title: Degradation of the bile salt export pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II.
abstract: The bile salt export pump (Bsep) represents the major bile salt transport system at the canalicular membrane of hepatocytes. When examined in model cell lines, genetic mutations in the BSEP gene impair its targeting and transport function, contributing to the pathogenesis of progressive familial intrahepatic cholestasis type II (PFIC II). PFIC II mutations are known to lead to a deficiency of BSEP in human hepatocytes, suggesting that PFIC II mutants are unstable and degraded in the cell. To investigate this further, we have characterized the impact of several PFIC II mutations on the processing and stability of rat Bsep. G238V, D482G, G982R, R1153C, and R1286Q all retain Bsep to the endoplasmic reticulum (ER) to different extents. Except for R1153C, the PFIC II mutants are degraded with varying half-lives. G238V and D482G are partially misfolded and can be stabilized by low temperature and glycerol. The proteasome provides the major degradation pathway for the PFIC II mutants, whereas the lysosome also contributes to the degradation of D482G. The PFIC II mutants appear to be more heavily ubiquitinated compared with the wild-type (wt) Bsep, and their ubiquitination is increased by the proteasome inhibitors. Overexpression of several E3 ubiquitin ligases, which are involved in ER-associated degradation (ERAD), lead to the decrease of both mutant and wt Bsep. Gene knockdown studies showed that the ERAD E3s Rma1 and TEB4 contribute to the degradation of G238V, whereas HRD1 contributes to the degradation of a mutant lacking the lumenal glycosylation domain (DeltaGly). Furthermore, we present evidence that G982R weakly associates with various components of the ER quality control system. These data together demonstrate that the PFIC II mutants except R1153C and DeltaGly are degraded by the ERAD pathway.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06510, USA. wang.lin@yale.edu
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Huiping
authors.initials: H
authors.lastname: Dong
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Soroka
authors.forename: Ning
authors.initials: N
authors.lastname: Wei
authors.forename: James L
authors.initials: JL
authors.lastname: Boyer
authors.forename: Mark
authors.initials: M
authors.lastname: Hochstrasser
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D001646:Bile / Q000378:metabolism; D002460:Cell Line; D002780:Cholestasis, Intrahepatic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D018450:Disease Progression; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007668:Kidney; D008565:Membrane Proteins / Q000235:genetics; D010957:Plasmids; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000235:genetics
chemical_list: C093491:ABCB11 protein, human; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; D008565:Membrane Proteins; C504816:MARCHF6 protein, human; C482345:SYVN1 protein, human; D044767:Ubiquitin-Protein Ligases
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