Human bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomers
JournalBiochimica et Biophysica Acta - Biomembranes
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AbstractThe 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. Copyright 2017 Elsevier B.V.
SponsorsThe research was funded by the National Institutes of Health, Institute of Diabetes, Digestive Diseases & Kidney grant # DK56631 to PWS.
Identifier to cite or link to this itemhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85038015983&doi=10.1016%2fj.bbamem.2017.11.016&partnerID=40&md5=54092e3ea76cca1c8853d7a58c1bbd44; http://hdl.handle.net/10713/8957