Cooh-Terminal Truncations Promote Proteasome-Dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments

Benharouga, Mohamed; Haardt, Martin; Kartner, Norbert; Lukacs, Gergely L.

To determine the expression levels of wt and truncated CFTR at the cell surface and early endosome, BHK cells were metabolically labeled to steady state overnight and rinsed with H buffer (154 mM NaCl, 10 mM Hepes, 3 mM KCl, 1 mM MgCl 2 , 0.1 mM CaCl 2 , 10 mM glucose, pH 7.6). Cells were biotinylated in the presence of 1 mg/ml sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (EZ-Link sulfo-NHS-SS-biotin; Pierce Chemical Co.) in H buffer at 37 Њ C . Cells were washed with ice-cold PBS, supplemented with 0.1% BSA, 1 mM MgCl 2 , and 0.1 mM CaCl 2 , and solubilized in RIPA buffer. Biotinylated CFTR was isolated by immunoprecipitation with M3A7 and L12B4 anti-CFTR Abs and then on Streptavidin-Sepharose (Sigma-Aldrich). Biotinylated CFTR was visualized with fluorography and its radioactivity was measured by PhosphorImage analysis.