs synaptobrevin 2, endobrevin/VAMP-8, and tomosyn. No accumulation of clustered vesicles is observed during the reaction. Rapid artificial clustering of SNARE-containing proteoliposomes enhances the fusion rate at low but not at saturating liposome concentrations. We conclude that the rate of liposome fusion is dominated by the intrinsic properties of the SNAREs rather than by the preceding docking step

Complications.

<p>Chi²-test; DGF, delayed graft function; CMV, cytomegalovirus</p><p>Complications.</p

Immunoadsorption (IA).

<p>^a Mann-Whitney-U test;</p><p>^b t-test;</p><p>^c Chi² test;</p><p>^d paired t-test of the platelet count before and after IA in the Glycosorb group;</p><p>^e paired t-test of the platelet count in the Immunosorba group; PPh, plasmapheresis; RTx, renal transplantation; BW, bodyweight.</p><p>Immunoadsorption (IA).</p

Biopsy results.

<p>^a Chi²-test;</p><p>^b t-test; ABMR, antibody-mediated rejection; HLA, human leucocyte antigen; DSA, donor specific antibodies; TCMR, T cell-mediated rejection; CNI calcineurin inhibitor</p><p>Biopsy results.</p

Costs for immunoadsorption.

<p>All prices are shown in Euro. Expenses do not include replacement fluids and labor costs.</p><p>Costs for immunoadsorption.</p