Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis.
Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule-plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP-23 co-localized with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule-associated SNARE vesicle-associated membrane protein (VAMP)-2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP-2 in these rafts were present in SNARE complexes containing SNAP-23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP-23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP-23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP-2 present in rafts increased twofold, and these proteins were now present in raft-associated phospho-SNAP-23/syntaxin 4/VAMP-2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.
Puri
Niti
N
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Roche
Paul A
PA
D000818:Animals; D002772:Cholera Toxin / Q000378:metabolism; D004140:Dinitrophenols / Q000276:immunology / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D016537:Gangliosidosis, GM1 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology / Q000494:pharmacology; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D050765:Qa-SNARE Proteins / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000819:agonists / Q000378:metabolism; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000276:immunology / Q000494:pharmacology; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
D004140:Dinitrophenols; D050765:Qa-SNARE Proteins; D017455:Receptors, IgE; D050600:SNARE Proteins; C498858:Snap23 protein, rat; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D050988:Vesicle-Associated Membrane Protein 2; D033921:Vesicular Transport Proteins; C034638:dinitrophenyl-bovine serum albumin; D012710:Serum Albumin, Bovine; D007073:Immunoglobulin E; D002772:Cholera Toxin