The COOH-terminal PDZ-interacting domain (TRL) is required for polarization of CFTR to the apical membrane. (a) Confocal fluorescence micrograph (xz plane) of MDCK cells stably expressing GFP-wt-CFTR in the apical membrane. (b) Confocal fluorescence micrograph (xz plane) of MDCK cells stably expressing GFP-CFTR-ΔTRL in the apical and lateral membranes. GFP fluorescence is green, and ZO-1, a protein in tight junctions that separates apical and basolateral membrane domains, is red. Scale bar = 10 μm. AP = location of apical membrane; BL = location of basal membrane. (c) Western blot of cells expressing wt-CFTR or CFTR-ΔTRL. Selective cell-surface biotinylation of the apical (AP) or basolateral membrane (BL). Whereas GFP-wt-CFTR was expressed primarily in the apical membrane (ratio of apical/basolateral membrane expression = 7.5), GFP-CFTR-ΔTRL was expressed nearly equally in the apical and basolateral membranes (ratio of apical/basolateral membrane expression = 0.6). Mature, glycosylated GFP-wt-CFTR band C is approximately 240 kDa. *High molecular weight form of CFTR that has been reported previously (33, 37). (d) Selective biotinylation of the apical membrane. (e) Selective biotinylation of the basolateral membrane. Biotin was detected with streptavidin-Texas-red as described (33). Images in d and e demonstrate that in our experiments, tight junctions were intact and the biotin regent applied to the apical membrane did not have access to CFTR present on the basolateral membrane and vice versa. In addition, the absence of core-glycosylated GFP-wt-CFTR (band B) in surface biotinylated samples indicates that cell integrity was not compromised and that biotin was not accessible to the endoplasmic reticulum and cis-Golgi apparatus, where core glycosylated CFTR band B is located.