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J Biol Chem, Vol. 273, Issue 28, 17732-17741, July 10, 1998

SNAP-23 Requirement for Transferrin Recycling in StreptolysinO-permeabilized Madin-Darby Canine Kidney Cells

Som-Ming LeungDagger , Dong Chen§, Bibhuti R. DasGupta, Sidney W. Whiteheart§, and Gerard ApodacaDagger

From the Dagger  Laboratory of Epithelial Cell Biology, the Renal-Electrolyte Division of the Department of Medicine and Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, the § Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536, and the  Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, Wisconsin 53706

Fusion of recycling and transcytotic vesicles with the apical and basolateral plasma membrane domains of Madin-Darby canine kidney (MDCK) cells requires the N-ethylmaleimide-sensitive factor and is sensitive to botulinum neurotoxin serotype E (BoNT/E). BoNT/E is thought to selectively proteolyze the 25,000-dalton synaptosomal associated protein (SNAP-25), a protein found in neurons or cells of neuroendocrine origin. However, SNAP-25 is not found in MDCK cells. One possible target for BoNT/E in MDCK cells is SNAP-23, a newly described SNAP-25 homolog that is found in several organs including kidney. Currently, the function of SNAP-23 is unknown. We have reconstituted transferrin recycling in permeabilized MDCK cells to assess the role of SNAP-23 in the endocytic traffic of this protein. We find that: (i) SNAP-23 is expressed in MDCK cells and is found both at the basolateral plasma membrane and associated with apical and basolateral vesicles, (ii) canine SNAP-23 is cleaved by BoNT/E, (iii) transferrin recycling is N-ethylmaleimide-sensitive factor-dependent and BoNT/E-sensitive, and (iv) addition of either exogenous SNAP-23 or anti-SNAP-23 antibodies inhibits ligand recycling. Our observations suggest that SNAP-23 may be required for fusion of recycling vesicles with the basolateral membrane of polarized MDCK cells.

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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