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First published online 23 March 2004
doi: 10.1242/jcs.01070

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Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity

Frédéric Bilan^1,*, Vincent Thoreau^1,*, Magali Nacfer¹, Renaud Dérand², Caroline Norez², Anne Cantereau², Martine Garcia³, Frédéric Becq² and Alain Kitzis^1,

¹ Laboratoire de Génétique Cellulaire et Moléculaire, UPRES EA 2622, CHU de Poitiers, BP 577, 86021 Poitiers CEDEX, France
² Laboratoire de Biomembranes et Signalisation Cellulaire, CNRS UMR 6558, Université de Poitiers, France
³ Laboratoire d'Immunologie et Interactions Moléculaires, UPRES EA 2224, Université de Poitiers, France

View larger version (46K): [in a new window]   Fig. 1. Syntaxin 8 and CFTR expression in the CHO-CFTR cell line and CHO-2T clone. (A) A monoclonal antibody raised against syntaxin 8 was used to study syntaxin 8 expression levels and localization in CHO cells expressing either CFTR (CHO-CFTR) or both CFTR and syntaxin 8 (CHO-2T). Specific labeling was visualized using a TRITC-conjugated secondary antibody. Bars, 30 µm. (B) Equal amounts of protein (10 µg) were subjected to SDS-PAGE followed by immunoblotting. Syntaxin 8 overexpression was confirmed in the CHO-2T clone. (C) Cell lysates containing an equal amount of protein were subjected to immunoprecipitation with CFTR antibody. The presence of CFTR in CHO-CFTR and in CHO-2T cells was revealed by immunoblotting.

View larger version (25K): [in a new window]   Fig. 2. Analysis by iodide efflux assay of CFTR chloride channel activity in the presence of syntaxin 8. (A-C) Iodide effluxes were measured using CHO-CFTR cells () or CFTR and Syn8 transfected CHO-2T cells (). Cells were treated with 2.5 µM forskolin (A), 5 µM forskolin (B) or 250 µM MPB-91 (C) during the interval time indicated by the bar at the top of each panel. (D) Summary of the data collected from 8-16 different experiments using CHO-CFTR cells (empty columns) or CHO-2T cells (filled columns). Data are expressed as a percentage of maximal activity in the presence of the corresponding agent in CHO-CFTR cells. ***P<0.0001.

View larger version (43K): [in a new window]   Fig. 3. Analysis of CFTR channel activity in the presence of different syntaxins. (A) Expression profile analysis of different syntaxins before (NT) or after plasmid transfection in CHO-CFTR cells (Syn8TM, Syn1A and Syn3). Bars, 20 µm. (B) Summary of data collected from four independent iodide efflux experiments using CHO-CFTR cells (empty columns) or CHO+CFTR+Syn8TM clone (filled columns). (C) Summary of data obtained from 12-16 independent iodide efflux experiments using CHO-CFTR, CHO-CFTR+Syn3 and CHO-CFTR+Syn1A stimulated by 5 µM forskolin. Data are expressed as a percentage of CFTR maximal activity. ***P<0.0001; ns, not significant.

View larger version (22K): [in a new window]   Fig. 4. Analysis by patch-clamp assay of the CFTR chloride channel activity in the presence of syntaxin 8. Typical whole-cell currents recorded from a CHO-CFTR cell (A) and from a Syn8 and CFTR transfected CHO-2T cell (B) in the absence or presence of 5 µM forskolin in the bath. Cells capacitances are 30 pS and 27 pS in A and B, respectively. (C) Averaged current-voltage relationships from 12 CHO-CFTR cells and seven CHO-2T cells in the presence of 5 µM forskolin. (D) Histograms showing current densities measured at +60 mV for the different experimental conditions indicated at the bottom of each column. The number of experiments is indicated on the graph. ***P<0.0001.

View larger version (144K): [in a new window]   Fig. 5. The impact of syntaxin 8 overexpression on GFP-CFTR cellular localization in COS-7 cells. (A-C) Thick optical sections acquired with 2, 0.6 or 0.8 µm steps (from top to bottom). (D-F) Projections of entire Z series. Cells were transfected with GFP-CFTR alone (A,D), cotransfected with GFP-CFTR and Syn8 (B,E) and cotransfected with GFP-CFTR and the cytosoluble form of syntaxin 8: Syn8TM (C,F). Green fluorescence resulting from GFP-CFTR expression shows strong and continuous localization of GFP-CFTR concentrated in plasma membrane of control cells (A,D). In doubly transfected cells, overexpression of Syn8 (as revealed by red fluorescence) is associated with a strong reduction of GFP-CFTR plasma membrane localization, while a partial colocalization is visualized in a juxtanuclear region as revealed by yellow fluorescence. By contrast, experiments using Syn8TM exhibit a large cytosolic staining of syntaxin 8 soluble form and a plasma membrane staining for GFP-CFTR (C,F). (G) Control experiments studying NCX2 localization in wild-type or Syn8-transfected COS-7 cells. Arrowheads indicate plasma membrane localization of NCX2 in both cases. Bars, 20 µm. Each image is representative of six independent experiments.

View larger version (71K): [in a new window]   Fig. 6. Syntaxin 8 must be anchored in the membrane to inhibit GFP-CFTR plasma membrane localization. We compared the intracellular localization of both proteins in COS-7 cells cotransfected with GFP-CFTR and Syn8 (A) or by GFP-CFTR and Syn8TM (B). Overexpressed (Aa) syntaxin 8 shows a predominantly perinuclear staining and also appears in a cytoplasm punctuated pattern. GFP-CFTR presents no plasma membrane localization (Ab), but is accumulated and colocalizes with syntaxin 8 only in the perinuclear region (arrows). (B) Soluble syntaxin 8 (Syn8TM) displays a continuous staining within the cell, but is predominantly accumulated in compartments where GFP-CFTR is present, notably at the plasma membrane. Bars, 10 µm.

View larger version (63K): [in a new window]   Fig. 7. GFP-CFTR and syntaxin 8 colocalize at least in the recycling endosome complexes in doubly transfected COS-7 cells. In each experiment (except D), COS-7 cells were cotransfected with GFP-CFTR and Syn8, then costained with anti-syntaxin 8 antibody and anti-Lamp-1 (A), anti-TfR (B) or anti-Rab11 (C) antibodies. All cell images present the projection of the entire Z series sections acquired by fluorescent confocal microscopy. Fluorograms were obtained as described in Materials and Methods. In each experiment, protein colocalizations were analyzed with three fluorograms. Syntaxin 8 (a in A-C) and GFP-CFTR (b in A-C) exhibit perinuclear staining as expected. These two proteins colocalize in the perinuclear region as shown in the merged images (d in A-C) and fluorograms (e in A-C). Lamp-1 immunostaining pattern (Ac) exhibits no colocalization with GFP-CFTR and syntaxin 8 both on merged image (Ad) and fluorograms (Af,g), whereas the immunostaining profile obtained with TfR (Bc) presents a partial colocalization on merged picture (Bd) and fluorograms (Bf,g). To further analyze colocalization compartments, we selected plots with high fluorescence intensity and placed them on the fluorogram bisector line (square in Bf,g) to generate images showing the colocalization patterns between CFTR and TfR (Bf') or Syn8 and TfR (Bg'). The vesicular pattern obtained in each case is very similar and shows a colocalization between the three proteins within these vesicles. By contrast, Rab11 immunostaining (Cc) closely matches in the perinuclear region with CFTR and syntaxin 8 stainings (Cd). This high degree of colocalization is sustained by fluorogram data (Cf,g) displaying Rab11/CFTR or Rab11/Syn8 fluorescent plots placed on the bisector line. As a control, we have studied endogenous localization between Rab11 and Lamp-1 or between Rab11 and TfR in wild type COS-7 cells. As expected, no overlap was found between Lamp-1 and Rab11 stainings (Da), whereas a partial colocalization was observed in the perinuclear region between Rab11 and TfR (Db). Bars, 20 µm. Each analysis is representative of three independent experiments.

View larger version (39K): [in a new window]   Fig. 8. CFTR and syntaxin 8 belong to the same protein complex. (A) Co-immunoprecipitation of CFTR with Syn8 in CHO-2T and HT29-CL19A cell lysates. Immunoprecipitated proteins were revealed after western blot with antibodies against CFTR. Bands B and C represent immature and mature forms of CFTR, respectively. CFTR antibody and nonimmune mouse IgG were used as positive and negative immunoprecipitation controls, respectively. (B) Co-immunoprecipitation of Syn8 with CFTR in CHO-2T and HT29-CL19A cell lysates. Immunoprecipitated proteins were revealed after western blot with antibodies against Syn8. CHO-2T and HT29 cells lysates were used as a positive control for western blot. Syn 8 antibody and nonimmune mouse IgG were used as positive and negative immunoprecipitation controls, respectively.

View larger version (48K): [in a new window]   Fig. 9. In vitro interactions between CFTR and endosomal SNARE proteins. (A) In vitro binding of GFP-CFTR with different recombinant cytosolic domains of syntaxin 8: GST-Syn8TM, GST-Syn8[1-99] and GST-Syn8[99-209]. (B) In vitro binding of GFP-CFTR with different recombinant endosomal SNARE proteins: GST-Syn7TM, GST-VAMP8TM and GST-vti1bTM. (A,B) Glutathione-sepharose-immobilized GST or GST-fusion proteins were incubated with COS-7+GFP-CFTR cell extracts. Bound proteins were studied by anti-GFP immunoblot. COS-7+GFP-CFTR lysate was used as a GFP-CFTR control; GST-Syn1ATM and GST alone were used, respectively, as positive and negative pull-down controls.