**3. From the Endoplasmic Reticulum to the Plasma Membrane**

Correctly folded canalicular ABC transporters leave the ER to reach the Golgi apparatus, where they undergo further post-translational modifications. However, little is known about the molecular players regulating their trafficking from the ER to the Golgi. Involvement of the coat protein complex II (COP II) machinery in ABCC7/cystic fibrosis transmembrane conductance regulator (CFTR) and ABCA1 exit from the ER has been documented [23,24]. Given the homology between these proteins, the same pathway may be involved in the sorting and traffic of canalicular ABC transporters. Once in the Golgi apparatus, canalicular ABC transporters undergo more complex glycosylation [25,26]; then, they are sorted and packaged into secretory vesicles and further delivered to the canalicular membrane [25,26].

Unlike other apical proteins in liver cells, canalicular ABC transporters do not undergo transcytosis after their sorting from the trans-Golgi network (TGN), but they are directly targeted to the canalicular membrane or subapical compartments (SACs) [27,28]. The labeling of newly synthesized ABC transporters has shown that ABCB1 is directly delivered to the canalicular membrane, whereas ABCB11 is targeted to the SAC before reaching the canalicular membrane [29]. Kipp and coworkers also described the involvement of many intracellular components, such as cyclic adenosine monophosphate (cAMP), taurocholate and Ca2+ in the vesicular trafficking of canalicular ABC transporters. Indeed, they showed that the administration of these components into the perfused liver or directly in cells increases the amount of ABC transporters present at the canalicular membrane as well as bile secretion [28].

In addition to these components, many interacting proteins, including specific GT-Pases, kinases, molecular motors and other factors, have been shown to associate with canalicular ABC transporters and promote their exocytosis and/or endocytosis. Indeed, CFTR-associated ligand (CAL), a Golgi-associated protein, has been found to interact with

ABCC2 and regulate its plasma membrane targeting [30]. Some members of the Ras-related in brain (RAB) GTPase family have also been identified as ABCB1-interacting proteins. The overexpression of RAB4, RAB5 or their constitutively active forms increases the presence of ABCB1 at the cell surface [31,32]. Moreover, the motor protein myosin II regulatory light chain (MLC2) was reported as a prominent regulator of canalicular ABC transporters. Using a yeast two-hybrid screen of a rat liver cDNA library, MLC2 was found to interact with the linker domains of ABCB1, ABCB4 and ABCB11 [33]. Based on immunofluorescence and biochemical experiments, Chan and colleagues showed that the inhibition of MLC2 or the expression of its dominant negative form leads to a decrease in ABCB11 levels at the apical membrane [33].

Furthermore, other studies have revealed that many kinases are important for the exocytosis of ABC transporters. These include the p38 mitogen-activated protein kinase (MAPK) [34,35], protein kinase A (PKA [36], protein kinase C (PKC) [37], proto-oncogene serine/threonine-protein kinase (Pim-1) [38] and phosphoinositide 3-kinase (PI3K) [39]. Misra and coworkers showed that the administration of wortmannin, a specific inhibitor of PI3K, resulted in a decrease in the amounts of ABCB11 and ABCC2 present at the canalicular membrane [39,40]. Another kinase, the liver kinase B1 (LKB1), was shown as a key regulator of ABCB11 trafficking. In LKB1 knockout (KO) mice, an altered distribution of ABCB11, as well as an impaired bile formation, was observed [41,42]. Ursodeoxycholic acid (UDCA), used as a treatment for patients with cholestasis, was also shown to stimulate the targeting of ABCC2 and ABCB11 transporters to the plasma membrane [43,44].
