A cis-acting five-amino-acid motif controls targeting of ABCC2 to the apical plasma membrane domain

Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo, Japan.
Journal of Cell Science (Impact Factor: 5.43). 03/2012; 125(Pt 13):3133-43. DOI: 10.1242/jcs.099549
Source: PubMed


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.

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    • "[50]–[52] In addition to the biosynthetic processes, ABC transporter localization and stability are regulated by multiple protein sequences. [24], [38], [53], [54] Among these, PDZ ligands have been shown to contribute to protein localization and stability at the plasma membrane. [29], [55]–[57] Given the apparent similarity of the ABCC6 C-terminal sequence to other PDZ-containing ABCC transporters and the presence of a disease-associated mutation within this region, we chose to evaluate the role of this sequence in the expression and regulation of ABCC6. "
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