Plasma membrane targeting of SNAP-25 increases its local concentration and is necessary for SNARE complex formation and regulated exocytosis.
SNAP-25 is an integral protein of the plasma membrane involved in neurotransmission and hormone secretion. The cysteine-rich domain of SNAP-25 is essential for membrane binding and plasma-membrane targeting. However, this domain is not required for SNARE complex formation and fusion of membranes in vitro. In this paper, we describe an 'intact-cell'-based system designed to compare the effect of similar amounts of membrane-bound and soluble SNAP-25 proteins on regulated exocytosis. In transfected neuroblastoma cells, Botulinum neurotoxin E (BoNT/E), a protease that cleaves SNAP-25, blocks regulated release of hormone. However, hormone release is rescued by expressing a wild-type SNAP-25 protein resistant to the toxin. BoNT/E-resistant SNAP-25 proteins lacking the cysteine-rich domain or with all the cysteines substituted by alanines do not form SNARE complexes or rescue regulated exocytosis when expressed at the same level as membrane-bound SNAP-25, which is approximately four-fold higher than the endogenous protein. We conclude that the cysteine-rich domain of SNAP-25 is essential for Ca(2+)-dependent hormone release because, by targeting SNAP-25 to the plasma membrane, it increases its local concentration, leading to the formation of enough SNARE complexes to support exocytosis.
Koticha
Darshan K
DK
Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.
McCarthy
Ellen E
EE
Baldini
Giulia
G
D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D001905:Botulinum Toxins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D017930:Genes, Reporter; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology*; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D014407:Tumor Cells, Cultured; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D011333:Pro-Opiomelanocortin; D001905:Botulinum Toxins; D003545:Cysteine; D002118:Calcium; C096326:botulinum toxin type E