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.