1. Trafficking of immature DeltaF508-CFTR to the plasma membrane and its detection by biotinylation
Yishan Luo, Ken McDonald, John W Hanrahan Biochem J. 2009 Apr 1;419(1):211-9, 2 p following 219.doi: 10.1042/BJ20081869.
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.
2. Monitoring MHC-II Endocytosis and Recycling Using Cell-Surface Protein Biotinylation-Based Assays
Kyung-Jin Cho, Paul A Roche Methods Mol Biol. 2019;1988:271-277.doi: 10.1007/978-1-4939-9450-2_19.
Most, if not all, plasma membrane proteins continuously undergo endocytosis and many rapidly recycle from endosomes back to the cell surface to maintain "stable" surface expression. We now describe a biochemical assay that is suited to follow the internalization and recycling kinetics of plasma membrane proteins. This assay involves biotinylation of plasma membrane proteins using sulfo-NHS-SS-biotin, a water-soluble, NHS-ester biotinylation reagent that contains a cleavable disulfide bond that allows for reversible labeling of proteins. Biotinylation is rapid and stable, and does not transfer from cell to cell, and the small size of the biotin probe does not affect cell function.
3. The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins
Kristine M Cihil, Agnieszka Swiatecka-Urban J Vis Exp. 2013 Dec 13;(82):e50867.doi: 10.3791/50867.
Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.
