Analysis of the Bile Salt Export Pump (ABCB11) Interactome Employing Complementary Approaches

Abstract

The bile salt export pump (BSEP, ABCB11) plays an essential role in the formation of bile. In hepatocytes, BSEP is localized within the apical (canalicular) membrane and a deficiency of canalicular BSEP function is associated with severe forms of cholestasis. Regulation of correct trafficking to the canalicular membrane and of activity is essential to ensure BSEP functionality and thus normal bile flow. However, little is known about the identity of interaction partners regulating function and localization of BSEP. In our study, interaction partners of BSEP were identified in a complementary approach: Firstly, BSEP interaction partners were co-immunoprecipitated from human liver samples and identified by mass spectrometry (MS). Secondly, a membrane yeast two-hybrid (MYTH) assay was used to determine protein interaction partners using a human liver cDNA library. A selection of interaction partners identified both by MYTH and MS were verified by in vitro interaction studies using purified proteins. By these complementary approaches, a set of ten novel BSEP interaction partners was identified. With the exception of radixin, all other interaction partners were integral or membrane-associated proteins including proteins of the early secretory pathway and the bile acyl-CoA synthetase, the second to last, ER-associated enzyme of bile salt synthesis.

Transmembrane insertases and N-glycosylation critically determine synthesis, trafficking, and activity of the nonselective cation channel TRPC6

“…WDR83OS is a ubiquitously expressed, predicted transmembrane protein evolutionarily conserved in Caenorhabditis elegans and Drosophila melanogaster. It is reported to interact with the Parkinson's disease P-type ATPase ATP13A2/PARK9 (101) and with the bile acid transporter ABCB11/BSEP (102). A homozygous splice site mutation in WDR83OS has been associated with a syndrome, including hypercholanemia, intractable itching, intellectual disability, and dysmorphic features (103).…”

RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic

“…However, using this method, we did not identify the few other known partners of ABCB4 [18]. This could be explained by: i) a weak sensitivity of the technique; ii) the stringent conditions of the immunoprecipitation; or iii) the poor overlap of the different techniques and studies, as already reported for the analysis of ABCB11 interactome [30]. RAB10 belongs to the RAB protein family, known as key players in intracellular traffic processes [19].…”

Molecular Regulation of Canalicular ABC Transporters

“…Numerous accessory factors were described to facilitate the translocation process, including the translocating chain-associated membrane protein (TRAM) and the translocon-associated protein (TRAP) [ 7 ]. Interestingly, TRAM and three subunits of the TRAP complex (SSR1, SSR3 and SSR4) were found to interact and coprecipitate specifically with ABCB11 [ 8 ].…”

“…Another key component of the quality control system is the B-lymphocyte receptor-associated protein (BAP), which was shown to control the folding state and sorting of many proteins in the ER [ 19 , 20 ]. The BAP29 and BAP31 isoforms were described to interact with the N-terminal domain of ABCB1 and ABCB11, respectively [ 8 , 21 ]. More interestingly, it has been shown that some mutations in the BAP31 gene are associated with liver dysfunction and cholestasis [ 22 ].…”

“…Using an immunoprecipitation assay combined with mass spectrometry analysis and yeast two-hybrid screens, Przybylla and colleagues identified several novel potential ER-resident partners for ABCB11, including the receptor expression-enhancing proteins (REEPs) involved in ER shaping, the immediate early response 3 interacting protein 1 (IER3IP1), the transmembrane proteins 205 (TMEM205) and 14A (TMEM14A) and the bile Acyl-CoA synthetase (BAC) [ 8 ]. However, their role in the regulation of the folding and/or the trafficking of ABCB11 has not been studied yet.…”