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.

Since recognition of most of the substrates destined for destruction by the proteasome requires polyubiquitin modification (Hershko and Ciechanover, 1998; Laney and Hochstrasser, 1999; Plemper and Wolf, 1999; Hirsh and Ploegh, 2000) , we examined whether T70 CFTR is subjected to ubiquitin attachments. COS-1 cells were transiently cotransfected with plasmids encoding for the wt or T70 CFTR and HA-tagged ubiquitin (HA-Ub). HA-Ub was detected with the monoclonal anti-HA Ab in immunoprecipitated T70 CFTR. The abundance of high molecular weight immunoreactive polypeptides was augmented by lactacystin and MG132, suggesting that proteasome activity is involved in the degradation of polyubiquitinated The complex-glycosylated truncated CFTR is stabilized by proteasome inhibitors. BHK cells expressing T70, T82, and T98 CFTR were metabolically labeled and chased in the presence of lactacystin (Lac; 10 M) or MG132 (MG; 10 M). The stability of the complex-glycosylated form was monitored by immunoprecipitation and fluorography. The radioactivity remaining in the complex-glycosylated CFTR was measured by Phosphor-Image analysis and expressed as the percentage of the amount after a 2-h chase (B). Data are means Ï® SEM, n Ï­ 3-4. (C and D) To rule out that lactacystin (10 M) delays the degradation of the complex-glycosylated form by promoting the biosynthesis or the conversion of the core-glycosylated form, cells were treated with lactacystin after the pulse labeling (C) or after the conversion of the core-to complex-glycosylated T70 (D). This protocol provided similar inhibition of the complex-glycosylated turnover as reported for A. When indicated, BFA (5 g/ ml) was included to prevent the conversion of the residual core-glycosylated form (D and E). (E-G) Comparison of the turnover rate of folded wt and T70 CFTR in the ER and post-Golgi compartments. After the pulse labeling of BHK transfectants, the fully mature core-glycosylated T70 and wt CFTR were accumulated in the ER by BFA (5 g/ml) during a 2-h chase. The disappearance of the folded core-glycosylated form was monitored during a subsequent 15-h chase in the presence of BFA and was expressed as the percentage of the amount detected after a 2-h chase (F and G). The stability of the complexglycosylated T70 and wt CFTR, representing post-Golgi pools, was determined as described for A. T70 CFTR (Fig. 7 A, top, lanes 8-10) . Similar results were obtained by monitoring the conjugation of endogenous Ub with a monoclonal anti-Ub Ab (Fig. 7 A, middle, lanes 6-10) . Ubiquitinated T70 CFTR could not be detected in cells expressing T70 CFTR or HA-Ub individually (Fig. 7 A, top, lanes 3-7) or after mock transfection (Fig. 7 A, lanes 1-3) , validating the specificity of the assay. To discriminate whether polyubiquitinated T70 CFTR was derived from the core-and/or the complex-glycosylated T70 CFTR (Jensen et al., 1995; Ward et al., 1995; Xiong et al., 1999) , double immunoprecipitation of metabolically labeled T70 CFTR was performed.