title: Reconstitution of transcytosis in SLO-permeabilized MDCK cells: existence of an NSF-dependent fusion mechanism with the apical surface of MDCK cells.
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)-alpha soluble NSF attachment protein (SNAP)-SNAP receptor (SNARE) complexes, while delivery from the TGN to the apical surface was independent of NSF-alpha SNAP-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 vesicle.
authors.affiliation: Department of Anatomy, University of California, San Francisco 94143-0452, USA.
authors.forename: G
authors.initials: G
authors.lastname: Apodaca
authors.forename: M H
authors.initials: MH
authors.lastname: Cardone
authors.forename: S W
authors.initials: SW
authors.lastname: Whiteheart
authors.forename: B R
authors.initials: BR
authors.lastname: DasGupta
authors.forename: K E
authors.initials: KE
authors.lastname: Mostov
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins; D001693:Biological Transport, Active / Q000187:drug effects; D001905:Botulinum Toxins / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D007070:Immunoglobulin A / Q000378:metabolism; D066298:In Vitro Techniques; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects; D008954:Models, Biological; D052066:N-Ethylmaleimide-Sensitive Proteins; D015739:Nocodazole / Q000494:pharmacology; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013301:Streptolysins; D033921:Vesicular Transport Proteins
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D007070:Immunoglobulin A; D008565:Membrane Proteins; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013301:Streptolysins; D033921:Vesicular Transport Proteins; C025964:streptolysin O; D019319:Okadaic Acid; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D001905:Botulinum Toxins; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D015739:Nocodazole