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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.





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