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Vol. 17, Issue 5, 2113-2124, May 2006

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A Second SNARE Role for Exocytic SNAP25 in Endosome Fusion

Department of Biochemistry, University of Wisconsin, Madison, WI 53706

Submitted January 25, 2006; Revised February 7, 2006; Accepted February 8, 2006
Monitoring Editor: Sandra Schmid

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca²⁺-dependent secretory vesicle–plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.

Abbreviations used: ARF6, ADP-ribosylation factor 6; BoNT, botulinum neurotoxin; CTxB, cholera toxin B; GFP, green fluorescent protein; LC, light chain; RE, recycling endosome; SE, sorting endosome; SNAP25, synaptosomal associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Tf, transferrin; VAMP, vesicle-associated membrane protein.

^* Present address: Department of Pharmaceutical Technology, Tokushima-bunri University, Saniki-city, Kagawa 769-2193, Japan.

Address correspondence to: Thomas F.J. Martin (tfmartin{at}wisc.edu).