SNAP-23 Requirement for Transferrin Recycling in StreptolysinO-permeabilized Madin-Darby Canine Kidney Cells*
- From the ‡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
Abstract
Fusion of recycling and transcytotic vesicles with the apical and basolateral plasma membrane domains of Madin-Darby canine kidney (MDCK) cells requires theN-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.
Footnotes
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↵* This work was supported during the tenure of an American Heart Association Minority Development Award and a grant from the National Institutes of Health RO1DK51970-01 (to G. A.) and a grant from the National Institutes of Health (to S. W. W.). Preliminary development of the Tf-permeabilized cell system was supported in part by National Institutes of Health Grants R01 AI25144 and AI36953 (to Keith Mostov).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ To whom correspondence should be addressed: Renal-Electrolyte Div., 982 Scaife Hall, University of Pittsburgh, Pittsburgh PA 15261. Tel.: 412-383-8893; Fax: 412-383-8955; E-mail:gla6{at}pop.pitt.edu.
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↵1 The abbreviations used are: NSF, NEM sensitive factor; ARE, apical recycling endosome; BoNT, botulinum neurotoxin; NEM, N-ethylmaleimide; pIgR, polymeric immunoglobulin receptor; SLO, streptolysin-O; SNAP, soluble NSF attachment protein; SNAP-25, 25,000 dalton synaptosomal associated protein; SNAP-23, 23,000 dalton SNAP-25 homolog; SNARE, SNAP receptor; t-SNARE, target membrane-associated SNAP receptor; Tf, transferrin; VAMP, vesicle-associated membrane protein; v-SNARE, vesicle membrane-associated SNAP receptor; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; MEM, minimal essential medium; BSA, bovine serum albumin; DTT, dithiothreitol.
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↵2 D. Chen and S. W. Whiteheart, submitted for publication.
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- Received February 2, 1998.
- Revision received April 27, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
