title: Involvement of a di-leucine motif in targeting of ABCC1 to the basolateral plasma membrane of polarized epithelial cells.
abstract: Localization of ATP-binding cassette transporter isoform C1 (ABCC1) to the basolateral membrane of polarized cells is crucial for export of a variety of cellular metabolites; however, the mechanism regulating basolateral targeting of the transporter is poorly understood. Here we describe identification of a basolateral targeting signal in the first cytoplasmic loop domain (CLD1) of human ABCC1. Comparison of the CLD1 amino acid sequences from ABCC1 to ABCC2 revealed that ABCC1 possesses a characteristic sequence, E(295)EVEALI(301), which is comprised of a cluster of acidic glutamate residues followed by a di-leucine motif. This characteristic sequence is highly conserved among vertebrate ABCC1 orthologs and is positioned at a site that is structurally equivalent to the apical targeting signal previously described in ABCC2. Alanine scanning mutagenesis of this sequence in full-length human ABCC1 showed that both L(300) and I(301) residues were required for basolateral targeting of ABCC1 in polarized HepG2 and MDCK cells. Conversely, E(295), E(296), and E(298) residues were not required for basolateral localization of the transporter. Therefore, a di-leucine motif within the CLD1 is a basolateral targeting determinant of ABCC1.
authors.affiliation: Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1205, Japan. Electronic address: emys@sci.u-hyogo.ac.jp.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Emi
authors.forename: Yasue
authors.initials: Y
authors.lastname: Harada
authors.forename: Masao
authors.initials: M
authors.lastname: Sakaguchi
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D007532:Isoleucine / Q000378:metabolism; D007930:Leucine / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008969:Molecular Sequence Data; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D007532:Isoleucine; D007930:Leucine; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D050505:Mutant Proteins; D021382:Protein Sorting Signals; C000715087:ABCC2 protein, human; C421109:multidrug resistance-associated protein 1
keywords: ABCC1; APN; ATP-binding cassette transporter; ATP-binding cassette transporter isoform C1; ATS; Basolateral plasma membrane; CLD; Di-leucine motif; HA; MCC; MSD; Manders’ correlation coefficient; NBD; Na(+),K(+)-ATPase α1 subunit; Na,K-ATPase; Targeting signal; aminopeptidase-N; apical targeting sequence; cytoplasmic loop domain; hemagglutinin; membrane spanning domain; nucleotide binding domain