Human bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomers

Paresh P. Chothe, Lindsay C. Czuba, Robyn H. Moore, Peter W. Swaan

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)645-653
Number of pages9
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1860
Issue number3
DOIs
StatePublished - Mar 2018

Bibliographical note

Publisher Copyright:
© 2017 Elsevier B.V.

Keywords

  • Bile acid
  • Epitope tag
  • Intestinal absorption
  • Transporter

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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