The SNARE proteins SNAP-25 and SNAP-23 display different affinities for lipid rafts in PC12 cells. Regulation by distinct cysteine-rich domains.
SNAP-25 and its ubiquitously expressed homologue, SNAP-23, are SNARE proteins that are essential for regulated exocytosis in diverse cell types. Recent work has shown that SNAP-25 and SNAP-23 are partly localized in sphingolipid/cholesterol-rich lipid raft domains of the plasma membrane and that the integrity of these domains is important for exocytosis. Here, we show that raft localization is mediated by a 36-amino-acid region of SNAP-25 that is also the minimal sequence required for membrane targeting; this domain contains 4 closely spaced cysteine residues that are sites for palmitoylation. Analysis of endogenous levels of SNAP-25 and SNAP-23 present in lipid rafts in PC12 cells revealed that SNAP-23 (54% raft-associated) was almost 3-fold more enriched in rafts when compared with SNAP-25 (20% raft-associated). We report that the increased raft association of SNAP-23 occurs due to the substitution of a highly conserved phenylalanine residue present in SNAP-25 with a cysteine residue. Intriguingly, although the extra cysteine in SNAP-23 enhances its raft association, the phenylalanine at the same position in SNAP-25 acts to repress the raft association of this protein. These different raft-targeting signals within SNAP-25 and SNAP-23 are likely important for fine-tuning the exocytic pathways in which these proteins operate.
Salaün
Christine
C
Henry Wellcome Laboratory of Cell Biology, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Davidson Building, University of Glasgow, G12 8QQ, Scotland, United Kingdom.
Gould
Gwyn W
GW
Chamberlain
Luke H
LH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D019308:Palmitic Acid / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D051381:Rats; 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; C100493:SNAP23 protein, human; D050600:SNARE Proteins; C107017:Snap23 protein, mouse; C494209:Snap25 protein, mouse; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D010649:Phenylalanine; D003545:Cysteine