The last exon of SNAP-23 regulates granule exocytosis from mast cells.
SNAP-25 and its ubiquitous homolog SNAP-23 are members of the SNARE family of proteins that regulate membrane fusion during exocytosis. Although SNAP-23 has been shown to participate in a variety of intracellular transport processes, the structural domains of SNAP-23 that are required for its interaction with other SNAREs have not been determined. By employing deletion mutagenesis we found that deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 to both the target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 binding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpressed SNAP-23 in cells possessing the capacity to undergo regulated exocytosis. Expression of human SNAP-23 in a rat mast cell line significantly enhanced exocytosis, and this effect was not observed in transfectants expressing the carboxyl-terminal VAMP-binding mutant of SNAP-23. Despite considerable amino acid identity, we found that human SNAP-23 bound to SNAREs more efficiently than did rat SNAP-23. These data demonstrate that the introduction of a "better" SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in mast cells.
Vaidyanathan
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VV
Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, MD 20892, USA.
Puri
N
N
Roche
P A
PA
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics*; D003594:Cytoplasmic Granules / Q000502:physiology*; D005089:Exocytosis; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D008407:Mast Cells / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D011817:Rabbits; D051381:Rats; D050600:SNARE Proteins; D014162:Transfection; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; C100493:SNAP23 protein, human; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins