RECONSTITUTION OF TRANSCYTOSIS IN SLO-PERMEABILIZED MDCK CELLS - EXISTENCE OF AN NSF-DEPENDENT FUSION MECHANISM WITH THE APICAL SURFACE OF MDCK CELLS

Recently, it was demonstrated that delivery from the trans-Golgi netwo rk (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-alp ha SNAP-SNARE. To determine if all traffic to the apical surface of th is cell lime was NSF independent, we reconstituted the transcytosis of pre-internalized IgA to the apical surface and recycling to the basol ateral 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. Botul inum neurotoxin serotype E, which is known to cleave the 25 000 Da syn aptosomal associated protein, inhibited both transcytosis and recyclin g, although incompletely, We conclude that membrane traffic to a targe t membrane is not determined by utilizing a single molecular mechanism for fusion. Rather, a target membrane, e.g. the apical plasma membran e of MDCK cells, may use multiple molecular mechanisms to fuse with in coming vesicles.