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 ((335)SFQE) and protein kinase C (PKC) ((339)TNK). Two-dimension NMR spectra analysis demonstrated that a tetramer, (340)NKGF, which overlaps with the potential PKC site within the 14-mer signal sequence, adopts a type I beta-turn conformation. Replacement of the potential phosphorylation residue Ser(335) and Thr(339) with alanine or deletion of either the 4 ((335)SFQE) or 10 aa (338-348, containing (339)TNKGF) 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 degrees 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.
Highlights
The results demonstrate that a tetrapeptide, 340NGKP, which overlaps with a potential protein kinase C (PKC) phosphorylation site in this 14-mer sequence, adopts a type I -turn structure
The Cytoplasmic Tail of Asbt-transfected cells (Asbt) Is Important for Both Membrane Localization and Substrate Binding Specificity—Previous studies (5) from our laboratory have demonstrated that a apical sorting motif is located on the cytoplasmic tail of Asbt and may transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells
The results show that compared with wild type Asbt, deletions of 14, 25, and 40 amino acid residues in the carboxyl tail of Asbt did not change the Naϩ dependence of taurocholate transport function but decreased the initial rates of TC transport activity by ϳ50, ϳ82, and Ͼ95%, respectively, in transfected COS 7 and MDCK cells (Fig. 1B and Fig. 2)
Summary
Rat ileal sodium-dependent bile acid transporter; Asbt-GFP, GFP-fused rat Asbt; COS 7, SV40-transformed monkey kidney fibroblast cells; Del, the C-end 4-amino acid (aa 335–338)-deleted Asbt; Del, the C-end 10-amino acid (aa 339 –348)deleted Asbt; Del, the C-end 14-amino acid (aa 335–348)-deleted Asbt; Del, the C-end 25-amino acid (aa 324 –348)-deleted Asbt; Del, the C-end 40-amino acid (aa 309 –348)-deleted Asbt; GFP, green fluorescent protein; MDCK, Madin-Darby canine kidney; Ntcp, rat liver Naϩ/taurocholate cotransporting polypeptide; Nt-GFP, 56 amino acids deleted from C-end of Ntcp and fused with GFP; NtA14-GFP, the COOH-terminal 56 amino acids of Ntcp were replaced with the 14amino acid cytoplasmic tail of Asbt and fused with GFP; PKC, protein kinase C; CK, casein kinase; TC, taurocholate; aa, amino acid; CFTR, cystic fibrosis transmembrane conductance regulator; CMV, cytomegalovirus; BSP, bromosulfophthalein; NOE, nuclear Overhauser effect. We have examined the sensitivity of the apical sorting of Asbt to drug treatment and low temperature shift.
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