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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adhesiveness
  • Cryoelectron Microscopy
  • Liposomes / chemistry*
  • Liposomes / metabolism*
  • Munc18 Proteins / chemistry*
  • Munc18 Proteins / genetics
  • Munc18 Proteins / metabolism
  • Munc18 Proteins / ultrastructure
  • Protein Binding
  • SNARE Proteins / chemistry*
  • SNARE Proteins / ultrastructure
  • Sensitivity and Specificity

Substances

  • Liposomes
  • Munc18 Proteins
  • SNARE Proteins