Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 7, Pages 2380-2385Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0712125105
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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 fluorescence microscopy. Vesicle SNARE (v-SNARE) proteins reconstituted into giant vesicles (approximate to 10 mu m) are fully mobile and functional. Through confocal microscopy, we observe that large vesicles (approximate to 100 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 uncouples 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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