A 14-amino acid sequence with a β-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, Frederick J. Suchy

Research output: Contribution to journalArticlepeer-review

58 Scopus citations

Abstract

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 β-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.

Original languageEnglish
Pages (from-to)4000-4009
Number of pages10
JournalJournal of Biological Chemistry
Volume278
Issue number6
DOIs
StatePublished - 7 Feb 2003

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