S-palmitoylation regulates biogenesis of core glycosylated wild-type and F508del CFTR in a post-ER compartment

Abstract

Defects in CFTR (cystic fibrosis transmembrane conductance regulator) maturation are central to the pathogenesis of CF (cystic fibrosis). Palmitoylation serves as a key regulator of maturational processing in other integral membrane proteins, but has not been tested previously for functional effects on CFTR. In the present study, we used metabolic labelling to confirm that wild-type and F508del CFTR are palmitoylated, and show that blocking palmitoylation with the pharmacologic inhibitor 2-BP (2-bromopalmitate) decreases steady-state levels of both wild-type and low temperature-corrected F508del CFTR, disrupts post-ER (endoplasmic reticulum) maturation and reduces ion channel function at the cell surface. PATs (protein acyl transferases) comprise a family of 23 gene products that contain a DHHC motif and mediate palmitoylation. Recombinant expression of specific PATs led to increased levels of CFTR protein and enhanced palmitoylation as judged by Western blot and metabolic labelling. Specifically, we show that DHHC-7 (i) increases steady-state levels of wild-type and F508del CFTR band B, (ii) interacts preferentially with the band B glycoform, and (iii) augments radiolabelling by [3H]palmitic acid. Interestingly, immunofluorescence revealed that DHHC-7 also sequesters the F508del protein to a post-ER (Golgi) compartment. Our findings point to the importance of palmitoylation during wild-type and F508del CFTR trafficking.

Figure 1. Palmitoylation of wild-type and F508del CFTR

(A) HEK293 cells transiently expressing CFTR were metabolically labeled with ³H-palmitic acid. Subsequent immunoprecipitation of CFTR indicated that both wild-type and F508del CFTR are palmitoylated (top panel). As a control, 2% of cell lysate was reserved for analysis by western blot prior to IP (lower panel). (B) Treatment with the palmitoylation inhibitor 2-BP (100 μM during labeling – 4 h) in stably transduced HeLa wild-type cells inhibits labeling of CFTR as indicated by ³H-palmitic acid (top panel). Cell lysate (2%) was studied by western blot prior to IP (lower panel).

Figure 2. Influence of palmitoylation on maturation of wild-type CFTR

(A) Western blot using HeLa cells stably transduced to express wild-type CFTR, CFBE (cystic fibrosis bronchial epithelial cells) stably expressing wild-type CFTR, Calu-3 (airway serous glandular) cells with high level CFTR under regulatory control of the endogenous promoter, and HEK293 cells encoding doxycycline-inducible CFTR in presence or absence of 2-BP (100–150 μM, 8 h). CFTR steady state levels decreased when palmitoylation was inhibited. (B) Pulse-chase analysis of HEK293 cells (expressing wild-type CFTR) labeled with ³⁵S methionine and cysteine followed by a chase in the presence or absence of 150 μM 2-BP. Treatment with 2-BP prevents proper CFTR maturation as shown by failure of band B progression to band C. Results were quantified (C) as a ratio of radiolabeled band C at each time point to starting levels of total (labeled bands B + C) CFTR immediately following the pulse. Halide efflux was measured (D) and quantified (E) by the SPQ fluorescence assay. Forskolin (20 μM) and genistein (50 μM) were added (solid arrow) to stimulate CFTR-dependent ion transport (rate of upward deflection tracks CFTR activity). The findings indicate a decrease in CFTR activity when palmitoylation is inhibited. Dotted arrow = addition of dequenching buffer; double arrow = addition of quenching buffer. Results are shown as mean ± SEM and normalized to control cells. *P < 0.05, **P < 0.005; n = 4.

Figure 3. Influence of palmitoylation on low temperature correction of F508del CFTR

(A) Western blot of F508del CFTR grown at low temperature (27°C). Treatment with 2-BP led to diminished correction of F508del CFTR as indicated by the absence of band C. Halide efflux was measured (B) and quantified (C) by the SPQ fluorescence assay. Forskolin (20 μM) and genistein (50 μM) were added (solid arrow) to stimulate CFTR-dependent ion transport. The findings indicate a decrease in surface activity of low temperature corrected F508del CFTR when palmitoylation is inhibited by 2-BP. Dotted arrow = addition of dequenching buffer; double arrow = addition of quenching buffer. Results are shown as mean ± SEM and normalized to control cells. *P < 0.005; n = 3.

Figure 4. Contribution of protein acyl transferases to CFTR biogenesis

Western blot of HEK293 cells co-transfected with HA-tagged DHHC proteins and either wild-type (A) or F508del (B) CFTR indicate an increase in band B following overexpression of DHHC-7. Co-immunoprecipitation (DHHC proteins used as anchor) identified a robust interaction between DHHC-7 and wild-type CFTR (C) as well as F508del CFTR (D). Metabolic labeling with ³H-palmitic acid in the presence of DHHC-7 resulted in enhanced CFTR palmitoylation (E), indicated by labeling after a 3–4 week exposure as compared to the 3–4 months required for CFTR labeling without DHHC-7. To verify palmitoylation, post-IP samples were split and treated with 1 M Tris-Cl (control) or 1 M hydroxylamine (pH 7.4) to remove palmitate via cleavage of the thioester bond.

Figure 5. Immunofluorescence of HeLa F508del cells in the presence or absence of DHHC-7

F508del CFTR was labeled in the absence (column I) and presence (columns II–IV) of overexpressed DHHC-7. Mutant CFTR and HA-tagged DHHC-7 were labeled with anti-NBD1 and anti-HA antibodies, respectively. Golgi (top panel IV) or ER (lower panel IV) compartments were detected with CellLight GFP (Molecular Probes). Immunofluorescence identified strong co-localization of F508del CFTR with DHHC-7 and Golgi markers. The data also describe increased levels of F508del CFTR following overexpression of DHHC-7.