title: Processing of CFTR bearing the P574H mutation differs from wild-type and deltaF508-CFTR.
abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) containing the deltaF508 mutation is retained in the endoplasmic reticulum (ER). This defect can be partially overcome by a reduction in temperature which allows some of the deltaF508 protein to exit the ER and move to the cell surface. Earlier studies showed that the CF-associated mutants, P574H and A455E, were also misprocessed. In this study, we found that processing of P574H and A455E was also temperature-sensitive; at 26 degrees C, some of the protein matured. In contrast to other CFTR mutants, P574H accumulated in punctate cytoplasmic bodies that colocalized with endoplasmic reticulum (ER) markers. At 26 degrees C, these bodies were no longer present. P574H showed a prolonged association with Hsp70 and also colocalized with Hsp70. We used brefeldin A (BFA) to determine which processing step(s) was altered by reduced temperature. Unlike wild-type CFTR, which was converted into an intermediate that was stable in the presence of BFA at 37 degrees C, deltaF508 and P574H produced the intermediate only when the temperature was reduced to 26 degrees C. Furthermore the wild-type intermediate was not associated with Hsp70. These data suggest that formation of the stable intermediate is a key temperature-sensitive step and appears to be coincident with release of the wild-type protein from Hsp70.
authors.affiliation: Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
authors.forename: L S
authors.initials: LS
authors.lastname: Ostedgaard
authors.forename: B
authors.initials: B
authors.lastname: Zeiher
authors.forename: M J
authors.initials: MJ
authors.lastname: Welsh
mesh_terms: D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D019556:COS Cells; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D017384:Sequence Deletion; D013696:Temperature
chemical_list: C505032:CFTR protein, human; D018840:HSP70 Heat-Shock Proteins; D011500:Protein Synthesis Inhibitors; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D020126:Brefeldin A