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 investigate the influence of proteasome inhibitors on the residence time of truncated CFTR at the cell surface, the turnover of biotinylated T70 CFTR was measured by the pulse-chase technique. The half-life of biotinylated T70 CFTR is sevenfold shorter (t 1/2 ‫ف‬ 1.9 h) than the wt CFTR (t 1/2 ‫ف‬ 14.5 h; Fig. 6, A and B ). Lactacystin and MG132 inhibited the turnover of biotinylated T70 CFTR by 2.6-and 1.9-fold, respectively. This observation provides direct support for our hypothesis that proteasomes, Figure 3 . The effect of leupeptin and pepstatin A on the metabolism of wt and T70 CFTR in lysosomes. (A) Separation of subcellular organelles on Percoll density gradient. Postnuclear supernatants of wt-and T70 CFTR-expressing BHK cells were fractionated on a 25% Percoll density gradient as described in Materials and Methods. Organellar distribution was established by the activity of specific marker enzymes (plasma membrane, alkaline phosphatase [A P]; Golgi, mannosidase II [Mann II]; and lysosomes, ␤-glucuronidase [␤-gluc]) as described previously . Enrichment of lysosomes in the high density fractions was confirmed by the accumulation of fluorescein-dextran (70 kD, 1.5 mg/ml) as well after an overnight labeling and a 3-h chase. (B) BHK cells expressing wt or T70 CFTR were treated with leupeptin and pepstatin A overnight (50 g/ml) and for 4 h (100 g/ml), respectively. Microsomes were isolated on Percoll density gradient, and fractions 5-13 and 15-18, comprising the majority of lysosomes and plasma membrane, Golgi regions, and endosomes, respectively, were pooled. Equal amounts (50 g) of protein were loaded and probed with NBD1 (L12B4) and NBD2 (M3A7) specific anti-CFTR Abs using ECL. directly or indirectly, are involved in the proteolysis of complex-glycosylated T70 CFTR from the cell surface and endosomal compartments (Fig. 6, C and D) .