CFTR

Cystic Fibrosis, the most common lethal genetic disorder among Caucasians, results from mutations in the CF gene that encodes the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). To date almost 1000 disease-causing mutations have been mapped in the CF gene. The most prevalent mutation, occurring in ~70% of the CF patients, results in the deletion of a phenylalanine at position 508 in the NBD1 of CFTR. The deltaF508 mutation causes misfolding and retention of the CFTR membrane protein in the ER that results in almost complete degradation of the protein. Since CFTR has 12 transmembrane segments that divide the protein in a small ER luminal part and a bulky cytosolic part it is of interest to investigate how both ER and cytosolic chaperones coordinate the quality control of this protein. Interestingly, even most of the wildtype CFTR protein fails to attain its native conformation since almost 70% of the newly synthesized protein is degraded, a process that already starts during synthesis.

Kleizen B, Braakman I & de Jonge HR Regulated trafficking of the CFTR chloride channel. Eur J Cell Biol. 2000;79:544-56. pdf

Currently, we investigate in detail the co-translational folding processes of the wildtype CFTR protein. To study CFTR folding we combine in vitro translation/translocation in semi-permeabilized cells that allows us to study CFTR nascent chain elongation by limited proteolysis. The extent of protease susceptibility of newly synthesized radiolabeled CFTR is a measure for folding and conformation. Furthermore, we use radioactive pulse-chase analysis, cell surface biotinylation, and GFP fusion proteins to determine localization and maturation of CFTR in intact cells.

In contrast to other model proteins studied in our lab we found that CFTR folds almost exclusively co-translationally. Furthermore we pinpointed several bottlenecks in CFTR folding. We also found that folding mutants CFTR deltaF508 and CFTR P205S are not immensly misfolded but only show very local conformational differences compared to wildtype CFTR structure. With these techniques we could investigate whether certain drugs could rescue the folding mutants by stabilizing local distortions in conformation.

• The CFTR crew
  Ineke Braakman
• Hanneke Hoelen
• Mieko Otsu
• Floor Peters
• Nune Kazaryan (bachelor student)
  
• Matthijs van den Berg (bachelor student)
  
• Elena Ganusova (alumna)
  
• Evi Charitou (master student)
  Bertrand Kleizen (alumnus)