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.

Membrane Topology of Human ASBT (SLC10A2) Determined by Dual Label Epitope Insertion Scanning Mutagenesis. New Evidence for Seven Transmembrane Domains

“…Combined, these data provide clear evidence that the moderately hydrophobic region between residues 255-280 is, in fact, part of an extramembranous protein domain of hASBT. These data are consistent with the absence of signal anchor and stop transfer properties that would allow membrane insertion of this sequence (17). Additionally, this explains the inability of this region, assigned TMD8 in the 9TM model (Fig.…”

“…Although computed hydrophobicity assignments can offer valuable insight, experimental data are essential to assign TMDs. In the case of hASBT, however, two opposing hypotheses have emerged based on empirical evidence; membrane insertion scanning technology has suggested a 9TM topology (17), whereas N-glycosylation analysis indicated 7 membrane-spanning regions (11). Since a single topology scanning technique rarely provides unequivocal results, the data presented here were designed to close the current controversy pertaining to the membrane topology of hASBT.…”

“…For example, mutants I and XII were constructed to corroborate the previously reported exofacial and cytosolic orientation of the N-and the C-terminal tails, respectively (1,3,17). To effectively distinguish between the divergent topology models, mutants IV-VIII were designed to localize on opposite sides of the membrane according to either the 7TM or the 9TM model ( Fig.…”

The solute carrier family SLC10: more than a family of bile acid transporters regarding function and phylogenetic relationships

“…Membrane insertion and sorting of NTCP and ASBT Based on bioinformatic predictions and experimental data, it has been shown that NTCP and ASBT have an extracellular N-terminus, an odd number of transmembrane helices (seven or nine) and a cytoplasmic Cterminus (Hagenbuch et al 1991;Hagenbuch and Meier 1994;Stieger et al 1994;Dawson and Oelkers 1995;Hallén et al 1999;Hallén et al 2002b;Zhang et al 2004). Several potential N-glycosylation sites are present in NTCP, ASBT and SOAT proteins, and site-directed mutagenesis has revealed that only N 5 and N 11 in rat Ntcp (Hagenbuch 1997), and N 10 in human ASBT (Zhang et al 2004) are glycosylated.…”

“…Using N-glycosylation-scanning mutagenesis, Zhang and coworkers strongly suggested a model with seven transmembrane domains for human ASBT (Zhang et al 2004). In contrast, a nine-transmembrane domain arrangement was found by Hallén and coworkers for human ASBT and NTCP (Hallén et al 1999(Hallén et al , 2000(Hallén et al , 2002b. However, it remains unclear whether all of these nine transmembrane segments cross the membrane or whether two of them fold as lipidburied re-entrance loops in the plasma membrane (Hallén et al 2002b;Zahner et al 2003).…”