**4. Membrane Stability of Canalicular ABC Transporters**

Membrane protein turnover through uninterrupted synthesis and degradation is essential to provide a functional set of proteins and ensure cell function. Tight regulation of protein stability at the plasma membrane is fundamental for cell homeostasis and relies on environmental signals and/or post-translational modifications such as phosphorylation/dephosphorylation and ubiquitination/deubiquitination cycles. Indeed, on the one hand, the accumulation of some proteins at the plasma membrane can be deleterious for cells and result, for instance, in a multidrug resistance (MDR) phenotype, a true obstacle in cancer treatment, caused by the development of chemoresistance [45]. On the other hand, defects in the expression level or stability of ABC transporters can also contribute to the development of human diseases, including cystic fibrosis [46], neuropathies [47] or cholestasis [48]. The regulation of the stability/turnover of proteins such as canalicular ABC transporters remains poorly understood, mostly due to technical limitations. ABC transporter stability is yet essential to regulate the spatiotemporal availability of a given protein at the bile canaliculi (e.g., between meals, the need for bile is reduced, and the amount of ABC transporters at the plasma membrane must be regulated accordingly). On the contrary, a decrease in the stability of numerous transporters at the plasma membrane, such as ABCB1, ABCC1 (MRP1) or ABCG2 (BCRP), would be necessary to improve the efficiency of cancer treatments facing ABC transporter-mediated MDR.

Several kinases are involved in the regulation of ABC transporter stability. The atypical Pim-1 kinase coimmunoprecipitates with and phosphorylates ABCB1. Pim-1 downregulation by siRNA diminishes ABCB1 maturation and favors its degradation through the ubiquitin–proteasome system, indicating that Pim-1 may stabilize ABCB1 at the plasma membrane [38]. A yeast two-hybrid screen using the linker domain of ABCB4 allowed the identification of receptor for activated C-kinase 1 (RACK1) as an interacting partner of this transporter. Moreover, RACK1 has been reported to activate two isoforms of PKC and be involved in the regulation of membrane stability for many proteins, thus playing a determinant role in fundamental cellular activities [49]. Following RACK1 knockdown, ABCB4 is no longer localized at the plasma membrane but mainly relocalized in cytosolic compartments [50].

PDZ (postsynaptic density protein (*PSD95*), Drosophila disc large tumor suppressor (*Dlg1*) and zonula occludens-1 protein (*ZO-1*))-domain-containing proteins are well

known for their function in protein stabilization at membranes. They act as scaffolds by linking transmembrane proteins to the cytoskeleton and thus regulate their subcellular localization and stability at the plasma membrane [51]. The PDZ-domain-containing protein ezrin–radixin–moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as sodium–hydrogen exchanger regulatory factor-1 (NHERF1), interacts with both ABCC2 and ABCB4 through their C-terminal PDZ-binding motif [52,53]. In the absence of EBP50, ABCB4 and ABCC2 are both targeted to the plasma membrane, but their presence is drastically reduced, therefore demonstrating that EBP50 plays a crucial role in the regulation of membrane stability for both ABCC2 and ABCB4 [52,53]. PDZK1 (NHERF3), another PDZ domain-containing protein, interacts with ABCC2 and increases its plasma membrane stability. Indeed, the expression of a dominant negative form of PDZK1 leads to a decrease in ABCC2 membrane expression and its accumulation in intracellular compartments [54,55].

Radixin is part of the ERM protein family, which is involved in actin cytoskeleton remodeling, e.g., to organize submembranous cortical actin or microvillosities [56]. Radixin KO mice develop a phenotype comparable to Dubin–Johnson syndrome. Indeed, these mice show a severe reduction in ABCC2 protein expression at bile the canaliculi without any change at the mRNA level. Importantly, this effect is specific to ABCC2 as no effect was observed for other canalicular ABC transporters such as ABCB1, ABCB11 or ABCB4 [57]. Moreover, a direct interaction between ABCC2 and radixin has been confirmed by GSTpulldown [57].
