title: Membrane insertion scanning of the human ileal sodium/bile acid co-transporter.
abstract: Mammalian sodium-dependent bile acid transporters (SBATs) responsible for bile salt uptake across the liver sinusoidal or ileal/renal brush border membrane have been identified and share approximately 35% amino acid sequence identity. Programs for prediction of topology and localization of transmembrane helices identify eight or nine hydrophobic regions for the SBAT sequences as membrane spanning. Analysis of N-linked glycosylation has provided evidence for an exoplasmic N-terminus and a cytoplasmic C-terminus, indicative of an odd number of transmembrane segments. To determine the membrane topography of the human ileal SBAT (HISBAT), an in vitro translation/translocation protocol was employed using three different fusion protein constructs. Individual HISBAT segments were analyzed for signal anchor or stop translocation (stop transfer) activity by insertion between a cytoplasmic anchor (HK M0) or a signal anchor segment (HK M1) and a glycosylation flag (HK beta). To examine consecutive HISBAT sequences, sequential hydrophobic sequences were inserted into the HK M0 vector or fusion vectors were made that included the glycosylated N-terminus of HISBAT, sequential hydrophobic sequences, and the glycosylation flag. Individual signal anchor (SA) and stop transfer (ST) properties were found for seven out of the nine predicted hydrophobic segments (H1, H2, H4, H5, H6, H7, and H9), supporting a seven transmembrane segment model. However, the H3 region was membrane inserted when translated in the context of the native HISBAT flanking sequences. Furthermore, results from translations of sequential constructs ending after H7 provided support for integration of H8. These data provide support for a SBAT transmembrane domain model with nine integrated segments with an exoplasmic N-terminus and a cytoplasmic C-terminus consistent with a recent predictive analysis of this transporter topology.
authors.affiliation: UCLA and Wadsworth Veterans Administration Hospital, Los Angeles, California 90073, USA.
authors.forename: S
authors.initials: S
authors.lastname: Hallén
authors.forename: M
authors.initials: M
authors.lastname: Brändén
authors.forename: P A
authors.initials: PA
authors.lastname: Dawson
authors.forename: G
authors.initials: G
authors.lastname: Sachs
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D007082:Ileum / Q000737:chemistry / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D029363:Organic Anion Transporters, Sodium-Dependent; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D012964:Sodium / Q000378:metabolism*; D027981:Symporters
chemical_list: D001647:Bile Acids and Salts; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D029363:Organic Anion Transporters, Sodium-Dependent; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D027981:Symporters; C071908:sodium-bile acid cotransporter; D012964:Sodium