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.

The disappearance of the complex-glycosylated, but not the core-glycosylated, T70, T82, and T98 CFTR was inhibited 2.3-, 2.6-, and 2.1-fold, respectively, by lactacystin when the drug was present during the pulse labeling and chase (Figs. 4, A and B, and 5 A) . MG132 exerted a less pronounced effect (Fig. 4 B) . Comparable inhibition was Figure 2 . The role of lysosomal proteases in the metabolism of truncated and wt CFTR. (A) After the pulse labeling of T70, T82, and T98 CFTR expressors, BHK cells were chased for 2 h to ensure the conversion of core-glycosylated form to complex-glycosylated CFTR at 37ЊC. Subsequent chase was performed in the presence of NH 4 Cl (15 mM). Bafilomycin B (Baf B; 2 M) and leupeptin plus pepstatin A (leuϩpep; 50 g/ml) were present during the entire pulse-chase. CFTR was immunoprecipitated and visualized by fluorography. (B) The radioactivity remaining in the complex-glycosylated T70, T82, and T98 CFTR after the chase was quantified by PhosphorImage analysis in experiments shown in A and expressed as the percentage of the initial amount present after 2 h of chase. Data represent means Ϯ SEM, n ϭ 3. (C) The effect of chathepsin inhibitors on the expression level of truncated CFTR. BHK transfectants were incubated with or without leupeptin and pepstatin A (50 g/ml) for 4 h. Equal amounts of protein were probed by immunoblotting as described in the legend to observed when lactacystin was added after the pulse labeling (Fig. 4 C) or after a 2-h chase (Fig. 4 D) . This latter protocol has ensured that augmented conversion of the core-to the complex-glycosylated form was not attributable to the lactacystin effect. Similarly, preventing ER to Golgi complex transport of the residual core-glycosylated T70 CFTR with brefeldin A (BFA) failed to influence the inhibitory effect of lactacystin (Fig. 4 D) , substantiating the notion that disposal of the complex-glycosylated mutants is delayed, rather than that the escape of core-glycosylated form is facilitated, by lactacystin. In fact, neither lactacystin nor MG 132 promoted the processing of the core-glycosylated T70 CFTR (Fig. 5 A) , similarly to other CFTR mutations identified in NBD1 (Jensen et al., 1995; Van Oene et al., 2000) . The maturation efficiency of T70 CFTR (20 Ϯ 2%, n ϭ 9) was negligibly increased in the presence of lactacystin (23 Ϯ 4%, n ϭ 4).