title: The forced aggresome formation of a bovine anion exchanger 1 (AE1) mutant through association with δF508-cystic fibrosis transmembrane conductance regulator upon proteasome inhibition in HEK293 cells.
abstract: The endoplasmic reticulum (ER)-associated degradation of various polytopic proteins, involving the most common mutant of cystic fibrosis transmembrane-conductance regulator (CFTR), deltaF508-CFTR, involves retrotranslocation of the polypeptide into the cytosol, leading to aggresome formation when the proteasome activity is attenuated. By contrast, an R664X nonsense mutant of the bovine anion exchanger 1 (AE1) is retained in the ER and does not form aggresomes upon proteasome inhibition in transfected HEK293 cells. Here, we report that R664X AE1 formed a large cytoplasmic aggregate when cells co-transfected with enhanced green fluorescence protein (EGFP)-deltaF508-CTR were exposed to the proteasome inhibitor lactacystin. R664X AE1 and EGFP-deltaF508-CFTR showed co-localization in the aggregates and signals of which coincided with gamma-tubulin and were caged by vimentin at the pericentriolar locus, demonstrating aggresome formation. On the other hand, EGFP-AnkN90, consisting of the N-terminal AE1 binding domain of ankyrin, a cytoplasmic protein, also exhibited co-localization with R664X AE1, but was found throughout the ER. Moreover, R664X-mutant protein was specifically immunoprecipitated with EGFP-deltaF508-CFTR from the cells co-expressing these proteins. These findings indicate that R664X AE1 is forcibly extracted from the ER to reside in aggresomes through association with deltaF508-CFTR.
authors: Laboratory of Molecular Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
authors: Hirokazu
authors: H
authors: Adachi
authors: Takao
authors: T
authors: Kurooka
authors: Wataru
authors: W
authors: Otsu
authors: Mutsumi
authors: M
authors: Inaba
mesh_terms: D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D046988:Proteasome Endopeptidase Complex / Q000235:genetics; D061988:Proteasome Inhibitors; D011817:Rabbits
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D061988:Proteasome Inhibitors; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D046988:Proteasome Endopeptidase Complex