Article

A second SARE role for exocytic SNAP25 in endosome fusion

Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin, United States
Molecular Biology of the Cell (Impact Factor: 4.47). 06/2006; 17(5):2113-24. DOI: 10.1091/mbc.E06-01-0074
Source: PubMed

ABSTRACT

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.

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    • "All constructs were checked by sequencing. shRNA plasmids targeting rat isoforms of synaptotagmin-1 and -9, and SNAP-25b were previously described (Aikawa et al., 2006; Lynch and Martin, 2007b). The plasmid CAPS-mKate2 was generated by subcloning rat CAPS from a CAPS-myc-his pcDNA3.1 plasmid into pmKate2-N1 from Evrogen using XhoI and KpnI. "
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