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The SNARE machinery is involved in apical plasma membrane trafficking in MDCK cells
S H Low1, S J Chapin, C Wimmer
1Department of Anatomy, Biochemistry and Biophysics, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0452, USA.
Abstract:
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.
Insights
Syntaxin 3, SNAP-23, and alpha-SNAP are key to apical membrane fusion in polarized cells. This SNARE machinery provides specificity for membrane traffic, distinct from basolateral transport mechanisms.
Area of Science:
- Cell Biology
- Molecular Biology
- Membrane Trafficking
Background:
- The SNARE machinery regulates membrane fusion in eukaryotic cells.
- Polarized epithelial cells exhibit distinct apical and basolateral membrane domains.
- The specific roles of SNARE components in apical targeting remain controversial.
Purpose of the Study:
- To investigate the involvement of SNARE proteins in apical membrane transport in polarized epithelial cells.
- To determine the specific roles of syntaxin 3, SNAP-23, and NSF in TGN to apical transport.
Main Methods:
- Overexpression of syntaxin 3 in MDCK cells.
- Inhibition of SNAP-23 using Botulinum neurotoxin E.
- Inhibition of alpha-SNAP using antibodies.
- Reconstituted in vitro transport assays.
- N-ethylmaleimide-sensitive factor (NSF) dependency assays.
Main Results:
- Syntaxin 3 overexpression inhibited TGN to apical transport and apical recycling, causing vesicle accumulation.
- Botulinum neurotoxin E and alpha-SNAP antibodies inhibited both TGN to apical and basolateral transport.
- N-ethylmaleimide-sensitive factor (NSF) was not involved in TGN to apical transport but was essential for basolateral transport.
Conclusions:
- Syntaxin 3, SNAP-23, and alpha-SNAP are crucial for apical membrane fusion.
- Apical membrane fusion utilizes specific SNARE machinery isoforms, ensuring polarized traffic specificity.
- Vesicle fusion with the apical plasma membrane employs a mechanism related to, but distinct from, other cellular fusion events.