THE SNARE MACHINERY IS INVOLVED IN APICAL PLASMA-MEMBRANE TRAFFICKINGIN MDCK CELLS

We have investigated the controversial involvement of components of th e SNARE (soluble N-ethyl maleimide-sensitive factor [NSF] attachment p rotein [SNAP] receptor) machinery in membrane traffic to the apical pl asma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN t o apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane, All other tes ted transport steps were unaffected by syntaxin 3 overexpression. Botu linum neurotoxin E, which cleaves SNAP-23, and antibodies against or-S NAP inhibit both TGN to epical and basolateral transport in a reconsti tuted in vitro system. In contrast, we find no evidence for an involve ment of N-ethyl maleimide-sensitive factor in TGN to apical transport, whereas basolateral transport is NSF-dependent, We conclude that synt axin 3, SNAP-23, and ct-SNAP are involved in apical membrane fusion. T hese results demonstrate that vesicle fusion with the apical plasma me mbrane does not use a mechanism that is entirely unrelated to other ce llular membrane fusion events, but uses isoforms of components of the SNARE machinery, which suggests that they play a role in providing spe cificity to polarized membrane traffic.