Sun A Q, Ananthanarayanan M, Soroka C J, Thevananther S, Shneider B L, Suchy F J
Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029, USA.
Am J Physiol. 1998 Nov;275(5):G1045-55. doi: 10.1152/ajpgi.1998.275.5.G1045.
The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the 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 structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter 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 approximately 75% of each protein was delivered to the basolateral surface compared with approximately 90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which 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 confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.
大鼠回肠顶端钠依赖性胆汁酸转运体（ASBT）和肝脏钠-牛磺胆酸盐共转运多肽（Ntcp）是一个新的阴离子转运体家族的成员。这些转运蛋白具有有限的序列同源性和几乎相同的预测二级结构，但分别定位于回肠肠上皮细胞的顶端表面和肝细胞的窦状表面。通过功能活性和免疫细胞化学定位研究评估，稳定转染的犬肾（MDCK）细胞将野生型ASBT和Ntcp分别正确定位于顶端和基底外侧。截短的和嵌合的转运体用于确定细胞质尾在胆汁酸转运活性和膜定位中的功能重要性。构建了两个cDNA，编码一种截短的转运体，其中Ntcp的56个氨基酸的COOH末端尾巴被去除或被一个8个氨基酸的表位FLAG替代。对于这两种突变体，基底外侧分选的保真度都有所下降，因为每种蛋白质约75%被转运到基底外侧表面，而野生型Ntcp蛋白约为90%。相反，ASBT细胞质尾的缺失导致转运活性完全丧失，且无法分选到顶端膜。基于胆汁酸转运活性的极性和共聚焦免疫荧光显微镜定位，一种Ntcp嵌合体（其中Ntcp的56个氨基酸的COOH末端尾巴被ASBT的40个氨基酸的细胞质尾替代）在很大程度上（8
