Reconstitution of transcytosis in SLO-permeabilized MDCK cells: Existence of an NSF-dependent fusion mechanism with the apical surface of MDCK cells

Gerard Apodaca, Michael H. Cardone, Sidney W. Whiteheart, Bibhuti R. DasGupta, Keith E. Mostov

Research output: Contribution to journalArticlepeer-review

72 Scopus citations

Abstract

Recently, it was demonstrated that delivery from the trans-Golgi network (TGN) to the basolateral surface of Madin-Darby canine kidney (MDCK) cells required N-ethylmaleimide-sensitive factor (NSF)-α soluble NSF attachment protein (SNAP)-SNAP receptor (SNARE) complexes, while delivery from the TGN to the apical surface was independent of NSF-αSNAP-SNARE. To determine if all traffic to the apical surface of this cell line was NSF independent, we reconstituted the transcytosis of pre-internalized IgA to the apical surface and recycling to the basolateral surface. Transcytosis and the recycling of IgA required ATP and cytosol, and both were inhibited by treatment with N-ethylmaleimide. This inhibition was reversed by the addition of recombinant NSF. Botulinum neurotoxin serotype E, which is known to cleave the 25 000 Da synaptosomal associated protein, inhibited both transcytosis and recycling, although incompletely. We conclude that membrane traffic to a target membrane is not determined by utilizing a single molecular mechanism for fusion. Rather, a target membrane, e.g. the apical plasma membrane of MDCK cells, may use multiple molecular mechanisms to fuse with incoming vesicles.

Original languageEnglish
Pages (from-to)1471-1481
Number of pages11
JournalEMBO Journal
Volume15
Issue number7
DOIs
StatePublished - Apr 1 1996

Keywords

  • MDCK cells
  • NSF-dependent fusion
  • SNARE
  • Transcytosis

ASJC Scopus subject areas

  • General Neuroscience
  • Molecular Biology
  • General Biochemistry, Genetics and Molecular Biology
  • General Immunology and Microbiology

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