Lipid raft association of SNARE proteins regulates exocytosis in PC12 cells.
SNAP25 and SNAP23 are plasma membrane SNARE proteins essential for regulated exocytosis in diverse cell types. Several recent studies have shown that these proteins are partly localized in lipid rafts, domains of the plasma membrane enriched in sphingolipids, and cholesterol. Here, we have employed cysteine mutants of SNAP25/SNAP23, which have modified affinities for raft domains, to examine whether raft association of these proteins is important for the regulation of exocytosis. PC12 cells were engineered that express the light chain of botulinum neurotoxin; in these cells all of the SNAP25 was cleaved to a lower molecular weight form, and regulated exocytosis was essentially absent. Exocytosis was rescued by expressing toxin-resistant SNAP25 or wild-type SNAP23, which is naturally toxin-resistant. Remarkably, a mutant SNAP25 protein with an increased affinity for rafts displayed a reduced ability to support exocytosis, whereas SNAP23 mutants with a decreased affinity for rafts displayed an enhancement of exocytosis when compared with wild-type SNAP23. The effects of the mutant proteins on exocytosis were dependent upon the integrity of the plasma membrane and lipid rafts. These results provide the first direct evidence that rafts regulate SNARE function and exocytosis and identify the central cysteine-rich region of SNAP25/23 as an important regulatory domain.
Salaün
Christine
C
Henry Wellcome Laboratory of Cell Biology, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, UK.
Gould
Gwyn W
GW
Chamberlain
Luke H
LH
D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011994:Recombinant Proteins; C100493:SNAP23 protein, human; C494208:SNAP25 protein, human; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D001905:Botulinum Toxins; D003545:Cysteine; C096326:botulinum toxin type E