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.

Stably transfected BHK cells were pulse labeled for 20 min and chased for 2 h to ensure the elimination of coreglycosylated T70 CFTR, verified by fluorography (Fig. 7 B, bottom, lanes 1 and 2; see also Fig. 4 D) . In a second set of samples, separation of ubiquitinated adducts was achieved by sequential immunoprecipitation with anti-CFTR and then with anti-Ub Ab. The results show that a negligible amount of polyubiquitinated and core-glycosylated T70 CFTR persisted after the initial 2-h chase (Fig. 7 B, top and bottom, lanes 1 and 2) . In contrast, radioactivity appeared in the high molecular weight, polyubiquitinated T70 CFTR when MG132 was present during the last 2 h of chase (Fig. 7 B, top, lane 4) . Accumulation of polyubiquitinated T70 CFTR was more pronounced in COS-1 cells transiently coexpressing T70 CFTR and HA-Ub (Fig. 7 C) . This is most likely due to the higher immunoprecipitation efficiency of the anti-HA Ab. Ubiquitinated conjugates were not detectable upon inhibition of lysosomal proteolysis, or in mock transfected cells (Fig. 7 B , top and middle, and C). Considering that the turnover of the complex-glycosylated, but not the core-glycosylated T70 CFTR, is sensitive to proteasome inhibitors and the core- Equal amounts of whole cell lysate, obtained from cells exposed to lactacystin (Lac; 10 M) and MG132 (10 M) for 3 h, were separated by SDS-PAGE and immunoblotted with the L12B4 anti-CFTR Ab. Complex-and core-glycosylated CFTR are indicated by black and white arrowheads, respectively. (E) Functional rescue of T70 CFTR by lactacystin. The cAMP-activated halide conductance of T70 CFTRexpressing BHK cells was measured by the iodide efflux assay in the absence (control) or presence of lactacystin treatment ( Ï©Lac; 10 M for 3 h). Data points are averages of triplicate determinations and show the difference of iodide release in the presence and absence of the protein kinase A agonist cocktail (10 M forskolin, 0.5 mM dibutyryl-cAMP, and 0.2 mM IBMX), added as indicated. glycosylated form has been largely eliminated before proteasome inhibition, we conclude that at least a fraction of polyubiquitinated T70 CFTR originates from the complex-glycosylated T70 CFTR. Although we cannot rule out that a small amount of polyubiquitinated T70 CFTR is derived from the core-glycosylated form, the following observations substantiate the notion that the complex-glycosylated T70 CFTR can form ubiquitinated adducts. Ubiquitinated complex-glycosylated form was visualized by anti-Ub Abs after the separation of the core-and complex-glycosylated forms by immunoprecipitation with anti-CFTR Abs and wheat germ agglutinin affinity chromatography (data not shown). Furthermore, coexpression of the dominant negative K48RUb with T70 CFTR delayed the disposal of the complex-glycosylated mutant CFTR (data not shown).