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The PEST sequence does not contribute to the stability of the cystic fibrosis transmembrane conductance regulator

DOI: 10.1186/1471-2091-3-29

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Using a web-based algorithm, PESTFind, we found a PEST sequence in the regulatory (R) domain of CFTR. The PEST sequence is found in many short-lived eukaryotic proteins and plays a role in their degradation. To determine its role in the stability and degradation of misprocessed CFTR, we introduced a number of site-directed mutations into the PEST sequence in the cDNA of ΔF508 CFTR, the most prevalent misprocessed mutation found in CF patients. Analysis of these mutants showed that the disruption of the PEST sequence plays a minor role in the degradation of the CFTR mutants. Multiple mutations to the PEST sequence within the R domain of CFTR inhibit maturation of CFTR and prevent the formation of a 100 kDa degradation product. The mutations, however, do not improve the stability of the mutant ΔF508 CFTR.These observations show that disruption of the structure of the R domain of CFTR can inhibit maturation of the protein and that the predicted PEST sequence plays no significant role in the degradation of CFTR.Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), the most prevalent fatal recessive genetic disease in the Caucasian population [1]. CFTR is a polytopic integral membrane protein synthesized in the endoplasmic reticulum (ER) and normally expressed on the apical surface of epithelial cells where it functions as a phosphorylation-stimulated and ATP-dependent chloride channel. The majority of CF patients express processing defective CFTRs that fail to mature to the cell surface; instead, the processing defective CFTRs are retained in the ER and are targeted for rapid degradation [2,3].The retention of processing defective CFTR is a response of the ER quality control system to misfolded proteins, which prevents the progression of misfolded or misassembled membrane and secretory proteins into later compartments of the secretory pathway [3]. During synthesis, nascent CFTR polypeptide chains are translated from


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