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J. Biol. Chem., Vol. 278, Issue 6, 4000-4009, February 7, 2003

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A 14-Amino Acid Sequence with a -Turn Structure Is Required for Apical Membrane Sorting of the Rat Ileal Bile Acid Transporter^*

An-Qiang Sun^§, Rachita Salkar^¶, Sachchidanand, Shuhua Xu^**, Lei Zeng, Ming-Ming Zhou, and Frederick J. Suchy

From the  Department of Pediatrics and Structural Biology Program,  Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029-6574 and the ^** Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520

The rat ileal sodium-dependent bile acid transporter (Asbt) is a polytopic membrane glycoprotein, which is specifically expressed on the apical domain of the ileal brush-border membrane. In the present study, an essential 14-amino acid (aa 335-348) sorting signal was defined on the cytoplasmic tail of Asbt with two potential phosphorylation sites motifs for casein kinase II (³³⁵SFQE) and protein kinase C (PKC) (³³⁹TNK). Two-dimension NMR spectra analysis demonstrated that a tetramer, ³⁴⁰NKGF, which overlaps with the potential PKC site within the 14-mer signal sequence, adopts a type I -turn conformation. Replacement of the potential phosphorylation residue Ser³³⁵ and Thr³³⁹ with alanine or deletion of either the 4 (³³⁵SFQE) or 10 aa (338-348, containing ³³⁹TNKGF) from the C terminus of Asbt resulted in a significantly decreased initial bile acid transport activity and increased the basolateral distribution of the mutants by 2-3-fold compared with that of wild type Asbt. Deletion of the entire last 14 amino acids (335-348) from the C terminus of Asbt abolished the apical expression of the truncated Asbt. Moreover, replacement of the cytoplasmic tail of the liver basolateral membrane protein, Na⁺/taurocholate cotransporting polypeptide, with the 14-mer peptide tail of Asbt redirected the chimera to the apical domain. In contrast, a chimera consisting of the 14-mer peptide of Asbt fused with green fluorescent protein was expressed in an intracellular transport vesicle-like distribution in transfected Madin-Darby canine kidney and COS 7 cells. This suggests that the apical localization of the 14-mer peptide requires a membrane anchor to support proper targeting. The results from biological reagent treatment and low temperature shift (20 °C) suggests that Asbt follows a transport vesicle-mediated apical sorting pathway that is brefeldin A-sensitive and insensitive to protein glycosylation, monensin treatment, and low temperature shift.

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