**7. Ubiquitination and Degradation of Canalicular ABC Transporters**

To target proteins for degradation, cells mostly use the endolysosomal pathway vs. proteasomal degradation, related to the monoubiquitination or polyubiquitination of their substrates, respectively [121,122].

The lysosomal pathway is the main way by which cells turn over plasma membrane proteins. Indeed, ABCB1 colocalizes with lysosomal-associated membrane protein 1 (LAMP1) in human colorectal cancer HTC15 cells [123]. In addition, the half-life of ABCB1 and ABCC2 is extended in cells treated with lysosomal inhibitors alone but not proteasomal inhibitors alone, suggesting the involvement of the lysosomal pathway in the degradation of these transporters [123,124]. However, ABCB11 expression is unaffected by treatment with lysosomal inhibitors, indicating that this transporter may use another degradation pathway [125,126]. Indeed, it has been shown that the inhibition of proteasomal degradation stabilizes wild-type (WT) and mutated ABCB11 in MDCK and HEK cells, suggesting that ABCB11 degradation involves the proteasome [125,127].

Several E3 ubiquitin ligases (E3 Ubl) may be involved in canalicular ABC transporter degradation. Ring finger protein 2 (RNF2) has E3 Ubl activity and may mediate the ubiquitination of ABCB1 [128]. E3 Ubl FBXO21 is involved in the proteasome-mediated degradation of ABCB1 [129]. Additionally, the E2-conjugating enzyme UBE2R1 (also named CDC34 or UBC3) and the E3 complex Skp1–Cullin–FBOX15 (SCFFbx15) are both implicated in ABCB1 ubiquitination [130]. Coprecipitation assays revealed that FBX015/Fbx15 (a member of the SCFFbx15 E3 complex) and UBE2R1 both interact with ABCB1, and their knockdown is

associated with a decrease in ubiquitination and subsequent degradation of ABCB1. By contrast, FBX015 expression enhances ABCB1 ubiquitination and degradation [130].

ABCB1 ubiquitination may be modulated by the MAPK pathway [131,132]. Indeed, the inhibition of MEK or the downregulation of its downstream effectors, such as ERK and p90 ribosomal S6 kinases (RSKs), lower ABCB1 protein expression in HTC15 cells [131,133]. Pulse-chase labeling experiments revealed that MEK inhibitor-mediated downregulation of ABCB1 is caused by the increase of its degradation [131]. The same team has shown that RSK1 induces self-ubiquitination of UBE2R1, followed by its proteasomal degradation in a phosphorylation-dependent manner, thus resulting in the protection of ABCB1 against degradation [132].

Some variations in ABC transporter genes are responsible for the production of an unstable protein which is retained in the ER and subsequently degraded in the cytosol by the ERAD system [134]. Some misfolded ABCB11 variants appear to be more ubiquitinated than the WT transporter [126]. The RING finger proteins Rma1, TEB4 and HRD1 are all E3 Ubl involved in the ubiquitination of ABCB11-WT and its variants but with a folding sensitivity as the knockdown of each E3 Ubl stabilizes different ABCB11 variants [126]. HRD1 targets proteins with defects in the ER lumen side, while TEB4 and Rma1 target proteins with defects in their moieties facing the cytosol. Likewise, E3 Ubl seems to exhibit sensitivity towards ABC transporters. GP78, rather than TEB4 and HDR1, plays an important role in the ubiquitination of ABCC2, as shown in patients with obstructive cholestasis and in rifampicin-treated HepG2 cells [63,135]. Proteins can escape ubiquitination through small ubiquitin-like modifier (SUMO) modification as both processes compete on the same residues. Using a protein–protein interaction assay, a number of SUMO-related proteins (including SUMO-1 and ubiquitin carrier protein 9/Ubc9) were pulled down using the linker region of the rat ABCC2 [136]. Moreover, the knockdown of SUMO-related enzymes in hepatoma cells reduces ABCC2 protein expression but not its mRNA expression or canalicular localization [136]. Proteins can escape degradation subsequent to their ubiquitination by reversing ubiquitination thanks to deubiquitinating enzymes (DUBs). As an example, the DUB ubiquitin-specific protease 19 (USP19), through TEB4 stabilization, negatively regulates the expression of a defective ABCB11 variant [137].

Manipulation of the ER quality control system might be combined with chemical or pharmacological chaperones to stabilize variants and restore the cell surface expression of ABC transporters. Indeed, cell surface biotinylation assays revealed that the most frequent ABCB11 variants found in patients with PFIC2, E297G and D482G are highly ubiquitinated [138], and this induces their internalization [73]. Additionally, the half-life of ABCC2 is extended in cells overexpressing a dominant negative form of ubiquitin due to the inhibition of ABCC2 degradation [73,124]. Therefore, by reducing susceptibility to ubiquitination, the chemical chaperone 4-phenylbutyrate (4-PB) extends the half-life of both ABCB11 and ABCC2 expressed at the cell surface [124,138,139]. However, since 4-PB has no effect on ABCB1 [139], the 4-PB mechanism of action would involve interaction with specific E3 Ubl or adaptor protein(s) for both ABCB11 and ABCC2. For instance, 4-PB downregulates Hsc70 (Hsp73) which plays a role in the lysosomal degradation of intracellular proteins and was shown to be required for the ubiquitin-dependent degradation of several proteins ([140]. and references therein).
