A 14-amino acid sequence with a beta-turn structure is required for apical membrane sorting of the rat ileal bile acid transporter.
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.
Sun
An-Qiang
AQ
Department of Pediatrics and Structural Biology Program, Mount Sinai School of Medicine, New York, New York 10029-6574, USA. An-Qiang.Sun@mssm.edu
Salkar
Rachita
R
Sachchidanand
Xu
Shuhua
S
Zeng
Lei
L
Zhou
Ming-Ming
MM
Suchy
Frederick J
FJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D020126:Brefeldin A / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases; D007082:Ileum / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D021381:Protein Transport; D051381:Rats; D017386:Sequence Homology, Amino Acid; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D020126:Brefeldin A; D002352:Carrier Proteins; D017931:DNA Primers; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; C037643:bile acid binding proteins; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)