1. Analysis of CFTR folding and degradation in transiently transfected cells
- Author
-
Richard L Watkins, Diane E. Grove, Meredith F.N. Rosser, and Douglas M. Cyr
- Subjects
Quality Control ,congenital, hereditary, and neonatal diseases and abnormalities ,Protein Folding ,Time Factors ,Ubiquitin-Protein Ligases ,Blotting, Western ,Cystic Fibrosis Transmembrane Conductance Regulator ,Cell Separation ,Endoplasmic Reticulum ,Transfection ,Article ,Humans ,Immunoprecipitation ,RNA, Small Interfering ,Cell Proliferation ,Sequence Deletion ,biology ,Base Sequence ,Endoplasmic reticulum ,HEK 293 cells ,Molecular biology ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,Folding (chemistry) ,Blot ,Kinetics ,HEK293 Cells ,Gene Knockdown Techniques ,biology.protein ,Protein folding ,Biogenesis - Abstract
Misfolding and premature degradation of F508del-CFTR is the major cause of cystic fibrosis. Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum to select misfolded proteins for degradation. The folding status of F508del-CFTR is monitored by at least two ER quality control checkpoints. The ER-associated Derlin-1/RMA1 E3 complex appears to recognize folding defects in CFTR that involve misassembly of NBD1 into a complex with the R-domain. In contrast, the cytosolic Hsp70/CHIP E3 complex appears to sense folding defects that occur after synthesis of NBD2. Herein we describe methods that allow for the study of how modulation of these ER quality control factors by siRNA impacts CFTR folding and degradation. The experimental system described employs transiently transfected HEK293 cells and is utilized to monitor the biogenesis of CFTR by both Western blot and pulse chase studies. Methods to detect complexes formed between CFTR folding intermediates and ER quality control factors will also be described.
- Published
- 2011