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.

Cells were washed with ice-cold PBS three times, resuspended in homogenization medium (0.25 M sucrose, 10 mM Hepes, 1 mM EDTA, 1 mM DTT, pH 7.2, supplemented with 5 g/ml leupeptin, 5 g/ml pepstatin A, and 2 mM PMSF). CHO-BQ1, CHO-K1, CaCo-2, and T84 cells were homogenized by nitrogen cavitation as described (Zhang et al., 1998) , whereas BHK cells were homogenized in a Dounce homogenizer. After the sedimentation of unbroken cells and nuclei (2,500 g , 5 min at 4 Њ C), mitochondria were pelleted by centrifugation (10,000 g , 10 min at 4 Њ C) from the postnuclear supernatant. Microsomes were fractionated on self-forming Percoll density gradient (25% Percoll in 0.25 M sucrose, 10 mM Hepes and 1 mM EDTA, 5 g/ml leupeptin, 5 g/ml pepstatin A, pH 7.3) at 28,000 g for 100 min. The density profile of the gradient was verified with density-marker beads (Sigma-Aldrich) and fractions were downloaded as described . In some experiments lysosomes were labeled with the fluid-phase marker, fluorescein-dextran (0.5 mg/ml, 70 kD; Molecular Probes), overnight and chased in full medium for 3 h. Alkaline phosphatase, ␤ -glucoronidase, and mannosidase II activity, specific markers of plasma membrane, lysosomes, and Golgi regions, respectively, were measured as described (Lukacs et al., 1994 . The fluorescence associated with the fractions was determined with fluorescence spectrophotometry in the presence of 0.2% Triton X-100.