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.

If the complex-glycosylated T70 CFTR was conformationally unstable, it was anticipated that the polypeptide in vivo thermostability would be compromised. The temperature dependence of the turnover of the complex-glycosylated wt and T70 CFTR was compared by metabolic pulse-chase studies. The turnover of the complex-glycosylated T70 CFTR was more than fivefold faster at 40ЊC (t 1/2 ‫ف‬ 0.5 h) than at 37ЊC (t 1/2 ‫ف‬ 2.7 h) (Fig. 9, A and C) . In sharp contrast, the turnover of the wt CFTR was only marginally affected at 40ЊC (t 1/2 ‫ف‬ 10.5 vs. 15 h at 37ЊC) (Fig. 9, Figure 6 . Residence time of cell surface biotinylated wt and T70 CFTR. (A) After metabolic labeling of the complex-glycosylated wt or T70 CFTR, cell surface resident proteins were biotinylated for 45 min and chased for the indicated time in complete medium. Biotinylated CFTR was immunoprecipitated by L12B4 and M3A7 anti-CFTR Abs and then isolated on Streptavidin-Sepharose (top). For comparison, 10% of the immunoprecipitate was loaded for fluorography (bottom). (B) Turnover rates of biotinylated wt and T70 CFTR. Radioactivity incorporated into the biotinylated CFTR was measured with Phos-phorImage analysis and expressed as the percentage of the initial label. Data represent means Ϯ SEM, n ϭ 3. (C and D) Proteasome inhibitors delay the degradation of the biotinylated T70 CFTR. Radioactivity remaining in the biotinylated T70 CFTR was measured in pulse-chase experiments after 4 h of chase in the absence or presence of lactacystin (Lac; 10 M) or MG132 (MG; 10 M) by PhosphorImage analysis. Data are expressed as a percentage of the amount remaining for the control T70 CFTR and represent means Ϯ SEM, n ϭ 3. B and D). The simplest interpretation of these data is that the intrinsic structural instability of the T70 CFTR becomes more apparent at elevated temperature, leading to thermodenaturation and proteolysis, whereas the compact tertiary structures of wt CFTR are resistant to unfolding.