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.

Several criteria were used to establish that proteasomes are involved in the accelerated metabolism of the truncated CFTR from post-Golgi compartments, including the cell surface. First, the degradation rates of the complexglycosylated T70, 82, and 98 CFTR were delayed in the presence of lactacystin, the most specific inhibitor of proteasome (Fenteany et al., 1995; Fig. 4 ). Second, a dramatic extension of the plasma membrane residence time of biotinylated T70 CFTR was evoked by lactacystin (Fig. 6) , implying that proteasomes are indispensable in the proteolysis of the truncated CFTR from the cell surface and/or endosomal compartment. Since proteasome inhibitors had no impact on the degradation of Tac-Lamp1, an indirect effect of lactacystin on lysosomal proteolysis could be precluded (Fig. 5) . Third, MG132 and lactacystin evoked the accumulation of polyubiqutinated adducts of the complexglycosylated T70 CFTR, consistent with the notion that a fraction of the T70 CFTR is tagged by polyubiquitin chain(s) before degradation (Fig. 7) . Finally, both MG132 and lactacystin partially restored the expression level of complex-glycosylated T70, T82, and T98 CFTR, as well as the plasma membrane cAMP-activated chloride conductance of T70 CFTR transfectants without facilitating the biogenesis of the mutants (Fig. 5, D and E) . Although these observations indicate that proteasome activity is one of the rate-limiting steps for the turnover of the full-length complex-glycosylated T70 CFTR, proteasome inhibitors failed to restore the mutant stability to that of the wt Figure 8 . Protease susceptibility of native wt and T70 CFTR. After treating the cells with cyclohexamide (100 g/ml) for 2.5 h at 37ЊC to deplete the core-glycosylated forms, microsomes were isolated with differential centrifugation from BHK-21 cells stably expressing wt or T70 CFTR. Microsomes were subjected to limited proteolysis at the indicated concentrations of trypsin for 15 min at 4ЊC. The proteolytic digestion pattern was probed by L12B4 anti-CFTR Ab and ECL. Figure 9 . Thermostability of the complex-glycosylated wt and T70 CFTR in vivo. (A and B) Temperature-dependent turnover of the complex-glycosylated T70 and wt CFTR. Metabolic stability of the pulse-labeled complexglycosylated T70 and wt CFTR was monitored as described in the legend to Fig. 4 . Chase was conducted at 26ЊC, 37ЊC, or 40ЊC. (C and D) The disappearance kinetics of complex-glycosylated T70 and wt CFTR were determined by PhosphorImage analysis in experiments shown in A and B. Data are means Ϯ SEM, n ϭ 3.