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dc.contributor.authorChothe, P.P.
dc.contributor.authorCzuba, L.C.
dc.contributor.authorMoore, R.H.
dc.date.accessioned2019-04-29T19:01:01Z
dc.date.available2019-04-29T19:01:01Z
dc.date.issued2018
dc.identifier.urihttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85038015983&doi=10.1016%2fj.bbamem.2017.11.016&partnerID=40&md5=54092e3ea76cca1c8853d7a58c1bbd44
dc.identifier.urihttp://hdl.handle.net/10713/8957
dc.description.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.en_US
dc.description.sponsorshipThe research was funded by the National Institutes of Health, Institute of Diabetes, Digestive Diseases & Kidney grant # DK56631 to PWS.en_US
dc.description.urihttps://dx.doi.org/10.1016/j.bbamem.2017.11.016en_US
dc.language.isoen_USen_US
dc.publisherElsevier B.V.en_US
dc.relation.ispartofBiochimica et Biophysica Acta - Biomembranes
dc.subjectbile aciden_US
dc.subjectepitope tagen_US
dc.subjecttransporteren_US
dc.subject.meshIntestinal Absorptionen_US
dc.titleHuman bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomersen_US
dc.typeArticleen_US
dc.identifier.doi10.1016/j.bbamem.2017.11.016
dc.identifier.pmid29198943


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