Organization of the membrane domain of the human liver sodium/bile acid cotransporter.
Mammalian sodium/bile acid cotransporters (SBATs) are glycoproteins with an exoplasmic N-terminus, an odd number of transmembrane regions, and a cytoplasmic C-terminus. Various algorithms predict eight or nine membrane-embedded regions derived from nine hydrophobic stretches of the protein (H1-H9). Three methods were used to define which of these were transmembrane or membrane-associated segments in the liver bile acid transporter. The first was in vitro translation/insertion scanning using either single hydrophobic sequences between the N-terminal domain of the alpha-subunit of the gastric H,K-ATPase and the C-terminal domain of the beta-subunit that contains five N-linked glycosylation exoplasmic flags or using constructs beginning with the N-terminus of the transporter of various lengths and again ending in the C-terminus of the H,K-ATPase beta-subunit. Seven of the predicted segments, but not the amphipathic H3 and H8 sequences, insert as both individual signal anchor and stop transfer sequences in the reporter constructs. These sequences, H3 and H8, are contained within two postulated long exoplasmic loops in the classical seven-transmembrane segment model. The H3 segment acts as a partial stop transfer signal when expressed downstream of the endogenous H2. In a similar manner, the other amphipathic segment, H8, inserts as a signal anchor sequence when translated in the context with the upstream transporter sequence in two different glycosylation constructs. Alanine insertion scanning identified regions of the transporter requiring precise alignment of sequence to form competent secondary structures. The transport activity of these mutants was evaluated either in native protein or in a yellow fluorescent protein (YFP) fusion protein construct. All alanine insertions in H3 and H8 abolished taurocholate uptake, suggesting that both these regions have structures with critical intramolecular interactions. Moreover, these insertions also prevented trafficking to the plasma membrane as assessed by confocal microscopy with a polyclonal antibody against either the C-terminus of the transporter or the YFP signal of the YFP-transporter fusion protein. Two glycosylation signals inserted in the first postulated loop region and four of five such signals in the second postulated loop region were not recognized by the oligosaccharide transferase, and the L256N mutation exhibited 10% glycosylation and was inactive. These findings support a topography with nine membrane-spanning or membrane-associated segments.
Hallén
S
S
Wadsworth Veterans Administration Hospital, West Los Angeles VA Medical Center, 11301 Wilshire Blvd., Los Angeles, CA 90073, USA.
Mareninova
O
O
Brändén
M
M
Sachs
G
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001647:Bile Acids and Salts / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D029363:Organic Anion Transporters, Sodium-Dependent; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D027981:Symporters
D001426:Bacterial Proteins; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D029363:Organic Anion Transporters, Sodium-Dependent; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D027981:Symporters; C054562:yellow fluorescent protein, Bacteria; C071908:sodium-bile acid cotransporter; D012964:Sodium