title: Differential roles of ubiquitination in the degradation mechanism of cell surface-resident bile salt export pump and multidrug resistance-associated protein 2.
abstract: We previously showed that ubiquitination, a reversible post-translational modification, facilitates degradation of cell surface-resident bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2), ABC transporters that are expressed at the canalicular membrane (CM) of hepatocytes. In the current study, its underlying mechanism was investigated by evaluating the role of ubiquitination in the processes of internalization and subsequent degradation of cell surface-resident BSEP and MRP2. Cell surface biotinylation analysis using Flp-In T-REx 293 cells showed that ectopic expression of Ub(Δ)(GG), which is ubiquitin (Ub) lacking the two C-terminal glycines essential for the Ub conjugation reaction, inhibited the internalization of 3× FLAG-BSEP, but not of MRP2, and the degradation of the internalized MRP2, but not of the internalized 3× FLAG-BSEP. Its inhibitory effect on BSEP internalization was also indicated by a time-lapse imaging analysis using the rat hepatoma cell line McA-RH7777 in which Ub(Δ)(GG) delayed the loss of fluorescence from photoactivated Dronpa-BSEP on the CM. The effect of Ub(Δ)(GG) on BSEP internalization in these experiments was abrogated by treatment with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, and the introduction of a Y1311A mutation into BSEP. This mutation eliminates the ability of BSEP to interact with the AP2 adaptor complex, an adaptor protein required for cargo selection in clathrin-mediated endocytosis. In conclusion, our data suggest that ubiquitination facilitates clathrin-mediated endocytosis of BSEP and the degradation of internalized MRP2, leading to the degradation of the cell surface-resident form of both transporters.
authors.affiliation: Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (K.A., H.H., K.I., T.M.); and Sugiyama Laboratory, RIKEN Innovation Center, Research Cluster for Innovation, RIKEN, Yokohama, Japan (Y.S.).
authors.forename: Kensuke
authors.initials: K
authors.lastname: Aida
authors.forename: Hisamitsu
authors.initials: H
authors.lastname: Hayashi
authors.forename: Kaori
authors.initials: K
authors.lastname: Inamura
authors.forename: Tadahaya
authors.initials: T
authors.lastname: Mizuno
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Sugiyama
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D001692:Biological Transport / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis / Q000235:genetics; D057809:HEK293 Cells; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D051381:Rats; D054875:Ubiquitination / Q000235:genetics*
chemical_list: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; D001647:Bile Acids and Salts; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C093491:ABCB11 protein, human; C000715087:ABCC2 protein, human