Isolation of CFTR: Chaperone Complexes by Co-Immunoprecipitation

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane glycoprotein that contains several large cytosolic subdomains. To progress through the secretory pathway, CFTR must fold and assemble its subdomains into stable conformations that permit exit from the endoplasmic reticulum (ER) (1 ). ER folding of both CFTR and mutant forms of CFTR (primarily CFTR ?F508) appears to be an inefficient step in CFTR biogenesis. Consequently, the majority of CFTR and nearly all CFTR ?F508 is retained in the ER and degraded via the ubiquitin-proteasome pathway (2 ,3 ). Molecular chaperones localized to the ER lumen (calnexin) and the cytosol (Hsp70, Hsc70, Hdj-2, Hdj-1, and Hsp90) have been shown to transiently associate with both CFTR and CFTR ?F508 (4 –7 ). As such, these chaperones may act to survey CFTR structure as part of the ER quality control system or they may serve to facilitate the normal folding of CFTR (8 ,9 ). To determine the role of chaperone proteins in CFTR biogenesis, we have developed methods to isolate complexes between CFTR and different classes of molecular chaperones. This chapter describes the methodology utilized to co-immunoprecipitate CFTR with cytosolic chaperones of the Hsp70 and Hsp40 class (6 ).