Human bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomers.
The human apical sodium-dependent bile acid transporter, hASBT/SLC10A2, plays a central role in cholesterol homeostasis via the efficient reabsorption of bile acids from the distal ileum. hASBT has been shown to self-associate in higher order complexes, but while the functional role of endogenous cysteines has been reported, their implication in the oligomerization of hASBT remains unresolved. Here, we determined the self-association architecture of hASBT by site-directed mutagenesis combined with biochemical, immunological and functional approaches. We generated a cysteine-less form of hASBT by creating point mutations at all 13 endogenous cysteines in a stepwise manner. Although Cysless hASBT had significantly reduced function correlated with lowered surface expression, it featured an extra glycosylation site that facilitated its differentiation from wt-hASBT on immunoblots. Decreased protein expression was associated with instability and subsequent proteasome-dependent degradation of Cysless hASBT protein. Chemical cross-linking of wild-type and Cysless species revealed that hASBT exists as an active dimer and/or higher order oligomer with apparently no requirement for endogenous cysteine residues. This was further corroborated by co-immunoprecipitation of differentially tagged (HA-, Flag-) wild-type and Cysless hASBT. Finally, Cysless hASBT exhibited a dominant-negative effect when co-expressed with wild-type hASBT which validated heterodimerization/oligomerization at the functional level. Combined, our data conclusively demonstrate the functional existence of hASBT dimers and higher order oligomers irrespective of cysteine-mediated covalent bonds, thereby providing greater understanding of its topological assembly at the membrane surface.
['Chothe|Paresh P|PP|', 'Czuba|Lindsay C|LC|', 'Moore|Robyn H|RH|', 'Swaan|Peter W|PW|']
D000595:Amino Acid Sequence D000818:Animals D001692:Biological Transport D019556:COS Cells D002462:Cell Membrane D002522:Chlorocebus aethiops D003545:Cysteine D003553:Cystine D005799:Genes, Dominant D006031:Glycosylation D006801:Humans D047468:Immunoprecipitation D016297:Mutagenesis, Site-Directed D029363:Organic Anion Transporters, Sodium-Dependent D010446:Peptide Fragments D046988:Proteasome Endopeptidase Complex D055503:Protein Multimerization D011499:Protein Processing, Post-Translational D055550:Protein Stability D011994:Recombinant Proteins D016415:Sequence Alignment D017386:Sequence Homology, Amino Acid D027981:Symporters D013656:Taurocholic Acid
Bile acid Transporter Epitope tag Intestinal absorption