Extope-CFTR remains at or is recycled to the cell surface on overexpression of Rab5 DN or Rab11. (A) Extope-CFTR is trapped at the cell surface on overexpression of Rab5DN. Cells were transfected with Rab5aDN and 24 h later labeled for 10 min with 12CA5 mAb. Extope-CFTR was detected 4 h after labeling with mAb in nonpermeabilized (NP) or permeabilized (P) cells. A typical nontransfected cell is shown as an inset in the panel with permeabilized cells. No intracellular Extope-CFTR could be detected in permeabilized cells when cell surface mAb label was removed by acidic wash before fixation and permeabilization. (B) Extope-CFTR is robustly recycled to the plasma membrane on overexpression of Rab11. Cells were transfected with Rab11 and 24 h later intact cells were labeled for 10 min with mAb 12CA5. The mAb bound to the cells surface pool of Extope-CFTR was then removed by an acidic wash. Cells were either fixed and immediately immunostained (0 h) or reincubated in media for 4 h (4 h). Extope-CFTR was then detected in permeabilized or nonpermeabilized cells.

Localization of internalized CFTR relative to organelle markers. Extope-CFTR was labeled on the cell surface with anti-HA antibody (16B12). Cells were reincubated and fixed after the indicated times. Cells were permeabilized, blocked, and internalized Extope-CFTR was immunostained as described in Figure 2A by using goat anti-mouse IgG Alexa Fluor 488 conjugate or goat anti-mouse Fab Alexa Fluor 568 for detection. Subcellular marker proteins were labeled as described in MATERIALS AND METHODS. EEA1 was detected with an antibody directly conjugated to fluorescein isothiocyanate, and γ-Adaptin was visualized using a rabbit antibody. RhoB was transiently expressed as a fusion to EGFP. Alexa Fluor 568-dextran conjugates were added to the medium for 1 h, and cells were either analyzed immediately or reincubated for another 3 h in regular medium (1 + 3 h). Transferrin Alexa Fluor 568 was added to the media for 1 h followed by a 30-min chase with regular media. Lysosomes were detected with Lysotracker Red.

Rescue of the cell surface pool of ΔF508 CFTR by Rab11 and Rab5DN. Cells were grown at 27°C in the presence of butyrate and different Rab GTPases were transiently overexpressed as EGFP (Rab4) or DsRed (Rab5, Rab5DN, Rab11, Rab11DN, and Rab7DN) fusions. Extope-ΔF508 CFTR was labeled with 16B12 mAb as described in Figure 6 and detected 24 h later with goat anti-mouse IgG Alexa Fluor 488 conjugate or goat anti-mouse IgG Alexa Fluor 568 conjugate. (A) Rab5DN stabilized Extope-ΔF508 CFTR at the plasma membrane. (B) Rab11 increased cell surface Extope-ΔF508 CFTR. (C) Rab4 did not significantly change the turnover of Extope-ΔF508 CFTR. (D) Rab7DN overexpression stabilized intracellular pools of Extope-ΔF508CFTR. (E) Images were analyzed with ImageJ 1.30v software to determine the mean fluorescence per cell on overexpression of different Rab GTPases. Each point represents the average of at least 10 cells, and standard deviations are indicated.

Turnover of ΔF508-CFTR at the cell surface differs from wild type CFTR. (A) Extope-ΔF508 CFTR matures at low temperature. Cells expressing Extope-CFTR and Extope-ΔF508 CFTR were grown at 37 or 27°C in the presence of 2 mM butyrate to increase expression, and cell lysates were analyzed by immunoblotting as described in Figure 1B by using anti-CFTR antibody 596. (B) Localization of Extope-CFTR and Extope-ΔF508 CFTR at different times after external labeling. BHK-21 cells stably expressing Extope-CFTR and Extope-ΔF508 CFTR were grown at 27°C in the presence of 2 mM butyrate. Extope-CFTR and the ΔF508 variant were labeled at the cell surface and visualized as described in MATERIALS AND METHODS at the times indicated. Nuclei were stained with propidium iodide. (C) Chemiluminescence detection of cell surface Extope-CFTR and Extope-ΔF508 CFTR. Extope-CFTR was labeled with anti-HA antibody 16B12 on cells, which had been grown at 27°C in the presence of butyrate. Cells were then reincubated at 37°C for the times indicated. Remaining Extope-CFTR (red circles) and Extope-ΔF508 (blue diamonds) on the cell surface were detected using sheep anti-mouse IgG horseradish peroxidase conjugate and chemiluminescence substrate as described in MATERIALS AND METHODS. Each point represents the average of eight wells and standard deviations are indicated.

Extope-CFTR is internalized and recycled. (A) Cell surface pools of Extope-CFTR are internalized. BHK-21 cells stably expressing Extope-CFTR were precooled, and cell surface pools of Extope CFTR were labeled for 30 min on ice with mouse monoclonal anti-HA antibody 16B12. Cells were then washed with ice-cold PBS and reincubated for the indicated times. Bars, 10 μm. (B) Internalized pools of CFTR are rapidly recycled. Cells were labeled for 10 min with monoclonal mouse anti-HA antibody 12CA5 and then washed with PBS, pH 3.7, to remove antibody from the cell surface. Reincubatiuon in prewarmed media for different times results in reappearance of CFTR at the cell edges. Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% saponin in PBS. Extope-CFTR labeled with 12CA5 mAb was detected with goat anti-mouse IgG conjugated to Alexa Fluor 488. Nuclei were stained with propidium iodide. (C) Recycled CFTR is redirected to the cell surface. Internalized Extope-CFTR is detectable at the cell surface of nonpermeabilized cells after 5-min reincubation in media. Extope-CFTR–expressing cells were labeled with 12CA5 and washed to remove external label as in Figure 2B. Cells were fixed immediately or after 5-min reincubation in warm media and either not permeabilized (NP) or permeabilized (P). Extope-CFTR labeled with 12CA5 mAb was immunostained by goat anti-mouse IgG conjugated to Alexa Fluor 488. (D) Quantification of CFTR recycling to the plasma membrane. The internalized pool of CFTR is almost completely redirected to the cell surface in 5 min. Experiments were performed as in Figure 2C, and mean green fluorescence per cell was determined as described in MATERIALS AND METHODS by using ImageJ 1.30v software. Each point represents the average of at least 10 cells and standard deviations are indicated.

External-epitope tagged CFTR (Extope-CFTR) matures and functions like unmodified wild type CFTR. (A) Schematic of strategy used to generate Extope-CFTR. Portions of EL1 and EL4 sequences incorporated into EL2 flanking a single HA-epitope (blue) are shown in green and red, respectively. (B) Western blot of external-epitope tagged CFTR (Extope-CFTR). Extope-CFTR matures like the unmodified wild type protein. Total cell lysates (25 μg) were separated by 6% SDS-PAGE, transferred to nitrocellulose, and Extope-CFTR was detected with monoclonal mouse anti-HA antibody 16B12 or monoclonal mouse anti-CFTR antibody 596. (C) cAMP stimulated ³⁶Cl^- efflux. Extope-CFTR functions as a chloride channel like the unmodified wild type protein. BHK-21 cells stably expressing wild type CFTR (blue diamonds) or Extope-CFTR (red squares) were loaded with Na³⁶Cl, transferred to chloride-free buffer, and cAMP-dependent chloride efflux was stimulated with stimulation cocktail as described in MATERIALS AND METHODS. An arrow indicates the stimulation at time 0. Extope-CFTR–expressing cells, which were not stimulated, are shown as control (black circles). Each point represents the average of three independent samples and standard deviations are indicated. (D) Immunofluorescence detection of total and cell surface derived CFTR. Extope-CFTR was labeled on the cell surface with monoclonal mouse anti-HA antibody and cells were reincubated for 2 or 48 h. Cells were fixed, permeabilized, and internalized pools of Extope-CFTR were detected with Alexa Fluor 568 conjugated to goat anti-mouse Fab fragments (left). Total pools of Extope-CFTR were subsequently labeled with anti-CFTR antibody 570 directly conjugated to Alexa Fluor 488 (right).

CFTR endocytosis is inhibited by low temperature and protease inhibitors. (A) Incubation of 16°C blocks endocytosis and allows intracellular detection of ΔF508-CFTR. Cells were grown at 27°C with 2 mM butyrate and incubated for 45 min at 16°C. Then, cell surface pools of Extope-CFTR and Extope-ΔF508 CFTR were labeled with 12CA5 mAb for 30 min at the same temperature. Subsequently cells were washed and reincubated in regular media for 1 h. Extope-CFTR was then detected in cells washed with regular PBS, pH 7.4 (no acidic wash), or after an acidic wash (acidic wash). Nuclei were stained with propidium iodide. (B) Lysosomal and proteasomal inhibitors inhibit endocytic turnover of ΔF508-CFTR. Cell surface Extope-CFTR or Extope-ΔF508 CFTR were labeled with 16B12 as described in Figure 2A. Cells were reincubated at 37°C for 7 h with either lysosomal inhibitors pepstatin-A and leupeptin (Pep + Leu, each 50 μg/ml) and E64 (3.5 μg/ml) or proteasomal inhibitors lactacystin (50 μM) and MG132 (50 μM) or without drug (control) and stained for immunofluorescence microscopy. Nuclei were stained with propidium iodide. (C) ΔF508 CFTR accumulates in lysosomes in the presence of the lysosomal inhibitors. Cells were treated with lysosomal inhibitors pepstatin-A and leupeptin as described in Figure 5B. Extope-ΔF508 CFTR was labeled with 16B12 at the cell surface and detected 7 h later by using goat anti-mouse IgG Alexa Fluor 488 conjugate. Lysosomes were labeled with Lysotracker Red (Molecular Probes). (D) Proteasomal inhibitor increases the amount of CFTR retained at the cellular surface. Extope-CFTR and ΔF508 CFTR were labeled on intact precooled cells on ice for 30 min with 16B12 mAb and cells were reincubated in the presence or absence of lactacystin (25 μM) for indicated times. Cell surface Extope-CFTR was detected in a chemiluminescence assay at indicated times by using sheep antimouse peroxidase conjugate and chemiluminescence (see MATERIALS AND METHODS). Each experiment was performed with 8 replicates and standard deviations are indicated.

Influence of Rab proteins on the endocytic routing of CFTR. (A) Cartoon indicating endocytic routes regulated by Rab proteins. Rab5 directs transport from the plasma membrane to endocytic vesicles and fusion of early endosomes. Rab7 mediates early-to-late endosome and late endosome-to-lysosome transport, and Rab9 is involved in transport from endosomes to the trans-Golgi network. Rab11 and Rab4 mediate recycling back to the plasma membrane: Rab4 controls a rapid direct recycling route from early endosomes to the cell surface and Rab11 catalyzes a recycling route through recycling endosomes to the plasma membrane. (B–F) Influence of wild type and dominant negative (DN) Rab variants. Rab proteins were transiently overexpressed as fusions with DsRed (Rab5, 7, 9, 11, and DN mutants) or as fusions with EGFP (Rab4 and Rab4DN). Extope-CFTR was labeled at the cell surface 24 h after transfection and detected 24 h later by immunofluorescence. Rab proteins overexpressed were Rab5a and Rab5aN133I (B), Rab7 and Rab7T22N (C), Rab9 and Rab9S21N (D), Rab11 and Rab11S25N (E), and Rab4a and Rab4aN121I (F). (G) Quantification of experiments shown in Figure 6, B–F: Rab GTPases effect cellular distribution of CFTR. Image analysis using ImageJ 1.30v software was performed to determine the ratio of fluorescence associated with plasma membrane to intracellular pools on overexpression of different Rab GTPases. Extope-CFTR was either labeled with secondary antibody conjugated to Alexa Fluor 488 (control green and on overexpression of Rab5, Rab7, Rab9, Rab11, and DN mutants) or Alexa Fluor 568 (control red, Rab4, and Rab4DN). Each point represents the average of at least 10 cells, and standard deviations are indicated. (H) Endocytosed CFTR colocalizes with Rab11, but not Rab4. Rab11 (DsRed fusion) and Rab4 (EGFP fusion) were transiently coexpressed in Extope-CFTR–expressing cells and labeling of Extope-CFTR was performed as described in Figure 6, B–F. Extope-CFTR, detected after 24 h by using a goat anti-mouse Alexa Fluor 633 conjugate, is shown in blue. Rab11 colocalizes on low expression with endocytosed CFTR, but not Rab4 (right cell), whereas high expression of Rab11 results in a bright staining of labeled CFTR at the cell surface (left cell).