Human bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomers.
Human bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomers.
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人胆汁酸转运蛋白ASBT（SLC10A2）形成功能性非共价均应二聚体和高阶低聚物。
DOI：
10.1016/j.bbamem.2017.11.016
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发表时间：
2018-03
期刊：
影响因子：
--
通讯作者：
Swaan PW
中科院分区：
文献类型：
--
作者：
Chothe PP;Czuba LC;Moore RH;Swaan PW
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.
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影响因子：
4.1
作者：
Chothe, Paresh P.;Swaan, Peter W.
通讯作者：
Swaan, Peter W.
影响因子：
--
作者：
Dawson, Paul A
通讯作者：
Dawson, Paul A
影响因子：
3.4
作者：
Javadpour, MM;Eilers, M;Smith, SO
通讯作者：
Smith, SO
影响因子：
2.9
作者：
HEBERT, DN;CARRUTHERS, A
通讯作者：
CARRUTHERS, A
影响因子：
4.8
作者：
Brast, Sabine;Grabner, Alexander;Ciarimboli, Giuliano
通讯作者：
Ciarimboli, Giuliano