SORTING OF RAT-LIVER AND ILEAL SODIUM-DEPENDENT BILE-ACID TRANSPORTERS IN POLARIZED EPITHELIAL-CELLS

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and th e liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary st ructures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transf ected Madin-Darby canine kidney (MDCK) cells appropriately localized w ild-type ASBT and Ntcp apically and basolaterally as assessed by funct ional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional import ance of the cytoplasmic tail in bile acid transport activity and membr ane localization. Two cDNAs were created encoding a truncated transpor ter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that simila r to 75% of each protein was delivered to the basolateral surface comp ared with similar to 90% of the wild-type Ntcp protein. In contrast, d eletion of the cytoplasmic tail of ASBT led to complete loss of transp ort activity and sorting to the apical membrane. An Ntcp chimera in wh ich the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 /- 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confoc al immuno fluorescence microscopy. These results indicate that a predo minant signal for sorting of the Ntcp protein to the basolateral domai n is located in a region outside of the cytoplasmic tail. These studie s have further shown that a novel apical sorting signal is localized t o the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface t o the apical domain of MDCK cells.