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 compare the conformational stability of the complex-glycosylated wt and T70 CFTR, in situ protease susceptibility and in vivo thermostability assays were performed. The conformation of the cytosolic domains was probed with limited proteolytic digestion in conjunction with immunoblotting, a method we have implemented to reveal conformational difference between the wt and the ⌬F508 CFTR (Zhang et al., 1998) . Intact microsomes were isolated by differential centrifugation from BHK cells, enriched in the complex-glycosylated wt or T70 CFTR by cyclohexamide chase, and subjected to limited proteolysis in the presence of an increasing concentration of trypsin. Immunoblot analysis of the proteolytic fragments with the L12B4 anti-CFTR Ab recognizing the NBD1 demonstrates a distinct proteolytic digestion pattern and a moderate, but reproducibly increased protease sensitivity of the complex-glycosylated T70 CFTR compared with wt CFTR (Fig. 8) . Similar results were obtained with proteinase K and using the NBD2-specific, M3A7 anti-CFTR Ab (data not shown), indicating that not only the NBD2, but also the NBD1 conformation was altered upon truncating the COOH terminus.