title: The vesicle- and target-SNARE proteins that mediate Glut4 vesicle fusion are localized in detergent-insoluble lipid rafts present on distinct intracellular membranes.
abstract: Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 ( approximately 35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-beta-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.
authors.affiliation: Henry Wellcome Laboratory for Cell Biology, Division of Biochemistry & Molecular Biology, Davidson Building, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom. l.chamberlain@bio.gla.ac.uk
authors.forename: Luke H
authors.initials: LH
authors.lastname: Chamberlain
authors.forename: Gwyn W
authors.initials: GW
authors.lastname: Gould
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003505:Cyclodextrins / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry*; D050356:Lipid Metabolism; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008861:Microsomes / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D009124:Muscle Proteins; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D015398:Signal Transduction; D013347:Subcellular Fractions; D033921:Vesicular Transport Proteins; D047392:beta-Cyclodextrins
chemical_list: D002352:Carrier Proteins; D003505:Cyclodextrins; D003902:Detergents; D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; C100493:SNAP23 protein, human; D050600:SNARE Proteins; C495693:Slc2a4 protein, mouse; C107017:Snap23 protein, mouse; D033921:Vesicular Transport Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D017830:Octoxynol; D005033:Ethylmaleimide