title: A C-terminal tyrosine-based motif in the bile salt export pump directs clathrin-dependent endocytosis.
abstract: The liver-specific bile salt export pump (BSEP) is crucial for bile acid-dependent bile flow at the apical membrane. BSEP, a member of the family of structurally related adenosine triphosphate (ATP)-binding cassette (ABC) proteins, is composed of 12 transmembrane segments (TMS) and two large cytoplasmic nucleotide-binding domains (NBDs). The regulation of trafficking of BSEP to and from the cell surface is not well understood, but is believed to play an important role in cholestatic liver diseases such as primary familial intrahepatic cholestasis type 2 (PFIC2). To address this issue, BSEP endocytosis was studied by immunofluorescence and a cell surface enzyme-linked immunosorbent assay (ELISA) endocytosis reporter system using a chimera of the interleukin-2 receptor α (previously referred to as Tac) and the C-terminal tail of BSEP (TacCterm). An autonomous endocytosis motif in the carboxyl cytoplasmic terminus of BSEP was identified. We define this endocytic motif by site-directed mutagenesis as a canonical tyrosine-based motif (1310) YYKLV(1314) (YxxØ). When expressed in HEK293T cells, TacCterm is constitutively internalized via a dynamin- and clathrin-dependent pathway. Mutation of the Y(1310) Y(1311) amino acids in TacCterm and in full-length human BSEP blocks the internalization. Subsequent sequence analysis reveals this motif to be highly conserved between the closely related ABCB subfamily members that mediate ATP-dependent transport of broad substrate specificity.
  
  
  CONCLUSIONS
  Our results indicate that constitutive internalization of BSEP is clathrin-mediated and dependent on the tyrosine-based endocytic motif at the C-terminal end of BSEP.
authors.affiliation: Liver Center, Yale University School of Medicine, New Haven, CT06520-8019, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Lam
authors.forename: Shuhua
authors.initials: S
authors.lastname: Xu
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Soroka
authors.forename: James L
authors.initials: JL
authors.lastname: Boyer
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000502:physiology*; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000502:physiology*; D034281:Dynamins / Q000502:physiology; D004705:Endocytosis; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D014443:Tyrosine; D020696:rab5 GTP-Binding Proteins / Q000502:physiology
chemical_list: C093491:ABCB11 protein, human; D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; D002966:Clathrin; D014443:Tyrosine; D020696:rab5 GTP-Binding Proteins; D034281:Dynamins