| Cystic fibrosis (CF) is caused by mutation of one protein, the cystic fibrosis transmembrane conductance regulator (CFTR), which normally functions as a chloride channel at the apical surface of epithelial cells. The most common CFTR mutation results in the deletion of a single amino acid (phenylalanine) at position 508, which causes the protein to fold improperly. The DeltaF508 CFTR is a temperature-sensitive (TS) processing mutant: At the restrictive temperature, 37°C, DeltaF508 CFTR misfolds in the endoplasmic reticulum (ER) and is degraded, but at the permissive temperature, 27°C, it is "rescued" from degradation (rDeltaF508). In particular, rDeltaF508 CFTR folds correctly enough to exit the ER and produces a chloride channel at the cell surface, similar to the wild-type (WT) protein. Unfortunately, rDeltaF508 is rapidly degraded after returning to the restrictive temperature, suggesting a surface stability defect. Therefore, it is clear that the DeltaF508 CFTR defect can be corrected under certain conditions, but first we must understand how the WT and DeltaF508 CFTR proteins are handled differentially by the ER and at the cell surface, which is the aim of the work presented herein. We first characterized an interaction between CFTR and a protein associated with ER degradation, valosin-containing protein (VCP), and found that it interacts with DeltaF508 CFTR, but not with the WT protein. This study provides further evidence that WT CFTR is processed efficiently in the ER. Second, with regard to DeltaF508 CFTR, we found that culture at the permissive temperature corrects not only the folding defect in the ER, but also the impaired surface stability of DeltaF508 CFTR. This result suggests that, like the folding defect, the surface stability defect of DeltaF508 CFTR is also TS. Additionally, we showed that two small molecular compounds known to mimic permissive temperature culture of DeltaF508 by correcting its ER processing defect also enhance its surface stability at 37°C. These studies add significantly to current CF knowledge and may aid in the design of future therapeutics for CF and other diseases resulting from protein folding mutations. Specifically, we identify specific differences between ER processing and surface trafficking of WT and DeltaF508 CFTR, and we reveal a novel mode of action for two known pharmacological interventions. |
