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SNAREpin/Munc18 promotes adhesion and fusion of large vesicles to giant membranes

Abstract : Exocytic vesicle fusion requires both the SNARE family of fusion proteins and a closely associated regulatory subunit of the Sec1/ Munc18 (SM) family. In principle, SM proteins could act at an early SNARE assembly step to promote vesicle-plasma membrane adhesion or at a late step to overcome the energetic barrier for fusion. Here, we use the neuronal cognates of each of these protein families to recapitulate, and distinguish, membrane adhesion and fusion on a novel lipidic platform suitable for imaging by fluores-cence microscopy. Vesicle SNARE (v-SNARE) proteins reconstituted into giant vesicles (Ϸ10 m) are fully mobile and functional. Through confocal microscopy, we observe that large vesicles (Ϸ100 nm) carrying target membrane SNAREs (t-SNAREs) both adhere to and freely move on the surface of the v-SNARE giant vesicle. Under conditions where the intrinsic ability of SNAREs to drive fusion is minimized, Munc18 stimulates both SNARE-dependent stable adhesion and fusion. Furthermore, mutation of a critical Munc18-binding residue on the N terminus of the t-SNARE syntaxin uncou-ples Munc18-stimulated vesicle adhesion from membrane fusion. We expect that the study of SNARE-mediated fusion with giant membranes will find wide applicability in distinguishing adhesion-and fusion-directed SNARE regulatory factors.
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David Tareste, Jingshi Shen, Thomas Melia, James Rothman. SNAREpin/Munc18 promotes adhesion and fusion of large vesicles to giant membranes. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 2008, 105 (7), pp.2380-2385. ⟨10.1073/pnas.0712125105⟩. ⟨inserm-02296589⟩



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