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

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.

A guide to plasma membrane solute carrier proteins

“…The best-studied members of this family, SLC10A1 and SLC10A2, play key roles in the regulation of BA levels in the body, helping reabsorb secreted BAs in the intestine and removing BAs from the liver's portal circulation [510]. Both SLC10A1 and SLC10A2 have been found to use homo-and heterodimeric and higher order oligomers as functional units on the cell membrane [511,512]. Due to the cytotoxic nature of BAs, both the expression and activity of these two proteins are regulated by their substrates, through the nuclear receptor like farnesoid X receptor [513].…”

“…Due to its location in the final portion of the small intestine, SLC10A2's main function is to reabsorb any BAs that were not already absorbed as a part of mixed micelles. This way, the body takes up to 95% of the BAs and minimizes the amount of BAs that must be synthesized again [512]. SLC10A6 (SOAT) is mainly expressed in the testis, where it localizes to the plasma membrane of primary spermatocytes [521].…”

Intestinal Absorption of Bile Acids in Health and Disease

“…Of particular interest is the important role of ASBT cysteine residues. Several studies have probed the 13 cysteine residues of ASBT and noted a number of these residues that are both highly conserved between ASBT and NTCP, and are crucial for transport function, trafficking, and stability (32,82,324,327,368). Notably, mutation of the highly conserved Cys51, Cys105, Cys132, and Cys255 to Ala or Thr nearly abolishes [ 3 H]-TC uptake (32).…”

“…Mutations at other sites, such as Cys69 or Cys314, somewhat reduce [ 3 H]-TC uptake. In an effort to understand the mechanisms underlying the role of cysteine residues, a recent study concluded that cysteine residues in ASBT are likely not important for its oligomerization (82). However, the precise role of many of these important residues requires further study.…”

S-acylation modulates the function of the apical sodium-dependent bile acid transporter in human cells

“…This has made prediction of protein S-acylation quite challenging, requiring experimental approaches in order to determine whether a protein is subject to S-acylation and which cysteine residues are involved. Interestingly, recent evidence has clearly demonstrated the importance of cysteine residues in ASBT function, though their potential role in maintaining ASBT function via Sacylation has yet to be fully investigated (17,18). We hypothesized that post-translational modification by S-acylation is critical for ASBT function and may modulate its rapid functional response to certain external cues, such as the availability of different types of fatty acids.…”