title: SNAREs prefer liquid-disordered over "raft" (liquid-ordered) domains when reconstituted into giant unilamellar vesicles.
abstract: Membrane domains ("rafts") have received great attention as potential platforms for proteins in signaling and trafficking. Because rafts are believed to form by cooperative lipid interactions but are not directly accessible in vivo, artificial phase-separating lipid bilayers are useful model systems. Giant unilamellar vesicles (GUVs) offer large free-standing bilayers, but suitable methods for incorporating proteins are still scarce. Here we report the reconstitution of two water-insoluble SNARE proteins into GUVs without fusogenic additives. Following reconstitution, protein functionality was assayed by confocal imaging and fluorescence auto- and cross-correlation spectroscopy. Incorporation into GUVs containing phase-separating lipids revealed that, in the absence of other cellular factors, both proteins exhibit an intrinsic preference for the liquid-disordered phase. Although the picture from detergent resistance assays on whole cells is ambiguous, reconstitutions of components of the exocytic machinery into GUVs by this new approach should yield insight into the dynamics of protein complex associations with hypothesized liquid-ordered phase microdomains, the correspondence between detergent-resistant membranes and liquid-ordered phase, and the mechanism of SNARE-mediated membrane fusion.
authors.affiliation: Dresden University of Technology, Institute of Biophysics, Tatzberg 47-51, 01307 Dresden, Germany.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Bacia
authors.forename: Christina G
authors.initials: CG
authors.lastname: Schuette
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Kahya
authors.forename: Reinhard
authors.initials: R
authors.lastname: Jahn
authors.forename: Petra
authors.initials: P
authors.lastname: Schwille
mesh_terms: D005089:Exocytosis; D008051:Lipid Bilayers; D008081:Liposomes; D011994:Recombinant Proteins / Q000378:metabolism; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; D011994:Recombinant Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins