衣斐 義一

ATP-binding cassette transporter isoform C7 (ABCC7), also designated as cystic fibrosis transmembrane conductance regulator (CFTR), is exclusively targeted to the apical plasma membrane of polarized epithelial cells. Although the apical localization of ABCC7 in epithelia is crucial for the Cl- excretion into lumens, the mechanism regulating its apical localization is poorly understood. In the present study, an apical localization determinant was identified in the N-terminal 80-amino acid long cytoplasmic region of ABCC7 (NT80). In HepG2 cells, overexpression of NT80 significantly disturbed the apical expression of ABCC7 in a competitive manner, suggesting the presence of a sorting determinant in this region. Deletion analysis identified a potential sorting information within a 20-amino acid long peptide (aa 41-60) of NT80. Alanine scanning mutagenesis of this region in full-length ABCC7 further narrowed down the apical localization determinant to four amino acids, W57DRE60. This WDRE sequence was conserved among vertebrate ABCC7 orthologs. Site-directed mutagenesis showed that W57 and E60 were critical for the apical expression of ABCC7, confirming a novel apical sorting determinant of ABCC7. Furthermore, a WXXE motif (tryptophan and glutamic acid residues with two-amino acid spacing) was found to be conserved among the N-terminal regions of apically localized ABCC members with 12-TM configuration. The significance of the WXXE motif was demonstrated for proper trafficking of ABCC4 to the apical plasma membrane.Key words: apical plasma membrane, sorting, ATP-binding cassette transporter, CFTR, MRP4.

Localization of ATP-binding cassette transporter isoform Cl (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 dileucine 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. (C) 2013 Elsevier Inc. All rights reserved.

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.

ATP-binding cassette transporter isoform C2 (ABCC2) localizes to the apical plasma membrane in polarized cells. Apical localization of ABCC2 in hepatocytes plays an important role in biliary excretion of endobiotics and xenobiotics, but the mechanism by which ABCC2 localizes to the apical membrane has not been conclusively elucidated. Here, we investigate the role of scaffolding proteins on ABCC2 localization with a focus on the function of PDZK1 (post-synaptic density 95/disk large/zonula occludens-1 domain containing 1) in regulating ABCC2 localization. The C-terminal 77 residues of ABCC2 were used to probe interacting proteins from HepG2 cells. Protein mass fingerprinting identified PDZK1 as a major interacting protein. PDZK1 associated with the plasma membrane, most likely at the apical vacuoles of HepG2 cells. Affinity pull-down assays confirmed that the C-terminal NSTKF of ABCC2 bound to the fourth PDZ domain of PDZK1. Removal of this PDZ-binding motif significantly reduced the normal apical localization of ABCC2. In HepG2 cells, overexpression of this fourth domain overcame endogenous PDZK1 and reduced the ABCC2 localization at the apical membrane with a reciprocal increase of intracellular accumulation of mislocalized ABCC2. These results suggest a possible role for an interaction between ABCC2 and PDZK1 in apical localization of ABCC2 in hepatocytes.

Most membrane proteins are recognized by a signal recognition particle and are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, whereas almost all peroxisomal membrane proteins are posttranslationally targeted to the destination. Here we examined organelle-targeting properties of the N-terminal portions of the peroxisomal isoform of the ABC transporter PMP70 (ABCD3) using enhanced green fluorescent protein (EGFP) fusion. When the N-terminal 80 amino acid residue (N80)-segment preceding transmembrane segment (TM) 1 was deleted and the TM1-TM2 region was fused to EGFP, the TM1 segment induced ER-targeting and integration in COS cells. When the N80-segment was fused to EGFP, the fusion protein was targeted to the outer mitochondrial membrane. When both the N80-segment and the following TM1-TM2 region were present, the fusion located exclusively to the peroxisome. The full-length PMP70 molecule was clearly located in the ER in the absence of the N80-segment, even when multiple peroxisome-targeting signals were retained. We concluded that the TM1 segment possesses a sufficient ER-targeting function and that the N80-segment is critical for suppressing the ER-targeting function to allow the TM1-TM2 region to localize to the peroxisome. Cooperation of the organelle-targeting signals enables PMP70 to correctly target to peroxisomal membranes.