Conformational maturation of CFTR but not its mutant counterpart (delta F508) occurs in the endoplasmic reticulum and requires ATP.

Abstract

Metabolic labeling experiments followed by immunoprecipitation were performed to investigate the kinetics, location and inhibitor sensitivity of degradation of both wild‐type (wt) and mutant (delta F508) cystic fibrosis conductance transmembrane regulator (CFTR). At the earliest stages of the biosynthetic process, both wt and delta F508 CFTR were found to be susceptible to degradation by endogenous proteases. Virtually all delta F508 CFTR and 45‐80% of wt CFTR were rapidly degraded with a similar half‐life (t1/2 approximately 0.5 h). The remaining wt CFTR attained a protease‐resistant configuration regardless of whether traffic between the endoplasmic reticulum (ER) and Golgi was operational. Metabolic energy is required for the conformational transition, but not to maintain the stability of the protease‐resistant wt CFTR. Intracellular degradation of delta F508 CFTR and of incompletely folded wt CFTR occurs in a non‐lysosomal, pre‐Golgi compartment, as indicated by the sensitivity of proteolysis to different inhibitors and temperature. Accordingly, products of the degradation of delta F508 CFTR could be detected by immunoblotting in isolated ER, but not in the Golgi. Together, these results suggest a dynamic equilibrium between two forms of wt CFTR in the ER: an incompletely folded, protease‐sensitive form which is partially converted by an ATP‐dependent process to a more mature form that is protease‐resistant and capable of leaving the ER. The inability delta F508 CFTR to undergo such a transition renders it susceptible to complete and rapid degradation in a pre‐Golgi compartment.

Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation

“…According to Western-blot analysis, the expression level of CFTR in CHO-BQ1 cells was similar to that of CaCo-2 epitheloid colon-carcinoma cells, expressing CFTR endogenously [24], whereas CHO-BQ2 cells displayed a higher ( 20-fold) level of CFTR expression (results not shown). ∆F508CFTR-expressing cells were cultured under the same conditions as CHO-BQ2 cells [25].…”

“…The anionic fluorescent probe bisoxonol (DiSBAC # [3]) was used to estimate the membrane potential (E m ) of cells in suspension at 37 mC, as described previously [24].…”

“…Extracellular transferrin and dextran were removed by extensive washes with ice-cold medium (see above). CHO-BQ1 cells were homogenized by nitrogen cavitation [25] and the postnuclear supernatant was fractionated on a self-forming Percoll-density gradient [25 % Percoll\0.25 M sucrose\10 mM Hepes\1 mM EDTA (pH 7.3)] for 100 min (28 000 g at 4 mC). The gradient was downloaded with a fraction collector (Auto DensiFlow ; Labconco) from the bottom of the tubes (see Figure 1).…”

“…Radioactive labelling of CHO-BQ2 cells was carried out with [$&S]methionine and [$&S]cysteine (0.1-0.2 mCi\ml ; Amersham) for 2-3 h and chased in the presence of excess cold methionine and cysteine [25]. Cell-surface proteins were biotinylated with sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-SS-biotin) (1 mg\ml in PBS) twice for 15 min on ice.…”

“…The efficiency of biotin stripping (92.2p2.3 %, n l 5) was evaluated by measuring the relative amount of biotinylated CFTR both before and after the reduction of the disulphide bonds on cells kept at 4 mC. Immunoprecipitation of the biotinylated CFTR was achieved with M3A7 and L12B4 monoclonal anti-CFTR antibodies [7] according to published protocol [25]. Immuno-isolated CFTR was eluted from protein G beads with 1 % (w\v) SDS.…”