Trafficking of immature DeltaF508-CFTR to the plasma membrane and its detection by biotinylation.
Recent studies suggest that immature, core-glycosylated DeltaF508-CFTR [the predominant mutant form of the CFTR (cystic fibrosis transmembrane conductance regulator)] can reach the plasma membrane under some conditions. In the present study we investigated this possibility since it has implications for understanding how therapeutics rescue the trafficking of mutant CFTR and perhaps other misfolded proteins. Core-glycosylated CFTR was labelled and pulled down on streptavidin beads after exposure to sulfo-NHS-SS-biotin [biotin attached to a reactive NHS (N-hydroxysuccinimide) ester with a disulfide spacer; molecular mass=606.7 Da]; however, intracellular proteins were also detected in the precipitates. When the R domain of CFTR was expressed in the cytosol of BHK (baby-hamster kidney) cells as a soluble polypeptide it was also labelled after surface biotinylation and pulled down on streptavidin beads. Intracellular biotinylation was reduced when cells were treated with sulfo-NHS-LC-biotin (biotin attached to a reactive NHS ester with an aminocaproic acid spacer) or sulfo-NHS-PEO(12)-biotin [biotin attached to a reactive NHS ester with a poly(ethylene glycol) spacer], but the reduction could be explained by the lower reactivity of these reagents. The R domain was detected on Western blots after loading <0.25% of the pulldown sample ( approximately 0.01% of total lysate protein), a fraction that could be ascribed to cells that were permeable to ethidium homodimer-1 (molecular mass=856.8 Da) and propidium iodide (molecular mass=668.6 Da). When BHK cells were incubated at 29 degrees C to rescue DeltaF508-CFTR trafficking, and then biotinylated and sorted to remove permeable cells, labelling of core-glycosylated DeltaF508-CFTR was no longer detected although a weak signal was still observed using CFBE (cystic fibrosis bronchial epithelial) cells. These results suggest that there is weak surface expression of immature DeltaF508-CFTR on airway epithelial cells and demonstrate the need to remove permeable cells when studying CFTR glycoforms by surface biotinylation.
Luo
Yishan
Y
Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec, Canada.
McDonald
Ken
K
Hanrahan
John W
JW
D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D005434:Flow Cytometry; D021381:Protein Transport / Q000502:physiology*
C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator