Post-translational disruption of the delta F508 cystic fibrosis transmembrane conductance regulator (CFTR)-molecular chaperone complex with geldanamycin stabilizes delta F508 CFTR in the rabbit reticulocyte lysate.
The DeltaF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) is a trafficking mutant, which is retained and degraded in the endoplasmic reticulum by the ubiquitin-proteasome pathway. The mutant protein fails to reach a completely folded conformation that is no longer a substrate for ubiquitination ("stable B"). Wild type protein reaches this state with 25% efficiency. In this study the rabbit reticulocyte lysate with added microsomal membranes has been used to reproduce the post-translational events in the folding of wild type and DeltaF508 CFTR. In this system wild type CFTR does not reach the stable B form if the post-translational temperature is 37 degrees C, whereas at 30 degrees C the behavior of both wild type and mutant proteins mimics that observed in the cell. Geldanamycin stabilizes DeltaF508 CFTR with respect to ubiquitination only when added post-translationally. The interaction of wild type and mutant CFTR with the molecular chaperones heat shock cognate 70 (hsc70) and heat shock protein 90 (hsp90) has been assessed. Release of wild type protein from hsc70 coincides with the cessation of ubiquitination and formation of stable B. Geldanamycin immediately prevents the binding of hsp90 to DeltaF508 CFTR, and after a delay releases it from hsc70. Release of mutant protein from hsc70 also coincides with the formation of stable B DeltaF508 CFTR.
Fuller
W
W
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, United Kingdom.
Cuthbert
A W
AW
D000818:Animals; D016227:Benzoquinones; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D047029:Lactams, Macrocyclic; D018832:Molecular Chaperones / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011809:Quinones / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism
D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D018832:Molecular Chaperones; D011809:Quinones; D014452:Ubiquitins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C001277:geldanamycin