Interplay between ER Exit Code and Domain Conformation in CFTR Misprocessing and Rescue

Multiple mutations in cystic fibrosis transmembrane conductance regulator (CFTR) impair its exit from the endoplasmic reticulum (ER). We compared two processing mutants: Delta F508 and the ER exit code mutant DAA. Although both have severe kinetic processing defect, DAA but not Delta F508 has substantial accumulation in its mature form, leading to higher level of processing at the steady state. DAA has much less profound conformational abnormalities. It has lower Hsp70 association and higher post-ER stability than Delta F508. The ER exit code is necessary for Delta F508 residual export and rescue. R555K, a mutation that rescues Delta F508 misprocessing, improves Sec24 association and enhances its post-ER stability. Using in situ limited proteolysis, we demonstrated a clear change in trypsin sensitivity in Delta F508 NBD1, which is reversed, together with that of other domains, by low temperature, R555K or both. We observed a conversion of the proteolytic pattern of DAA from the one resembling Delta F508 to the one similar to wild-type CFTR during its maturation. Low temperature and R555K are additive in improving Delta F508 conformational maturation and processing. Our data reveal a dual contribution of ER exit code and domain conformation to CFTR misprocessing and underscore the importance of conformational repair in effective rescue of Delta F508.