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.

Although cathepsin inhibitors stabilized the complexglycosylated wt CFTR by 7-10-fold in lysosomes as well as in the light-density fraction, only a modest accumulation of T70 CFTR could be documented by immunoblot analysis (Fig. 3 B) . Similar results were obtained in lysosomes isolated by the calcium-precipitation method (data not shown; Kawashima et al., 1998) . Surprisingly, leupeptin and pepstatin A provoked the accumulation of degradation intermediates of both wt and T70 CFTR predominantly in lysosomes (Fig. 3 B) . These inhibitors also provoked the appearance of breakdown products in isolated lysosomes of intestinal epithelia (CaCo-2 and T84) expressing CFTR endogenously, implying that lysosomal proteolysis cannot be attributed to heterologous overexpession (data not -21 cells expressing wt, T26, T70, T82 , or T98 CFTR were metabolically labeled overnight with [ 35 S]methionine and [ 35 S]cysteine. Plasma membrane proteins biotinylated with 1 mg/ml sulfo-NHS-SSbiotin for 60 min at 37ЊC were isolated by immunoprecipitation with L12B4 and M3A7 anti-CFTR Abs and then on Streptavidin-Sepharose (top). 10% of the immunoprecipitate was directly loaded (bottom). To avoid clonal variations, this, and the experiments shown in C, were carried out on a mixture of clones. The sulfo-NHS-SS-biotin remains membrane impermeant during the labeling, indicated by the lack of biotinylated core-glycosylated forms. Complex-and core-glycosylated CFTR are indicated by black and white arrowheads, respectively. (B) The expression level of wt and truncated CFTR at the cell surface (biotinylated) and at post-ER compartments (complex glycosylated). The radioactivity of biotinylated CFTR was measured by Phos-phorImage analysis in experiments as described in the legend to A. The level of the complex-glycosylated CFTR was determined by densitometry of immunoblots (C). Data represent means Ϯ SEM (n ϭ 3), and are expressed as the percentage of the wt, designated as 100%. (C) Expression of wt, T26, T70, T82, and T98 CFTR in BHK-21 cells. CFTR expression in cell lysate was assessed by immunoblotting using the L12B4 and M3A7 anti-CFTR Abs. BHK lane contains a lysate of the parental cells. (D) Glycosidase sensitivity of complex-and core-glycosylated wt and T70 CFTR. Cell lysates were incubated in the presence or absence of endoglycosidase H (endo H) or peptide-N-glycosidase F (PNGase F) for 3 h at 37ЊC. Polypeptides were separated by SDS-PAGE and probed by the L12B4 anti-CFTR Ab. Complex-, core-, and deglycosylated CFTR are indicated by black, white, and hatched arrowheads, respectively. (E) Targeting efficiency of newly synthesized CFTR to the cell surface. After the pulse labeling of wt or T70 CFTR for 20 min, plasma membrane insertion of the channels was determined by biotinylation during a 1-h chase at 37ЊC using freshly dissolved sulfo-NHS-SS-biotin (1 mg/ml) every 15 min. Biotinylated CFTR was precipitated with L12B4 and M3A7 Abs and then isolated on Streptavidin-Sepharose (biot). Labeled CFTR was visualized by fluorography. To monitor the pulse labeling of total CFTR pools, 10% of the precipitate was loaded directly (lysate).