**4. Processing and Trafficking of BSEP**

Membrane insertion and folding occur at the level of the endoplasmic reticulum (ER) [27]. Insertion into the ER membrane is facilitated by the protein transport protein SEC61, which assists transmembrane portions of nascent proteins to adopt helicity prior to domain folding. Correct positioning of domains or subdomains relative to each other typically occurs late in the folding trajectory of a multidomain membrane protein. Of all ABC proteins, the folding trajectory of cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7) has been studied the most [28,29]. The intracellular loops (ICLs) play a critical role in transporter folding by contributing to the formation of the functionally important TMD/NBD coupling interface [30]. Furthermore, the involvement of molecular chaperons is required, as they sense the presence of hydrophobic helices in the cytosol, and thus contribute to obtaining the folding endpoint [31].

In the ER, newly synthesized and correctly folded BSEP undergoes N-linked core glycosylation. The sugar moieties are added at four conserved asparagine residues in extracellular loop 1 (ECL1), namely Asn109, 116, 122 and 125, and then are subject to subsequent modifications while traveling through the Golgi stacks. N-linked core glycosylation in the ER lumen plays a pivotal role in ER protein folding by mediating interactions with the lectin chaperones calnexin and calreticulin and by increasing the folding efficiency [32]. Only correctly folded proteins are trafficked to the Golgi apparatus in clathrin-coated COPII vesicles. Aberrantly folded proteins are identified by the endoplasmic-reticulum-associated degradation (ERAD) machinery and retro-translocated to the cytoplasm for degradation in the 26S proteasome following ubiquitination [33]. A number of BSEP mutants, including G238V, D482G, G982R, R1153C and R1286Q, are predominantly degraded by ERAD, thus leading to a PFIC2 phenotype [27,34,35]. Different ERAD E3 ubiquitin ligases are thought to recognize and ubiquitinate different mutants of BSEP [27]. Before trafficking to the canalicular membrane, BSEP is fully glycosylated in the Golgi apparatus through trimming to the core structure and extension from the core [34]. Glycosylation directly impacts protein stability and at least two of the four glycans are required for BSEP trafficking to the canalicular membrane [35]. Using enhanced green fluorescent protein (EGFP)-tagged mouse BSEP, it was shown that the partial glycosylation of the PFIC2-related mutant D482G causes an unstable BSEP protein and reduces levels of the mature protein at the canalicular membrane [36].

The majority of integral plasma membrane proteins of polarized hepatic cells are distributed from the basolateral membrane to the appropriate apical cell surface location via transcytosis. In contrast, ABC transporters targeted to the canalicular membrane use the non-transcytotic direct route from the Golgi apparatus via Rab11a-positive apical endosomes [37,38]. Under physiological conditions, the apical pool of BSEP is strictly regulated by the demand for biliary excretion of bile salts. The intracellular endosomal pool is thought to exceed that at the canalicular membrane [39] by at least 6-fold. Internalization of BSEP is mediated by clathrin-coated vesicles and is dependent on the highly conserved endocytic cargo motif (Trp-Lys-Leu-Val) [40]. This trafficking motif is recognized by adaptor protein 2 (AP-2), which modulates the internalization process and expression of cell-surfaceresident BSEP through direct interaction [41]. Moreover, trafficking of BSEP through the endosomal system to the canalicular membrane is a microtubule-dependent process and requires the myosin light chain [42], myosin Vb [43] and Rab11a. The latter two components were shown to also be associated with canalicular biogenesis by maintaining proper trafficking of Rab11a–myosin Vb-containing membranes to the canalicular membrane in polarized WIF-B9 cells [43].

Continuous cycling of BSEP between the apical and intracellular pools is disrupted in most human cholestatic liver diseases. Shifting the balance towards endocytic internalization results in impaired bile salt secretion [44]. A causative role of enhanced retrieval into the subapical endosomal compartment was demonstrated for estradiol 17 β-D-glucuronide (E17G)-induced cholestasis, an experimental model for pregnancy-related cholestasis [45,46]. In this model, BSEP was found to co-localize with clathrin, AP-2

and Rab5 as evidence for clathrin-mediated endocytosis [46]. Classical (Ca2+-dependent) protein kinase C (cPKC)–p38, mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)–ERK1/2 signaling pathways are thought to be involved in (E17G) induced cholestasis [47–49].

TCA, the major bile acid in mammals, as well as cyclic adenosine monophosphate (cAMP) are known to increase the apical pool of BSEP within minutes by promoting its cellular relocation [37]. Moreover, TCA was demonstrated to induce the formation of bile canaliculi in mice via the liver kinase B1 (LKB1)–AMP-activated protein kinase (AMPK) pathway [50]. A subsequent publication showed that knocking out LKB1, the upstream serine–threonine kinase, which is implicated in regulation of cellular energy metabolism, impairs both canalicular biogenesis and intracellular trafficking of BSEP. On the other hand, cAMP induces BSEP trafficking through a PKA-mediated pathway, which does not involve AMPK activation [51]. Unlike TCA-mediated trafficking, this process is PI3Kindependent [52]. Similar to TCA, the conjugated bile salt tauroursodeoxycholate (TUDCA) also promotes the relocation of BSEP to the canalicular membrane through activation of the p38 MAPK [53,54]. Similar to other conjugated bile salts, TUDCA stimulates the ATPase activity of BSEP [55].

Ubiquitination is another modification, which changes the expression of cell-surfaceresident BSEP. The half-life of BSEP in the canaliculi is shortened by modification with two to three ubiquitin molecules. This induces the removal of the protein from the cell surface, whereby the rates are governed by the degree of ubiquitination. While the PFIC2-related mutations E297G and D482G cause short-chain ubiquitination, thereby shortening the halflife of cell-surface-resident BSEP, the chemical chaperone 4-phenylbutyrate (4-PB) reduces its degradation rate [56]. In a later study, ubiquitination of canalicular BSEP was shown to act as a signal for internalization by promoting clathrin-mediated endocytosis. After internalization, BSEP is either recycled back to the canalicular membrane in a Rab11-dependent manner or degraded through a ubiquitination-independent pathway [57]. Degradation was suggested to be lysosome-mediated and dependent on a sorting signal from within the endosomal compartment [58].
