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© The Rockefeller University Press, 0021-9525/1998//1503 $5.00
The Journal of Cell Biology, Volume 141, Number 7, , 1998 1503-1513

Articles

The SNARE Machinery Is Involved in Apical Plasma Membrane Trafficking in MDCK Cells



Seng Hui Low*, Steven J. Chapin*, Christian Wimmer§, Sidney W. Whiteheart, László G. Kömüves{ddagger}, Keith E. Mostov*, and Thomas Weimbs*

* Department of Anatomy, Department of Biochemistry and Biophysics, Cardiovascular Research Institute, {ddagger} Department of Dermatology and Veteran Administration Medical Center, University of California, San Francisco, California 94143-0452; § Department of Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center, New York 10021; || Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide–sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against {alpha}-SNAP inhibit both TGN to apical and basolateral transport in a reconstituted in vitro system. In contrast, we find no evidence for an involvement of N-ethyl maleimide–sensitive factor in TGN to apical transport, whereas basolateral transport is NSF-dependent. We conclude that syntaxin 3, SNAP-23, and {alpha}-SNAP are involved in apical membrane fusion. These results demonstrate that vesicle fusion with the apical plasma membrane does not use a mechanism that is entirely unrelated to other cellular membrane fusion events, but uses isoforms of components of the SNARE machinery, which suggests that they play a role in providing specificity to polarized membrane traffic.

Abbreviations used in this paper: BoNT, botulinum neurotoxins; GPI, glycosylphosphatidylinositol; NEM, N-ethyl maleimide; NSF, N-ethyl maleimide–sensitive factor; pIgR, polymeric immunoglobulin receptor; SL, Signal-less; SLO, streptolysin-O; SNAP, soluble NSF attachment protein; SNAP-25, synaptosomal-associated protein of 25 kD; SNARE, SNAP receptor; WT, wild-type.

S.H. Low and T. Weimbs were supported by the Irvington Institute for Immunology; T. Weimbs by a Feodor-Lynen-Fellowship of the Alexander von Humboldt-Foundation; S. Chapin by an American Cancer Society postdoctoral fellowship (PF3666) and an National Institutes of Health (NIH) institutional NRSA (T32HL07731); C. Wimmer by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. This work was supported by NIH grants R01 AI25144, AI39161 to K.M. and NIH grants HL56652 to S.W.

Address all correspondence to Keith Mostov, University of California, San Francisco, Department of Anatomy, San Francisco, CA 94143-0452. Tel.: (415) 476-6048. Fax: (415) 476-4845. E-mail: mostov{at}itsa.ucsf.edu


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