Abstract
Soluble secreted proteins and membrane proteins are subjected to protein quality control pathways during their synthesis in the endoplasmic reticulum (ER) and delivery to other destinations. Foremost among these quality control pathways is the selection of misfolded proteins for ER-associated degradation (ERAD). A growing number of diseases, including Cystic Fibrosis, are linked to the ERAD pathway. In most cases, a membrane protein known as the Cystic Fibrosis Transmembrane Conductance Regulator, or CFTR, is prematurely degraded by ERAD. Cell-based assays and in vitro studies have elucidated factors required for the recognition and degradation of CFTR, yet mechanistic details on how these factors target specific disease-causing variants is limited. Given the possibility that variants might exhibit unique susceptibilities to ubiquitin modification, which is required for proteasome-mediated degradation, we devised an assay that recapitulates this event. Here, we demonstrate that ER-enriched membranes from transfected human cells support CFTR ubiquitination when combined with radiolabeled ubiquitin and isolated enzymes in the ubiquitination cascade. We also show that select disease-causing variants are ubiquitinated more extensively than wild-type channels and to varying degrees. Our system provides a platform to examine how other purified factors impact CFTR ubiquitination and the ubiquitination of additional disease-associated membrane proteins.