title: A cis-acting five-amino-acid motif controls targeting of ABCC2 to the apical plasma membrane domain.
abstract: ATP-binding cassette transporter isoform C2 (ABCC2) is exclusively targeted to the apical plasma membrane of polarized cells. Although apical localization of ABCC2 in hepatocytes is crucial for the biliary excretion of a variety of metabolites, the mechanism regulating its apical targeting is poorly understood. In the present study, an apical targeting signal was identified in the first cytoplasmic loop domain (CLD1) of ABCC2 in HepG2 cells. Overexpression of CLD1 significantly disturbed the apical targeting of FLAG-ABCC2 in a competitive manner, suggesting the presence of a saturable sorting machinery in HepG2 cells. Next, deletion analysis identified a potential targeting sequence within a 20-amino-acid long peptide (aa 272-291) of CLD1. Alanine scanning mutagenesis of this region in full-length ABCC2 further narrowed down the apical targeting determinant to five amino acids, S(283)QDAL(287). Of these, S(283) and L(287) were found to be conserved among vertebrate ABCC2 orthologs. Site-directed mutagenesis showed that both S(283) and L(287) were crucial for the targeting specificity of ABCC2. Introducing this apical targeting sequence into the corresponding region of ABCC1, an exclusively basolateral protein, caused the hybrid ABCC1 to partially localize in the apical membrane. Thus, the CLD1 of ABCC2 contains a novel apical sorting determinant, and a saturable sorting machinery is present in polarized HepG2 cells.
authors.affiliation: Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo, Japan. emys@sci.u-hyogo.ac.jp
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Emi
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yasuda
authors.forename: Masao
authors.initials: M
authors.lastname: Sakaguchi
mesh_terms: D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D016764:Cell Polarity; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D056945:Hep G2 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008969:Molecular Sequence Data; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D020033:Protein Isoforms; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D057900:Transcytosis; D014162:Transfection
chemical_list: D000409:Alanine; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D020033:Protein Isoforms; D021382:Protein Sorting Signals; C000715087:ABCC2 protein, human