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 examine whether impaired plasma membrane targeting of the complex-glycosylated truncated CFTR can attribute to accelerated degradation, the efficacy of cell surface delivery of the mutant was determined first. BHK cells constitutively expressing wt or truncated CFTR were metabolically labeled with [ 35 S]methionine and [ 35 S]cysteine overnight and cell surface-exposed CFTR was covalently tagged with sulfo-NHS-SS-biotin. After the solubilization and immunoprecipitation of CFTR, biotinylated polypeptides were isolated on Streptavidin-Sepharose and visualized by fluorography after separation by SDS-PAGE ( Fig. 1 A) . PhosphorImage analysis revealed that the abundance of biotinylated T70, T82, and T98 CFTR was only 5-8% relative to wt or T26 CFTR in the mixture of clones ( Fig. 1 B) . The steady state level of the complex-glycosylated T70, T82, and T98 CFTR pool was also diminished to 5-10% relative to that of wt or T26 CFTR, as determined by quantitative Western blotting using L12B4 or M3A7 anti-CFTR Abs (Fig. 1 , B and C). The complex and core-glycosylated forms of wt and truncated CFTR could be distinguished by their distinct electrophoretic mobility as well as their endoglycosidase H sensitivity as described previously and shown on Fig. 1 , C and D (Cheng et al., 1990) . The parallel decrease in the size of the complex-glycosylated and biotinylated pools of the truncated CFTR suggests that the plasma membrane targeting is largely preserved. To confirm this notion, the cell surface delivery of newly synthesized T70 CFTR was measured.