A C‐terminal tyrosine‐based motif in the bile salt export pump directs clathrin‐dependent endocytosis

The bile salt export pump (BSEP, ABCB11) is an ATP-dependent bile salt pump that functions at the liver canalicular membrane. Mutations in BSEP that result in defective trafficking can cause cholestatic disorders including progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis and cholestasis of pregnancy (1-3). The amount of BSEP on the canalicular membrane is regulated by postprandial demand for the enterohepatic circulation of bile salts (4). Pulse-chase experiments revealed a large intracellular pool of Bsep in rat liver that is mobilized for targeting and recycling of Bsep to and from the canalicular membrane (5). Furthermore, Bsep constitutively recycles between the canalicular membrane and Rab11a positive endosomes in WIF-B9 cells (6). Thus, the maintenance and retrieval of BSEP on the apical membrane is crucial for its function (4, 6). The retrieval of Bsep also plays an important role in the pathophysiology of rat cholestatic models. Bsep protein is internalized in isolated perfused rat liver and rat hepatocyte couplets after estradiol 17β-D-glucuronide administration, a process blocked by protein kinase inhibitors or dibutyrl cAMP (7, 8). Cholestasis due to lipopolysaccharide administration, as well as oxidative stress in rats results in internalization of Bsep (9). However, the mechanism by which Bsep is retrieved from the canalicular membrane remains largely unknown. In MDCK cells, dominant negative expression of Eps15 increases the apical membrane expression of rat Bsep suggesting that a clathrin-dependent mechanism may play a role in regulating cell surface Bsep expression (10). Clathrin has previously been shown to be involved in apical endocytosis in rat hepatocytes (11). Targeting and trafficking of membrane proteins depend on sequence motifs and protein-protein interaction with various trafficking machinery (12). Targeting of a number of membrane proteins to coated pits and their traffic through endocytic compartments are generally mediated by endocytic signals located at the cytoplasmic domain of proteins (13-16). Two major types of signals have been described to regulate targeting and trafficking of a number of receptor proteins: 1. a tyrosine-based YXX∅ sequence where ∅ is an amino acid with a bulky hydrophobic group, and 2. dileucine-based LL motifs where the latter leucine may be replaced by a hydrophobic amino acid residue (16-19). Depending on the primary sequence context and the relative position of the motif to the membrane, these motifs serve as plasma membrane targeting signals (20, 21). They are also utilized for efficient sorting to the endosomal system where the protein may be recycled or transported to the lysosome for degradation (22). The adaptor AP2 is a plasma membrane localized clathrin adaptor whose subunits bind directly to tyrosine-based Yxx∅ or NPXY internalization motifs within cytoplasmic or transmembrane regions of proteins to mediate clathrin-dependent endocytosis (23-27). For example, the cytosolic domain of another ABC transporter, Cystic Fibrosis Transmembrane Regulator (CFTR) has numerous consensus endocytic motifs that regulate the clathrin-dependent endocytosis (14, 28). However, the presence of discrete sorting signals in the cytosolic tail of BSEP has not been elucidated. In this study, we tested whether the C-terminus of human BSEP has a targeting/trafficking signal and whether it contributes to the cell surface expression of BSEP. We demonstrated by domain swapping using the C-terminal region of BSEP attached to Tac (IL2Rα) that an internalization motif is present in BSEP. Tac is a cell surface type 1 transmembrane protein that is targeted to the plasma membrane by default. Tac chimeras have been extensively used for the identification of trafficking signals because of the monomeric nature of the Tac protein and the existence of highly specific N-terminal antibodies to track the chimeric protein (29). We identify by mutagenesis analysis the exact motif in BSEP as a YYKLV sequence. By transferring this YYKLV motif directly to Tac, we further showed that this motif is sufficient for internalization. Finally, we demonstrated that this motif is functional in the full length human BSEP.