title: 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 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.
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, 1150 St. Nicholas Avenue, New York, NY 10032, USA.
authors.forename: David
authors.initials: D
authors.lastname: Tareste
authors.forename: Jingshi
authors.initials: J
authors.lastname: Shen
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Melia
authors.forename: James E
authors.initials: JE
authors.lastname: Rothman
mesh_terms: D000268:Adhesiveness; D020285:Cryoelectron Microscopy; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D051938:Munc18 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D050600:SNARE Proteins / Q000737:chemistry* / Q000648:ultrastructure; D012680:Sensitivity and Specificity
chemical_list: D008081:Liposomes; D051938:Munc18 Proteins; D050600:SNARE Proteins