SNAREs Prefer Liquid-disordered over “Raft” (Liquid-ordered) Domains When Reconstituted into Giant Unilamellar Vesicles*[boxs]

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.

[1]  M. Wheeler,et al.  Disruption of Pancreatic β-Cell Lipid Rafts Modifies Kv2.1 Channel Gating and Insulin Exocytosis* , 2004, Journal of Biological Chemistry.

[2]  C. Seidel,et al.  Determinants of liposome fusion mediated by synaptic SNARE proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[3]  K. Kumakura,et al.  Site of Docking and Fusion of Insulin Secretory Granules in Live MIN6 β Cells Analyzed by TAT-conjugated Anti-syntaxin 1 Antibody and Total Internal Reflection Fluorescence Microscopy* , 2004, Journal of Biological Chemistry.

[4]  S. Munro.  Lipid Rafts Elusive or Illusive? , 2003, Cell.

[5]  D. Saslowsky,et al.  Syntaxin Is Efficiently Excluded from Sphingomyelin-enriched Domains in Supported Lipid Bilayers Containing Cholesterol , 2003, The Journal of Membrane Biology.

[6]  Petra Schwille,et al.  Probing Lipid Mobility of Raft-exhibiting Model Membranes by Fluorescence Correlation Spectroscopy* , 2003, Journal of Biological Chemistry.

[7]  A. Shevchenko,et al.  Resistance of cell membranes to different detergents , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Petra Schwille,et al.  A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy. , 2003, Methods.

[9]  Sarah L Veatch,et al.  Organization in lipid membranes containing cholesterol. , 2002, Physical review letters.

[10]  H. Heerklotz.  Triton promotes domain formation in lipid raft mixtures. , 2002, Biophysical journal.

[11]  Petra Schwille,et al.  Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis. , 2002, Biophysical journal.

[12]  Thomas C. Südhof,et al.  Snares and munc18 in synaptic vesicle fusion , 2002, Nature Reviews Neuroscience.

[13]  R. Jahn,et al.  Rapid and Selective Binding to the Synaptic SNARE Complex Suggests a Modulatory Role of Complexins in Neuroexocytosis* , 2002, The Journal of Biological Chemistry.

[14]  Ken Jacobson,et al.  Partitioning of Thy-1, GM1, and cross-linked phospholipid analogs into lipid rafts reconstituted in supported model membrane monolayers , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[15]  A. V. Samsonov,et al.  Characterization of cholesterol-sphingomyelin domains and their dynamics in bilayer membranes. , 2001, Biophysical journal.

[16]  D. Wiersma,et al.  Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion. , 2001, Biophysical journal.

[17]  G. Gould,et al.  SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[18]  D. Bruns,et al.  SNAREs are concentrated in cholesterol‐dependent clusters that define docking and fusion sites for exocytosis , 2001, The EMBO journal.

[19]  R. Jahn,et al.  Homo- and Heterooligomeric SNARE Complexes Studied by Site-directed Spin Labeling* , 2001, The Journal of Biological Chemistry.

[20]  E Gratton,et al.  Lipid rafts reconstituted in model membranes. , 2001, Biophysical journal.

[21]  Richard H. Scheller,et al.  SNARE-mediated membrane fusion , 2001, Nature Reviews Molecular Cell Biology.

[22]  K. Jacobson,et al.  Lipid rafts in model and biological membranes , 2001 .

[23]  Kai Simons,et al.  Lipid rafts and signal transduction , 2000, Nature Reviews Molecular Cell Biology.

[24]  M. L. Wagner,et al.  Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker. , 2000, Biophysical journal.

[25]  W. Huttner,et al.  Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane , 2000, Nature Cell Biology.

[26]  D. Brown,et al.  Structure and Origin of Ordered Lipid Domains in Biological Membranes , 1998, The Journal of Membrane Biology.

[27]  A. T. Brunger,et al.  Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly. , 1998, Biochemistry.

[28]  Benedikt Westermann,et al.  SNAREpins: Minimal Machinery for Membrane Fusion , 1998, Cell.

[29]  Deborah A. Brown,et al.  Cholesterol and Sphingolipid Enhance the Triton X-100 Insolubility of Glycosylphosphatidylinositol-anchored Proteins by Promoting the Formation of Detergent-insoluble Ordered Membrane Domains* , 1998, The Journal of Biological Chemistry.

[30]  E. Ikonen,et al.  Functional rafts in cell membranes , 1997, Nature.

[31]  P. Schwille,et al.  Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. , 1997, Biophysical journal.

[32]  E. London,et al.  Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Deborah A. Brown,et al.  Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface , 1992, Cell.

[34]  G van Meer,et al.  Lipid sorting in epithelial cells. , 1988, Biochemistry.

[35]  P. Saffman,et al.  Brownian motion in biological membranes. , 1975, Proceedings of the National Academy of Sciences of the United States of America.