Identification and characterization of a novel member of the heterodimeric amino acid transporter

56 Pages·2001·0.55 MB·English

JBC Papers in Press. Published on October 8, 2001 as Manuscript M107517200
Submitted to the Journal of Biological Chemistry: M1:07517
Revised on October 1, 2001

Identification and characterization of a novel member of
the heterodimeric amino acid transporter family presumed
to be associated with an unknown heavy chain

Arthit Chairoungdua1, Yoshikatsu Kanai1,3,*, Hirotaka Matsuo1,2,
Jun Inatomi1, Do Kyung Kim1 and Hitoshi Endou1
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1Department of Pharmacology and Toxicology, Kyorin University lo
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School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, 2First fro
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Department of Physiology, National Defense Medical College, 3-2 ttp://w
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Namiki, Tokorozawa, Saitama 359-8513 and 3PRESTO, Japan Science and w.jb
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Technology Corporation (JST), JAPAN rg
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Running Title: A novel member of heterodimeric amino acid A
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transporter associated with unknown heavy chain , 2
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*Address correspondence and proofs to:
Dr.Yoshikatsu Kanai, Department of Pharmacology and Toxicology,
Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka,
Tokyo 181-8611, Japan
Tel: [phone], Ext.3453,
Fax: [phone]
e-mail: [email protected]
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Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
SUMMARY
We identified a novel amino acid transporter designated Asc-2
(asc-type amino acid transporter 2). Asc-2 exhibited relatively
low but significant sequence similarity to the members of the
heterodimeric amino acid transporters. The cysteine residue
responsible for the disulfide bond formation between transporters
(light chains) and heavy chain subunits in the heterodimeric amino
acid transporters is conserved for Asc-2. Asc-2 is, however, not
colocalized with the already known heavy chains such as 4F2hc (4F2
heavy chain) or rBAT (related to b0,+ amino acid transporter) in D
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mouse kidney. Because Asc-2 solely expressed or co-expressed with lo
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4F2hc or rBAT did not induce functional activity, we generated fro
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fusion proteins in which Asc-2 is connected with 4F2hc or rBAT. ttp://w
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The fusion proteins were sorted to the plasma membrane and .jb
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expressed the function corresponding to the Na+-independent rg
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transport system asc. Distinct from the already identified system ue
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asc transporter Asc-1 which is associated with 4F2hc, Asc-2- A
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mediated transport is less stereoselective and did not accept some , 2
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of the high affinity substrates of Asc-1 such as α-aminoisobutyric
acid and β-alanine. Asc-2 message was detected in kidney, placenta,
spleen, lung and skeletal muscle. In kidney, Asc-2 protein was
present in the epithelial cells lining collecting ducts. In the
Western blot analysis on mouse erythrocytes and kidney, Asc-2 was
detected as multiple bands in the nonreducing condition, whereas
the bands shifted to a single band at lower molecular weight,
suggesting the association of Asc-2 with other protein(s) via a
disulfide bond. The finding of Asc-2 would lead to the
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establishment of a new subgroup of heterodimeric amino acid
transporter family which includes transporters associated not with
4F2hc or rBAT but with other unknown heavy chains.
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INTRODUCTION
Amino acid transport across the plasma membrane is mediated
by various amino acid transport systems which have been classified
based on the substrate selectivity and Na+-dependence (1).
Recently, amino acid transporters corresponding to most of the
classical amino acid transport systems have been identified by
means of molecular cloning approaches (2-9). Among them,
heterodimeric amino acid transporters are unique, because they are
associated with single membrane spanning type II membrane
glycoproteins such as 4F2hc (4F2 heavy chain) or rBAT (related to D
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b0,+ amino acid transporter) (4). lo
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4F2hc is the heavy chain of the 4F2 antigen (CD98) originally fro
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identified as a cell-surface antigen upregulated upon lymphocyte ttp://w
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activation (10, 11). Structurally, the 4F2 antigen is a .jb
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heterodimeric protein composed of two subunits, an ~80-kDa rg
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glycosylated heavy chain and a ~40-kDa nonglycosylated light chain ue
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(10, 11). Now, the 4F2 light chain has been revealed to be an A
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amino acid transporter. The transporter corresponding to the amino , 2
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acid transport systems L, y+L, x- and asc has been shown to be the
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4F2 light chain, which requires 4F2hc for its functional
expression (12-21). By now, 6 proteins which belong to SLC7 family
have been identified to be 4F2 light chains (12-21). In addition,
a protein structurally related to the 4F2hc-associated
transporters has been identified, which also belongs to the SLC7
family but couples with the other type II membrane glycoprotein, a
heavy chain subunit rBAT, to form a system b0,+ amino acid
transporter (22-24), thereby establishing a family of amino acid
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transporters associated with type II membrane glycoproteins (the
heterodimeric amino acid transporter family). It has been shown
that a conserved cysteine residue in the predicted extracellular
loop of the transporter proteins (light chains) is responsible for
the disulfide bond formation with the heavy chains (25). In the
heterodimeric amino acid transporters, the heavy chain subunits
are proposed to assist the light chain subunits to be sorted to
the plasma membrane (13, 23, 26).
In the characterization of system b0,+ transporter b0,+AT in
Xenopus oocytes, Pfeiffer et al. generated a fusion protein in D
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which the C-terminus of the light chain b0,+AT was connected with lo
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the N-terminus of its associating heavy chain rBAT (24). They fro
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showed that the fusion protein was functional and exhibited the ttp://w
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identical properties to those obtained by the coexpression of b0,+AT w.jb
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and rBAT. In addition, the mutant fusion protein whose light chain rg
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portion was mutated was not functional, confirming that the ue
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detected transport activity was due to the fusion protein itself A
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and not to the associated oocyte endogenous light chain (24). In , 2
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the present study, although we identified a novel transporter-like
protein which exhibits relatively low but significant structural
similarity to the light chain subunits of the heterodimeric amino
acid transporters, we could not functionally express it in Xenopus
oocyte plasma membrane. Therefore, we constructed fusion proteins
in which the C-terminus of the transporter protein was connected
with the N-terminus of rBAT or 4F2hc to expect that the fusion
proteins were sorted to the plasma membrane. We show that they
were in fact sorted to the plasma membrane and exhibited the
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functions of a transporter subserving system asc, yet distinct
from those of the already identified system asc transporter Asc-1
(21). The transporter is proposed to be associated with the
unknown heavy chain through the conserved cysteine residue to be
functional in the plasma membrane.
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EXPERIMENTAL PROCEDURES
cDNA for Asc-2- The cDNA for a mouse expressed sequence tag
(GenBankTM/EBI/DDBJ accession no. AI875555) showing nucleotide
sequence similarity to LAT1 (12) was obtained from the Integrated
and Molecular Analysis of Genomes and their Expression (IMAGE cDNA
clone no.1972372). The cDNA insert was subcloned into the
mammalian expression vector pcDNA3.1(+) (Invitrogen) at EcoRI and
NotI restriction enzyme cleavage sites. The cDNA was sequenced in
both directions by the dye terminator cycle sequencing method
(Perkin Elmer and Applied Biosystems). Transmembrane regions of D
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proteins were predicted based on SOSUI algorithm (27). lo
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In vitro translation- In vitro translation of cRNAs for Asc-2 and ttp://w
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mouse 4F2hc (21) were performed by using a rabbit reticulocyte .jb
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lysate system with or without canine pancreatic microsome membrane rg
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(Promega) and endoglycosidase H (Boehringer Mannheim), as ue
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described elsewhere (12, 28-30). A
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Construction of fusion proteins- Fusion proteins were constructed
as described elsewhere with some modifications (24). To generate a
Asc-2-rBAT fusion protein, Asc-2 cDNA fragment was amplified by
PCR using a sense primer corresponding to the nucleotides 17-36 of
Asc-2 cDNA sequence extended at its 5’ end by adding HindIII and
EcoRI restriction sites and GCGC (5’-
GCGCGAATTCAAGCTTGAACACCCTGTTTGACAGGG-3’), and a reverse primer
corresponding to the end of the coding sequence extended at its 5’
end by adding SpeI and EcoRI restriction sites and GCGC (5’-
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GCGCGAATTCACTAGTATTTTTCTGTTCTTCTGGAT-3’). The PCR products was
digested with HindIII and EcoRI and ligated to HindIII and EcoRI
sites of a mamalian expression vector pcDNA3.1(+) (Invitrogen).
Mouse rBAT cDNA was amplified using a sense primer corresponding
to the coding sequence starting just after the start codon (ATG)
extended at its 5’ end by adding EcoRI restriction site and GCGC
(5’-GCGCGAATTCGATGAGGACAAAGGCAAGAG-3’) and a reverse primer
corresponding to nucleotides 2240-2259 of mouse rBAT cDNA sequence
(GenBank/EMBL/DDBJ Data Bank accession number NM009205 (31))
extended at its end by NotI restriction site and GCGC (5’- D
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GCGCGCGGCCGCCATATTTAAATGCTTTAGTA-3’). The PCR products was lo
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digested with EcoRI and NotI and then introduced into the vector fro
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containing Asc-2 (see above) precleaved with EcoRI and NotI. ttp://w
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For Asc-2-4F2hc fusion protein, Asc-2 cDNA fragment was .jb
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amplified using a sense primer corresponding to the nucleotides rg
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17-36 of Asc-2 cDNA sequence extended at its 5’ end by adding ue
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HindIII and EcoRI restriction sites and GCGC (5’- A
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GCGCGAATTCAAGCTTGAACACCCTGTTTGACAGGG-3’) and a reverse primer , 2
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corresponding to the end of the coding sequence extended at its 5’
end by adding EcoRI and ClaI restriction sites and GCGC ( 5’-
GCGCGAATTCATCGATATTTTTCTGTTCTTCTGGAT-3’). The PCR products was
digested with HindIII and EcoRI and ligated to HindIII and EcoRI
restriction sites of pcDNA3.1(+) (Invitrogen). Mouse 4F2hc cDNA
was amplified using a sense primer corresponding to the coding
sequence starting just after the start codon (ATG) extended at its
5’ end by adding ClaI restriction site and GCGC (5’-
GCGCATCGATAGCCAGGACACCGAAGTGGA-3’) and a reverse primer
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corresponding to nucleotides 1820-1839 of mouse 4F2hc cDNA
sequence (GenBank/EBI/DDBJ accession number AB023408 ((21)))
extended at its end by adding NotI restriction site and GCGC (5’-
GCGCGCGGCCGCTGAGGCAGGGGTGATGTTTT-3’). The PCR products was
digested with ClaI and NotI and introduced into the vector
containing Asc-2 (see above) predigested with ClaI and NotI.

Xenopus oocyte expression- cRNAs were obtained by in vitro
transcription using T7 RNA polymerase for cDNAs of Asc-2, Asc-2-
rBAT fusion protein and Asc-2-4F2hc fusion protein subcloned in D
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pcDNA3.1(+) (Invitrogen) and linearized with XbaI, as described lo
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elsewhere (31). The Xenopus oocyte expression studies and uptake fro
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measurements were performed as described previously (32, 33). The ttp://w
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uptake of 14C-labeled amino acids were measured 3 days after w.jb
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injection of cRNAs. Twenty-five nanogram of cRNAs were injected to rg
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each oocyte. For coexpression of Asc-2 and mouse 4F2hc or mouse ue
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rBAT, 12 ng of Asc-2 cRNA and 13 ng of mouse 4F2hc cRNA (21) or A
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mouse rBAT cRNA (31) were injected into oocytes. , 2
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Amino acid uptake measurements in Xenopus oocytes- Groups of six
to eight oocytes were incubated in 500 µl of standard uptake
solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl , 1 mM MgCl , 10 mM
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HEPES and 5 mM Tris, pH 7.4) or Na+-free uptake solution in which
NaCl in the standard uptake solution was replaced by choline-Cl,
containing 0.5-3.0 µCi of the radiolabeled compounds (17).
Preliminary experiments to determine the time-course of [14C]L-
serine (100 µM) uptake into oocytes expressing the Asc-2-rBAT and
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Asc-2-4F2hc fusion proteins indicated that the uptake was linearly
dependent on incubation time up to 30 min (data not shown).
Therefore, in all the subsequent experiments, the uptake levels
were measured over 30 min and the values were expressed as
pmol/oocyte/min.
K and V of amino acid substrates were determined using
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Eadie-Hofstee equation based on the amino acid uptakes mediated by
the Asc-2-rBAT fusion protein measured at 0.3, 1, 3, 10, 30 and
100 µM for L-serine, L-alanine, L-threonine, L-cysteine and L-
glycine, and at 1, 3, 10, 30, 100, and 300 µM for L-valine and L- D
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leucine. The amino acid uptakes mediated by the Asc-2-rBAT fusion lo
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protein or Asc-2-4F2hc fusion protein were calculated as fro
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differences between the means of the uptakes by the oocytes ttp://w
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injected with cRNA for the fusion proteins and those of the .jb
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control oocytes injected with water. rg
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For the efflux measurement, 100 nl (10 nCi) of [14C]L-serine ue
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(600 µM) was injected into the oocytes with fine-tipped glass A
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micropipette as described elsewhere (9, 21, 34). The individual , 2
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oocytes were incubated for 5 min in ice-cold Na+-free uptake
solution, and then transferred to Na+-free uptake solution, with or
without 100 µM nonradiolabeled L-serine, kept at room temperature
(18 ~ 22oC). The radioactivity in the medium and the radioactivity
remaining in the oocytes were measured. The values were expressed
as % radioactivity (radioactivity in medium or that in
oocytes/(radioactivity in medium + radioactivity in oocytes) x
100%) (9, 21, 34).
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