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First published online 2 March 2004
doi: 10.1242/jcs.00974

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Regions of human kidney anion exchanger 1 (kAE1) required for basolateral targeting of kAE1 in polarised kidney cells: mis-targeting explains dominant renal tubular acidosis (dRTA)

Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK

View larger version (84K): [in a new window]   Fig. 1. Colocalisation of kAE1(R589H), kAE1(S613F) and kAE1(R901Stop) proteins with intracellular markers in non-polarised MDCKI cells. (A-C) MDCKI-kAE1(R589H), (D-F) MDCKI-kAE1(S613F) and (G-M) MDCKI-kAE1(R901Stop). Cells were treated with Bric 170 anti-AE1, followed by anti-calnexin or anti-LAMP1 antibodies and then incubated with TRITC-conjugated anti-mouse secondary antibody and FITC-conjugated anti-rabbit secondary antibody. (A,D,G,K) Visualising anti-AE1. (B,E,H) Visualising anti-calnexin. (L) Visualising anti-LAMP1. (C,F) Merged images show calnexin and kAE1(R589H) and kAE1(S613F) staining (overlaps in yellow). (I,M) Merged images show that kAE1(R901Stop) staining partially overlaps with calnexin (I) and that kAE1(R901Stop) also localises to a perinuclear region that overlaps with LAMP1 (M). Scale bar, 15 µm.

View larger version (76K): [in a new window]   Fig. 2. Surface labelling of non-polarised MDCKI-kAE1 cells with FITC-Bric 6 anti-AE1 antibody. MDCKI cells stably expressing p3.1 vector (A,B), kAE1 (C,D) or kAE1(R901Stop) (E-H) were incubated with FITC-Bric 6, which recognises an extracellular epitope on AE1. (A,C,E,G) bright field images. (B,D,F,H) fluorescence images. The majority of kAE1 cells bind FITC-Bric 6 (D), whereas the majority of kAE1(R901Stop) cells do not bind Bric 6, (F) with only a small number of obvious positive cells (H). Scale bar, 15 µm.

View larger version (53K): [in a new window]   Fig. 3. In polarised MDCKI cells, kAE1 is localised to the basolateral membrane. MDCKI cells stably expressing kAE1 were seeded at high density and allowed to polarise. (A) Cells were incubated with anti-AE1 Bric 170, then a biotin-conjugated anti-mouse antibody and visualised using a FITC-labelled anti-biotin antibody. The upper and lower images show focal planes parallel and perpendicular to the cell monolayer, respectively. (B,C) MDCKI-kAE1 cells were double-stained with anti-AE1-Bric 170, and either a tight junction marker anti-ZO-1 antibody (B) or an anti-Na+/K+ATPase antibody (C). Merged images show kAE1 located on the lateral side of the ZO-1 (B) and colocalises with the basolateral marker Na+/K+-ATPase (C). Scale bars, 10 µm. AP, apical; BL, basolateral.

View larger version (70K): [in a new window]   Fig. 4. Polarised expression of dominant dRTA kAE1 mutants. MDCKI-kAE1(R589H) cells (A), MDCKI-kAE1(S613F) cells (B), MDCKI-kAE1(R901Stop) cells (C,E) and MDCK1-kAE1 cells (D) were polarised on filters. (A-C) Cells were stained for AE1 using Bric 170 (green), (A-C, bottom panels) tight junctions were localised with anti-ZO-1 antibody (red). (D,E) FITC-Bric 6 antibody was added to apical and basolateral surfaces of the intact cell monolayer. (Top panels) Focal planes taken parallel to the epithelium near the centre of the cells (A,B,D) and at the apical surface (C,E). (Bottom panels) Focal planes perpendicular to the epithelium. (A-C, top) Staining with anti-AE1 Bric 170; (A-C, bottom) merged images of anti-AE1 (green) and anti-ZO-1 staining (red). Proteins kAE1(R589H) and kAE1(S613F) are both distributed throughout the polarised cells (A,B, respectively), whereas kAE1(R901Stop) is at the apical membrane (C) and was detected exclusively at the apical surface by FITC-Bric 6 (E). kAE1 was detected exclusively at the basolateral surface by FITC-Bric 6 on filters (D). Scale bar in C, 12 µm (for A-C); scale bar in E, 15 µm (for D,E); AP, apical; BL, basolateral.

View larger version (68K): [in a new window]   Fig. 5. Radiolabelling of kAE1 and kAE1(R901Stop) in polarised (A,E) or non-polarised (B,C,D) MDCKI cells. Cells were preincubated with sodium butyrate and labelled with 200 µCi/ml Easy-TagTM [35S]methionine for 30 minutes (A,C,D) and pulse chased, or steady-state labelled for 4 hours (B,E). Immunoprecipitates were prepared using anti-AE1 Bric 155 (A) or Bric 170 (B-E) as described in Materials and Methods. Immunoprecipitates were treated with endo H or PNGase F where indicated and analysed by SDS-PAGE. (A) shows a pulse-chase experiment on MDCKI-kAE1 cells polarised on filters. The p3.1 vector control is shown in lane 1. (B) Steady-state labelled non-polarised MDCKI-kAE1 and MDCKI-kAE1(R901Stop) cells, respectively. (C,D) Pulse chase studies on non-polarised MDCKI-kAE1 cells (C) or MDCKI-kAE1(R901Stop) cells (D). (E) Selective biotinylation of the apical or basolateral surfaces of kAE1 and kAE1(R901Stop) cells polarised on filters. The apical and basolateral surface was selectively biotinylated, total AE1 immunoprecipitated and the biotinylated fraction selected using streptavidin beads. AP, apical; BL, basolateral.

View larger version (55K): [in a new window]   Fig. 6. Polarised MDCKI cells expressing kAE1 are permeable to biotin reagent. MDCKI (A), MDCKI-kAE1(R901Stop) (B) and MDCKI-kAE1 (C) cells were selectively biotinylated on the apical surface. Biotinylated proteins were detected using a FITC-conjugated anti-biotin antibody. (D) MDCKI-kAE1 cells stained for biotinylated proteins (green, top panel) and kAE1 (red, middle panel). kAE1 cells have lateral staining (C, top panel D) suggesting that they are permeable to the usually impermeant biotinylation reagent. Polarised MDCKI-kAE1 cells biotinylated on the lateral membranes (green) clearly correspond to cells expressing kAE1 (red) (D, bottom panel). Scale bar, 15 µm

View larger version (78K): [in a new window]   Fig. 7. Effects of kAE1 C-terminal mutations and the deletion of the kAE1 N-terminus in polarised MDCKI and LLC-PKI cells. Filter-polarised cells MDCKI-kAE1 (Y904A,V907A) (A), MDCKI-kAE1(V907A) (B), MDCKI-kAE1(Y904F) (C), MDCKI-kAE1(V911Stop) (D) and MDCKI-AE1mem (E) were stained with Bric 170 for AE1. Tight junctions were stained with ZO-1 antibody. (B-D, top panels) Focal planes parallel to the epithelium near the centre of the cells. (A,E, top panels) Focal planes at the apical surface. (All bottom panels) Focal planes perpendicular to the epithelium. (A-E, top and middle panels) Staining with anti-AE1 Bric 170. (A-E, bottom panels) Merged images of anti-AE1 (green) and anti-ZO-1 staining (red). The double mutant kAE1(Y904AV907A) and also kAE1Y904F were mis-targeted in MDCKI cells (A and C, respectively) whereas kAE1(V907A) had identical localisation to normal kAE1 (B). This suggests that the tyrosine residue at position 904 is crucial for basolateral targeting of kAE1. Removal of the N-terminus in the construct AE1mem also resulted in the mis-targeting of kAE1 to the apical membrane. (F,G) Polarised LLC-PK1 cells transiently transfected with normal kAE1 (F) or kAE1(R901Stop) (G). Cells were stained for AE1 using Bric 170. localisation of kAE1 was basolateral in LLC-PK1 cells, while kAE1(R901Stop) showed apical and basolateral location. (H) Sequence alignment of C-termini of AE1 from different species. Scale bars in E, 20 µm (for A-E), in F and G, 10 µm. AP, apical; BL, basolateral.