Synaptic proteins and SNARE complexes are localized in lipid rafts from rat brain synaptosomes.
The biochemical characterization of the SNARE proteins present in lipid microdomains, also known as "lipid rafts," has been addressed in earlier studies, with conflicting data from different laboratories. In this study, we use rat brain synaptosomes as a model with which to examine the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM), also known as 'lipid rafts.' By means of buoyancy analysis in sucrose gradients of Triton X-100-solubilized synaptosomes, we identified a pool of SNARE proteins (SNAP 25, syntaxin 1, and synaptobrevin2/VAMP2) significantly associated with DRM. Furthermore, Munc18, synaptophysin, and high amounts of the isoforms I and II of synaptotagmin were also found in DRM. In addition, SDS-resistant and temperature-dependent SNARE complexes were also detected in DRM. Treatment of synaptosomes with methyl-beta-cyclodextrin resulted in persistence of the proteins present in the DRM isolated using Triton X-100, whilst strongly impairing calcium-dependent glutamate release. The results from the present work show that lipid microdomains are sites where SNARE proteins and complexes are actually present, as well as important elements in the control of regulated exocytosis.
Gil
Carles
C
Departament de Bioquímica i Biologia Molecular and Institut de Neurociències, Universitat Autònoma de Barcelona, Cerdanyola 08193, Catalunya, Spain. carles.gil@uab.es
Soler-Jover
Alex
A
Blasi
Juan
J
Aguilera
José
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D018698:Glutamic Acid / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D050683:R-SNARE Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins; D013569:Synapses / Q000378:metabolism*; D016708:Synaptophysin / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000378:metabolism; D050827:Syntaxin 1; D013696:Temperature; D013997:Time Factors; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D003902:Detergents; D008055:Lipids; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D016708:Synaptophysin; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D018698:Glutamic Acid; D017830:Octoxynol; D002784:Cholesterol