**2. Folding and Glycosylation of Canalicular ABC Transporters**

Protein folding is a highly regulated process that is mediated by numerous factors, including folding proteins and molecular chaperones [5]. Some of these proteins have been shown as interactors of canalicular ABC transporters, controlling their biosynthesis and folding. As most transmembrane proteins, ABC transporter biosynthesis starts with their cotranslational translocation and insertion into the endoplasmic reticulum (ER) membrane through the Sec61 translocon complex [6]. 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].

Once in the ER lumen, nascent ABC transporters are N-glycosylated. Glycans are added to their extracellular asparagine residues [9] and play a critical role in protein folding, stability and interaction with some chaperones [10]. Two main chaperone families exist in the ER: the heat shock protein (HSP) family, which promotes the folding of a wide variety of proteins, and the lectin chaperones, which recognize and fold specifically glycosylated proteins [11]. Calnexin (CNX) and its soluble homolog calreticulin are lectin chaperones

that were shown to bind both ABCG5 and ABCG8 and stimulate their folding and assembly. Okiyoneda and colleagues showed that the silencing of either CNX or calreticulin decreases the expression of ABCG5/G8 [12]. It has also been reported that CNX and the heat shock cognate 71 kDa protein (Hsc70) interact with ABCB1 [13,14].

In addition to their folding function, chaperones are central players in the quality control process. They evaluate the folding state of proteins and regulate their ER retention or ER exit [15]. They allow only properly folded proteins to exit the ER, and contrariwise, they retain abnormally folded proteins longer before their targeting to the ER-associated degradation (ERAD) pathway. Some variations in canalicular ABC transporter-encoding genes were described to affect the folding of these transporters, thereby leading to their retention in the ER. Indeed, a prolonged association between misfolded ABCB1 variants and CNX has been observed [13]. Interestingly, we and others have demonstrated that several small molecules known as pharmacological and chemical chaperones can facilitate the folding and exit of defective ABCB4 and ABCB11 variants from the ER [16–18]. 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.
