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 pr ocesses, the structural domains of SNAP-23 that are required for its intera ction with other SNAREs have not been determined. By employing deletion mut agenesis we found that deletion of the aminoterminal 18 amino acids of SNAP -23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 t o both the target SNARE syntaxin and the vesicle SNARE vesicle-associated m embrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 am ino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 bin ding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpres sed SNAP-23 in cells possessing the capacity to undergo regulated exocytosi s. Expression of human SNAP-23 in a rat mast cell line significantly enhanc ed 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 effi ciently than did rat SNAP-23, These data demonstrate that the introduction of a "better" SNARE binder into secretory cells augments exocytosis and def ines the carboxyl terminus of SNAP-23 as an essential regulator of exocytos is in mast cells.