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 Delta F508 mutation of cystic fibrosis transmembrane conductance regula tor (CFTR) is a trafficking mutant, which is retained and degraded in the e ndoplasmic reticulum by the ubiquitin-proteasome pathway. The mutant protei n fails to reach a completely folded conformation that is no longer a subst rate for ubiquitination ("stable B"). Wild type protein reaches this state with 25% efficiency. In this study the rabbit reticulocyte lysate with adde d microsomal membranes has been used to reproduce the post-translational ev ents in the folding of wild type and Delta F508 CFTR. In this system wild t ype CFTR does not reach the stable B form if the post-translational tempera ture is 37 degreesC, whereas at 30 degreesC the behavior of both wild type and mutant proteins mimics that observed in the cell. Geldanamycin stabiliz es Delta F508 CFTR with respect to ubiquitination only when added post-tran slationally. The interaction of wild type and mutant CFTR with the molecula r 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 im mediately prevents the binding of hsp90 to Delta F508 CFTR, and after a del ay releases it from hsc70. Release of mutant protein from hsc70 also coinci des with the formation of stable B Delta F508 CFTR.