Peripheral protein quality control removes unfolded CFTR from the plasma membrane

Abstract

Therapeutic efforts to restore biosynthetic processing of the cystic fibrosis transmembrane conductance regulator lacking the F508 residue (DeltaF508CFTR) are hampered by ubiquitin-dependent lysosomal degradation of nonnative, rescued DeltaF508CFTR from the plasma membrane. Here, functional small interfering RNA screens revealed the contribution of chaperones, cochaperones, and ubiquitin-conjugating and -ligating enzymes to the elimination of unfolded CFTR from the cell surface, as part of a peripheral protein quality-control system. Ubiquitination of nonnative CFTR was required for efficient internalization and lysosomal degradation. This peripheral protein quality-control mechanism probably participates in the preservation of cellular homeostasis by degrading damaged plasma membrane proteins that have escaped from the endoplasmic reticulum quality control or are generated by environmental stresses in situ.

Fig. 1

Conformationally destabilized rΔF508CFTR is ubiquitinated and targeted for ESCRT-dependent degradation. (A) CFTR PM stability was determined by enzyme-linked immunosorbent assay (ELISA). ΔF508CFTR was rescued at 26°C for 36 hours, followed by 37°C incubation for 2.5 hours (rΔF) or at 26°C. (B) (Left) CFTR internalization and (right) endocytic recycling were measured by ELISA. Unfolding of rΔF508CFTR was accomplished at 37°C (2.5 hours). (C) Trypsin susceptibility of CFTR variants in microsomes was measured by quantifying the remaining full-length CFTR with immunoblotting and densitometry. Bands B and C indicate core- and complex-glycosylated CFTR, respectively. (D) rΔF508CFTR ubiquitination in post-Golgi compartments was measured by d-IP and normalized for CFTR in precipitates. Nonrescued ΔF508CFTR (ΔF) was used as a positive control. (E and F) Metabolic stability of rΔF508CFTR was measured by CHX chase at 37°C and immunoblotting with an antibody against HA in ESCRT KD HeLa cells (E). Remaining complex-glycosylated CFTR was quantified by densitometry and expressed as a percentage of the initial amount (F). SMARTpool (p) or single siRNA (1 and 2) were used. siNT; nontargeted siRNA.

Fig. 2

CHIP mediates rΔF508CFTR elimination from PM. (A) Functional siRNA screen to identify Ub ligases responsible for rΔF508CFTR elimination from PM in HeLa cells. Ub ligases were knocked down by 50 nM SMARTpool siRNA, and rΔF508CFTR PM stability was measured at 37°C by ELISA. TSG101 siRNA; positive control. Data are means ± SEM from two or three independent quadruplicate experiments. (B) Effect of Ub ligase or UbcH5c KD on rΔF508CFTR PM stability in IB3 cells. (C) rΔF508CFTR metabolic stability in post-Golgi compartments was measured by immunoblotting with CHX chase upon CHIP KD by SMARTpool siRNA. Remaining complex-glycosylated CFTR was plotted. (D) Internalization of CFTRs (5 min), TfR (5 min), and CD4Tcc-Ub(AllRΔG) (2.5 min) were measured at 37°C in CHIP-KD cells. (E) CHIP KD enhanced rΔF508CFTR endocytic recycling. (F) rΔF508CFTR PM stability in E2-KD cells was measured as in (A).

Fig. 3

rΔF508CFTR ubiquitination is regulated by chaperones and co-chaperones. (A) rΔF508CFTR ubiquitination in post-Golgi compartments was measured in SMARTpool siRNA-transfected HeLa cells as in Fig. 1D. (B) Quantification of CFTR ubiquitination in immunoblots (A) and (G). Signal was normalized for CFTR in precipitates and expressed as percentages of rΔF508CFTR ubiquitination in siNT-treated cells after unfolding (37°C, 2.5 hours) as in Fig. 1D. (C) (Left) PM stability and (right) internalization of rΔF508CFTR were measured by ELISA in COS7 cells coexpressing myc-CHIP variants. (D) CHIP interacts with core- and complex-glycosylated rΔF508CFTR in HeLa cells transfected with myc-CHIP variants. (E) Interaction of chaperones with complex-glycosylated rΔF508CFTR was shown by co-IP in HeLa cells after in vivo cross-linking by 0.1 mM dithiobis[succinimidyl propionate] (DSP). Core-glycosylated wt and rΔF508CFTR were minimized by CHX chase (lanes 3 and 4). (F) rΔF508CFTR PM stability was evaluated upon KD of chaperone or cochaperone by SMARTpool siRNA as in Fig. 2A. Data are means ± SEM from two or three independent quadruplicate experiments. (G) rΔF508CFTR ubiquitination in post-Golgi compartments was measured in SMARTpool siRNA-transfected HeLa cells as in (A).

Fig. 4

Internalization and lysosomal targeting of rΔF508CFTR are regulated by distinct sets of chaperones and cochaperones. (A) Internalization of unfolded rΔF508CFTR (5 min), TfR (5 min) and CD4Tcc-Ub(AllRΔG) (2.5 min) was determined as in Fig. 2D. (B and C) Effect of chaperone or cochaperone KD on lysosomal sorting of (B) rΔF508CFTR and (C) Lamp2/CD63 was examined by measuring the acidification rate from early endosomes to lysosomes (see fig. S9). (D) Metabolic stability of (left) core-glycosylated ΔF508CFTR and (right) complex-glycosylated rΔF508CFTR in SMARTpool siRNA-transfected HeLa cells was determined by immunoblotting with CHX chase. (E) CFTR disappearance was quantified by densitometry and expressed as percentages of initial amount (T-0) in experiments shown in (D). Bar graphs of each sample represent the data (top) at 0- to 3-hour and (bottom) 0- to 6-hour chase. MG132 and bafilomycin A1 (BafA1) were positive controls for ERAD and lysosomal degradation, respectively.

Fig. 5

The peripheral QC machinery ubiquitinates the unfolded CFTR and dictates the PM level. (A) Reconstitution of rΔF508CFTR ubiquitination in vitro. Purified complex-glycosylated rΔF508CFTR-His₁₀ was incubated with E1, UbcH5c, CHIP, Hsc70, Ub-Myc, and ATP at 30°C for 2 hours. rΔF508CFTR was isolated by d-IP and analyzed by immunoblotting. (B) CHIP-dependent ubiquitination of purified rΔF508CFTR from PM-enriched vesicles was stimulated by recombinant Hsc70. (C) Wt and rΔF508 CFTR-His₁₀ were purified and ubiquitinated as in (B). (D) Classification of chaperones/cochaperones based on their effect on rΔF508CFTR PM density (fig. S5E) and stability (Figs. 2A and 3F). Components of the peripheral QC machinery identified are indicated by larger type. (E) Working model for the peripheral protein QC of rΔF508CFTR.