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Plasma membrane targeting of SNAP-25 increases its local concentration and is necessary for SNARE complex formation and regulated exocytosis

Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA

^* Author for correspondence (e-mail: gb74{at}columbia.edu )

Accepted 24 May 2002

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 Ca²⁺-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.

Key words: SNAP-25, Regulated exocytosis, Cysteine-rich domain

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