Journal of Cell Science
A cis-acting five-amino-acid motif controls targeting of
ABCC2 to the apical plasma membrane domain
Yoshikazu Emi*, Yuki Yasuda and Masao Sakaguchi
Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1205, Japan
*Author for correspondence (email@example.com)
Accepted 7 March 2012
Journal of Cell Science 125, 3133–3143
? 2012. Published by The Company of Biologists Ltd
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
FLAG2ABCC2 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,
S283QDAL287. Of these, S283and L287were found to be conserved among vertebrate ABCC2 orthologs. Site-directed mutagenesis
showed that both S283and L287were 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.
Key words: Apical plasma membrane, ATP-binding cassette transporter, Sorting signal
Epithelial cells form a boundary between different extracellular
environments, and eventually exhibit polarity by differentiating
their plasma membranes into apical and basolateral domains. In
polarized epithelial cells, newly synthesized plasma membrane
proteins are sorted into distinct carrier vesicles at the trans-Golgi
network (TGN) and asymmetrically distributed into the apical or
basolateral plasma membrane (Rodriguez-Boulan and Mu ¨sch,
2005; Fo ¨lsch et al., 2009). The plasma membrane is also involved
in rapid endocytosis and recycling, during which endocytosed
proteins are sorted to either endosomes for recycling or to
lysosomes for degradation. In spite of these dynamic movements,
a significant fraction of membrane proteins, such as transporter
molecules, are maintained at constant levels on the plasma
membrane. To accomplish this, a complex array of protein–
protein interactions governs many regulatory aspects that
determine the localization of these proteins and, thus, plasma
membrane proteins contain sorting information that specifies
their destination (Mellman and Nelson, 2008).
functionally distinct domains on plasma membranes, and thus
exhibit characteristics of polarized epithelial cells (Arias et al.,
1994). In the liver, the canalicular membranes of adjacent
hepatocytes form the bile canaliculi and form the apical domain,
whereas the sinusoidal membranes of individual hepatocytes
encompass the Disse space and form the basolateral domain. The
bile canaliculi represent the excretory pole of hepatocytes and
function as an important route of elimination of potentially toxic
metabolites. The formation and maintenance of a polarized
distribution of proteins in these two domains are crucial for the
hepatocytes to perform various activities such as vectorial
transport of a wide variety of metabolites. Thus, retaining
polarity of hepatocytes is critical for the maintenance of
ATP-binding cassette (ABC) transporters comprise a large
family of multi-spanning transmembrane proteins and are divided
into seven subfamilies, from A to G. These transporters are found
in various membranous organelles, where they vectorially
transport a variety of endogenous and exogenous compounds
by utilizing the energy released from ATP hydrolysis (Dean et al.,
2001). Most transporter proteins that are trafficked to the plasma
membranes of polarized epithelial cells are sorted to different
membrane domains, and this trafficking is regulated by distinct
pathways. Human ABC transporter subfamily C (ABCC)
includes 13 isoforms of plasma membrane associated cellular
export pumps (Deeley et al., 2006). Of these, ABCC2 was
originally identified as an organic anion transporter situated on
the apical surface of hepatocytes, and transports glucuronidated
bilirubin into the bile canaliculi (Nies and Keppler, 2007). The
activity of ABCC2 is regulated by transcriptional, translational
and post-translational mechanisms (Nies and Keppler, 2007).
Obviously, correct targeting of ABCC2 to the apical membrane is
of immense physiological importance. However, despite its
importance, molecular machinery responsible for the apical
targeting of newly synthesized ABCC2 in polarized epithelial
cells is poorly understood.
Several attempts to identify the distinct apical target-
determining regions of ABCC2 using mutagenesis strategies or
Journal of Cell Science
by analyzing chimeras formed between ABCC2 and basolaterally
localized ABCC1 have produced varying results (Nies et al.,
2002; Ferna ´ndez et al., 2002; Konno et al., 2003; Bandler et al.,
2008). Although sequential truncation or substitution of specific
amino acid residues has been used as a strategy to determine the
sorting signals of many membrane proteins, unfolded or
incompletely assembled polytopic transporter proteins are often
trapped in the ER following synthesis, and are subsequently
destroyed by the stringent ER-associated quality control
mechanisms. For example, a number of mutations that cause
cystic fibrosis actually produce functional, but slightly misfolded,
forms of CFTR/ABCC7 that are retained in the ER where they
are degraded prior to their arrival at the plasma membrane
(Bertrand and Frizzell, 2003). Similar impairment in protein
trafficking of hereditary defective ABCC2 is believed to occur in
Dubin-Johnson syndrome, in which abnormal accumulation of
glucuronidated bilirubin causes hyperbilirubinemia (Keitel et al.,
2000; Hashimoto et al., 2002; Nies and Keppler, 2007). Thus, the
extent of deletion or amino acid substitution introduced into the
primary sequence of a protein could potentially become a
deleterious determinant for the precise intracellular targeting of
the modified protein. Accordingly, deletions created in the
ABCC2 protein often result in their unexpected retention in the
ER (Nies et al., 2002; Ferna ´ndez et al., 2002).
A complex array of protein–protein interactions generally
governs various regulatory aspects related to the polarized
localization of newly synthesized proteins in epithelial cells
(Mellman and Nelson, 2008; Gonzalez and Rodriguez-Boulan,
2009;Weisz and Rodriguez-Boulan,2009). Webegan this study by
postulating that proteins that have entered the ER and are destined
for the Golgi apparatus and, eventually, the plasma membrane
might display sorting signals on their cytosolic surfaces, and that
the cellular machinery would recognize such signals to control the
delivery of proteins to their correct subcellular compartments down
the biosynthetic-secretory pathway. To circumvent the unfavorable
effects that could compromise the trafficking of genetically
modified ABCC2, we set up a nondestructive assay in which the
apical localization of full-length ABCC2 was disturbed under
competitive conditions by overproducing a certain domain of
ABCC2. In the present study, we used this competition assay and
identified an apical targeting signal within the first cytoplasmic
loop domain (CLD1) of ABCC2. We further analyzed this region
by alanine scanning mutagenesis and found that five amino acids,
S283QDAL287, comprise an important apical targeting determinant
for ABCC2 in polarized HepG2 cells.
Confirmation of polarized distribution of FLAG–ABCC2 in
Like hepatocytes in liver tissues, hepatocarcinoma-derived
HepG2 cells are structurally and functionally polarized. In
monolayer culture, HepG2 cells reorganize to form sealed
vacuoles between the plasma membranes of adjacent polarized
cells (illustrated in supplementary material Fig. S2). The plasma
membranes of the vacuoles in HepG2 cells and the bile canaliculi
of hepatocytes represent the apical domain (Sormunen et al.,
1993). Aminopeptidase-N (APN) and the Na+,K+-ATPase a1
subunit (Na,K-ATPase) are marker proteins for the apical and
basolateral membranes, respectively (Sztul et al., 1987; Lian
et al., 1999). Since actin filaments are abundant just beneath the
plasma membrane, the apical vacuoles and cell perimeters are
readily visualized in polarized cells under a fluorescence
microscope using Rhodamine-conjugated phalloidin (van der
Wouden et al., 2002). As shown in (supplementary material Fig.
S1A), the Rhodamine-derived fluorescent signals (F-actin)
delineating the apical vacuoles and cell perimeters of HepG2
cells overlapped with the immunostained APN and Na,K-
ATPase, respectively; thus, indicating that the vacuoles and cell
perimeters of HepG2 cells conveniently serve as models for
apical and basolateral domains, respectively.
To ensure accurate topology of transiently expressed ABCC2, a
preprotrypsin signal peptide was placed just in front of three
repeated FLAG tags (Fig. 1A). This signal peptide allows the N-
terminus of the following part to situate on the extracellular side of
the plasma membrane. Without permeabilization, only N-terminal
FLAG-taggedproteins located on the external sideof thebasolateral
plasma membrane were detectable by immunofluorescence. If the
tagged proteins were directed towards the apical vacuoles of HepG2
cells, the immunofluorescence signal was seen only when the cell
membrane was permeabilized with nn X-100. In permeabilized
cells, immunostaining of FLAG–ABCC2 produced a single
punctuated signal between the neighboring cells, which merged
with the apical vacuoles (supplementary material Fig. S1A,
Fig. S2A). In contrast, no cell surface staining was observed in
(supplementary material Fig. S2B). ABCC1, a closely related
member of the ABCC protein family, is sorted to the basolateral
membrane of polarized epithelial cells (Deeley et al., 2006).
Consistent with this, we observed FLAG-tagged ABCC1 (FLAG–
cells (supplementary material Fig. S1A, Fig. S2A). In addition,
immunostaining of non-permeabilized cells showed extracellular
labeling of FLAG–ABCC1,
localization of FLAG–ABCC1 (supplementary material Fig. S2B).
These results suggested that fusion of the FLAG tag, along with the
preceding signal peptide, to ABCC2 or ABCC1 did not affect their
localization, as both fusion proteins were targeted in a manner
identical to that of their endogenous counterparts. These results also
suggested that a monolayer culture of HepG2 cells can serve as a
useful model system to study the polarized localization of these
transporters in hepatocytes.
Apical targeting information resides in the first
cytoplasmic loop domain of ABCC2
ABC transporters typically comprise two tandemly arranged
polytopic membrane spanning domains (MSD1 and MSD2) and
two cytoplasmic nucleotide binding domains (NBD1 and NBD2)
(Dean et al., 2001). An additional membrane spanning domain,
MSD0, exists at the N-terminus of ABCC2 and the first
cytoplasmic loop domain (CLD1) connects MSD0 to MSD1
(Fig. 1A). Many proteins that are trafficked from the ER to the
plasma membrane might display sorting signals on their cytosolic
surface, and the cellular trafficking machinery would recognize
such signals to navigate them along the biosynthetic-secretory
pathway. ABCC2 contains three long cytoplasmic loop domains
(CLD1, NBD1 and NBD2) and six short cytoplasmic loops
(Fig. 1A), and the former loop domains are more likely to contain
apical targeting determinants. NBD1 and NBD2 are peptides
approximately 300 amino acids long and are required for ATP-
binding. CLD1, on the other hand, comprises a comparatively
short polypeptide containing 127 amino acids (amino acid
residues 185 to 311 from the initiation methionine), and its
Journal of Cell Science 125 (13)3134
Journal of Cell Science
Transcytosis assays for protein trafficking were performed according to a
previously published protocol (Bastaki et al., 2002). Briefly, after five washes
with ice-cold serum-free medium (SFM), HepG2 cells were incubated for 15 min
on ice with antibodies (1:100 dilution) in SFM/1% fetal calf serum. After surface
decoration, cells were extensively washed with SFM and chased in growth medium
at 37˚C. Chased cells were washed, fixed, permeabilized, blocked, and incubated
with fluorescently labeled secondary antibodies and phalloidin. Fluorescence was
examined as described above.
We thank Shintaro Tsuji and Yuri Kotani for technical assistance.
This work was supported by a Grant-in-aid for Scientific Research
from The Ministry of Education, Culture, Sports, Science and
Technology of Japan [grant number 20370041 to M.S.], and grants
from the Foundation of Himeji Institute of Technology [grant
number 2003 to Y.E.]. This work is also supported by the Global
Center of Excellence Program from The Ministry of Education,
Culture, Sports, Science and Technology of Japan (‘Picobiology:
Life science at the atomic level’) to the Graduate School of Life
Science, University of Hyogo.
Supplementary material available online at
Altschuler, Y., Hodson, C. and Milgram, S. L. (2003). The apical compartment:
trafficking pathways, regulators and scaffolding proteins. Curr. Opin. Cell Biol. 15,
Arias, I. M., Boyer, J. L., Fausto, N., Schachter, D. and Shafritz, D. A. (1994). The
Liver: Biology and Pathobiology. New York, NY: Raven Press Ltd.
Bandler, P. E., Westlake, C. J., Grant, C. E., Cole, S. P. C. and Deeley, R. G. (2008).
Identification of regions required for apical membrane localization of human
multidrug resistance protein 2. Mol. Pharmacol. 74, 9-19.
Bastaki, M., Braiterman, L. T., Johns, D. C., Chen, Y. H. and Hubbard, A. L.
(2002). Absence of direct delivery for single transmembrane apical proteins or their
‘‘Secretory’’ forms in polarized hepatic cells. Mol. Biol. Cell 13, 225-237.
Bertrand, C. A. and Frizzell, R. A. (2003). The role of regulated CFTR trafficking in
epithelial secretion. Am. J. Physiol. Cell Physiol. 285, C1-C18.
Dean, M., Rzhetsky, A. and Allikmets, R. (2001). The human ATP-binding cassette
(ABC) transporter superfamily. Genome Res. 11, 1156-1166.
Deeley, R. G., Westlake, C. and Cole, S. P. C. (2006). Transmembrane transport of
endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance
proteins. Physiol. Rev. 86, 849-899.
Dunn, K. W., Kamocka, M. M. and McDonald, J. H. (2011). A practical guide to
evaluating colocalization in biological microscopy. Am. J. Physiol. Cell Physiol. 300,
Ellis, M. A., Potter, B. A., Cresawn, K. O. and Weisz, O. A. (2006). Polarized
biosynthetic traffic in renal epithelial cells: sorting, sorting, everywhere. Am. J.
Physiol. Renal Physiol. 291, F707-F713.
Emi, Y., Ikushiro, S. and Kato, Y. (2007). Thyroxine-metabolizing rat uridine
diphosphate-glucuronosyltransferase 1A7 is regulated by thyroid hormone receptor.
Endocrinology 148, 6124-6133.
Emi, Y., Nomura, S., Yokota, H. and Sakaguchi, M. (2011). ATP-binding cassette
transporter isoform C2 localizes to the apical plasma membrane via interactions with
scaffolding protein. J. Biochem. 149, 177-189.
Ferna ´ndez, S. B. M., Hollo ´, Z., Kern, A., Bakos, E´., Fischer, P. A., Borst, P. and
Evers, R. (2002). Role of the N-terminal transmembrane region of the multidrug
resistance protein MRP2 in routing to the apical membrane in MDCKII cells. J. Biol.
Chem. 277, 31048-31055.
Fo ¨lsch, H., Mattila, P. E. and Weisz, O. A. (2009). Taking the scenic route:
biosynthetic traffic to the plasma membrane in polarized epithelial cells. Traffic 10,
Fushimi, K., Sasaki, S. and Marumo, F. (1997). Phosphorylation of serine 256 is
required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water
channel. J. Biol. Chem. 272, 14800-14804.
Gafvelin, G., Sakaguchi, M., Andersson, H. and von Heijne, G. (1997). Topological
rules for membrane protein assembly in eukaryotic cells. J. Biol. Chem. 272, 6119-
Gonzalez, A. and Rodriguez-Boulan, E. (2009). Clathrin and AP1B: key roles in
basolateral trafficking through trans-endosomal routes. FEBS Lett. 583, 3784-3795.
Hashimoto, K., Uchiumi, T., Konno, T., Ebihara, T., Nakamura, T., Wada, M.,
Sakisaka, S., Maniwa, F., Amachi, T., Ueda, K. et al. (2002). Trafficking and
functional defects by mutations of the ATP-binding domains in MRP2 in patients with
Dubin-Johnson syndrome. Hepatology 36, 1236-1245.
Katsura, T., Gustafson, C. E., Ausiello, D. A. and Brown, D. (1997). Protein kinase A
phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected
LLC-PK1 cells. Am. J. Physiol. 272, F817-F822.
Keitel, V., Kartenbeck, J., Nies, A. T., Spring, H., Brom, M. and Keppler, D. (2000).
Impaired protein maturation of the conjugate export pump multidrug resistance
protein 2 as a consequence of a deletion mutation in Dubin-Johnson syndrome.
Hepatology 32, 1317-1328.
Kikuchi, S., Hata, M., Fukumoto, K., Yamane, Y., Matsui, T., Tamura, A.,
Yonemura, S., Yamagishi, H., Keppler, D., Tsukita, S. et al. (2002). Radixin
deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile
canalicular membranes. Nat. Genet. 31, 320-325.
Kocher, O., Comella, N., Gilchrist, A., Pal, R., Tognazzi, K., Brown, L. F. and
Knoll, J. H. (1999). PDZK1, a novel PDZ domain-containing protein up-regulated in
carcinomas and mapped to chromosome 1q21, interacts with cMOAT (MRP2), the
multidrug resistance-associated protein. Lab. Invest. 79, 1161-1170.
Konno, T., Ebihara, T., Hisaeda, K., Uchiumi, T., Nakamura, T., Shirakusa, T.,
Kuwano, M. and Wada, M. (2003). Identification of domains participating in the
substrate specificity and subcellular localization of the multidrug resistance proteins
MRP1 and MRP2. J. Biol. Chem. 278, 22908-22917.
Li, M., Wang, W., Soroka, C. J., Mennone, A., Harry, K., Weinman, E. J. and
Boyer, J. L. (2010). NHERF-1 binds to Mrp2 and regulates hepatic Mrp2 expression
and function. J. Biol. Chem. 285, 19299-19307.
Lian, W.-N., Tsai, J.-W., Yu, P.-M., Wu, T. W., Yang, S.-C., Chau, Y.-P. and Lin,
C.-H. (1999). Targeting of aminopeptidase N to bile canaliculi correlates with
secretory activities of the developing canalicular domain. Hepatology 30, 748-760.
Matter, K. and Mellman, I. (1994). Mechanisms of cell polarity: sorting and transport
in epithelial cells. Curr. Opin. Cell Biol. 6, 545-554.
Mellman, I. and Nelson, W. J. (2008). Coordinated protein sorting, targeting and
distribution in polarized cells. Nat. Rev. Mol. Cell Biol. 9, 833-845.
Nies, A. T. and Keppler, D. (2007). The apical conjugate efflux pump ABCC2 (MRP2).
Pflugers Arch. 453, 643-659.
Nies, A. T., Ko ¨nig, J., Cui, Y., Brom, M., Spring, H. and Keppler, D. (2002).
Structural requirements for the apical sorting of human multidrug resistance protein 2
(ABCC2). Eur. J. Biochem. 269, 1866-1876.
Rodriguez-Boulan, E. and Mu ¨sch, A. (2005). Protein sorting in the Golgi complex:
shifting paradigms. Biochim. Biophys. Acta 1744, 455-464.
Sakaguchi, M. (1997). Mutational analysis of signal-anchor and stop-transfer sequences
in membrane proteins. In Membrane Protein Assembly (ed. G. von Haijine), pp. 135-
150. Austin, TX: Landes Company.
Sormunen, R., Eskelinen, S. and Lehto, V. P. (1993). Bile canaliculus formation in
cultured HEPG2 cells. Lab. Invest. 68, 652-662.
Sztul, E. S., Biemesderfer, D., Caplan, M. J., Kashgarian, M. and Boyer, J. L.
(1987). Localization of Na+,K+-ATPase a-subunit to the sinusoidal and lateral but not
canalicular membranes of rat hepatocytes. J. Cell Biol. 104, 1239-1248.
Tsuchida, M., Emi, Y., Kida, Y. and Sakaguchi, M. (2008). Human ABC transporter
isoform B6 (ABCB6) localizes primarily in the Golgi apparatus. Biochem. Biophys.
Res. Commun. 369, 369-375.
van der Wouden, J. M., van IJzendoorn, S. C. and Hoekstra, D. (2002). Oncostatin
M regulates membrane traffic and stimulates bile canalicular membrane biogenesis in
HepG2 cells. EMBO J. 21, 6409-6418.
Wang, J. and Wilkinson, M. F. (2001). Deletion mutagenesis of large (12-kb) plasmids
by a one-step PCR protocol. Biotechniques 31, 722-724.
Weisz, O. A. and Rodriguez-Boulan, E. (2009). Apical trafficking in epithelial cells:
signals, clusters and motors. J. Cell Sci. 122, 4253-4266.
Yang, Q., Onuki, R., Nakai, C. and Sugiyama, Y. (2007). Ezrin and radixin both
regulate the apical membrane localization of ABCC2 (MRP2) in human intestinal
epithelial Caco-2 cells. Exp. Cell Res. 313, 3517-3525.
Apical targeting signal of ABCC23143