Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells.
The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.
Bilan
Frédéric
F
Institut de Physiologie et de Biologie Cellulaires, CNRS UMR6187, Université de Poitiers, France; CHU de Poitiers, BP577, 86021 Poitiers cedex, France.
Nacfer
Magali
M
Fresquet
Fleur
F
Norez
Caroline
C
Melin
Patricia
P
Martin-Berge
Alice
A
Costa de Beauregard
Marie-Alyette
MA
Becq
Frédéric
F
Kitzis
Alain
A
Thoreau
Vincent
V
D000818:Animals; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007454:Iodides / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050766:Qb-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050683:R-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D007454:Iodides; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C112938:VAMP8 protein, human; C491416:VTI1B protein, human