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.

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.