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Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2380-5. doi: 10.1073/pnas.0712125105. Epub 2008 Feb 11.

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

Author information

1
Department of Physiology and Cellular Biophysics, Columbia University, 1150 St. Nicholas Avenue, New York, NY 10032, USA.

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 fluorescence microscopy. Vesicle SNARE (v-SNARE) proteins reconstituted into giant vesicles ( approximately 10 mum) are fully mobile and functional. Through confocal microscopy, we observe that large vesicles ( approximately 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.

PMID:
18268324
PMCID:
PMC2268145
DOI:
10.1073/pnas.0712125105
[Indexed for MEDLINE]
Free PMC Article

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