Expression of the aspartate/glutamate mitochondrial carriers aralar1
and citrin during development and in adult rat tissues
Araceli del Arco
1,3
, Julia
´
n Morcillo
2
, Juan Ramon Martı
´
nez-Morales
2
, Carmen Galia
´
n
1
, Vera Martos
1
,
Paola Bovolenta
2
and Jorgina Satru
´
stegui
1
1
Departamento de Biologı
´
a Molecular, Centro de Biologı
´
a Molecular Severo Ochoa, Universidad Auto

whereas the characteristic expression of aralar1 in skeletal
muscle was detected at E18 and that in the heart began early
in development (E11) and was preferentially localized to
auricular myocardium in late embryonic stages. Aralar1 was
also expressed in bone marrow, T-lymphocytes and
macrophages, including Kupffer cells in the liver, indicating
that this is the major AGC isoform present in the hemato-
poietic system. Both aralar1 and citrin were expressed in fetal
gut and adult stomach, ovary, testis, and pancreas, but only
aralar1 is enriched in lung and insulin-secreting b cells. These
results show that aralar1 is expressed in many more tissues
than originally believed and is absent from hepatocytes,
where citrin is the only AGC isoform present. This explains
why citrin deficiency in humans (type II citrullinemia) only
affects the liver and suggests that aralar1 may compensate
for the lack of citrin in other tissues.
Keywords: aspartate/glutamate carrier; calcium; citrulline-
mia; development; mitochondria.
Metabolites are transported through the inner mitochondrial
membrane by proteins belonging to the mitochondrialcarrier
(MC) superfamily [1]. The structure of these carriers
(molecular mass  30 kDa) consists of a threefold repetition
of a sequence of about 100aminoacids [2,3] with two putative
transmembrane domains. In the last few years, a number of
new MCs have been identified [3–5], including a subfamily of
Ca
2+
-binding mitochondrial carriers (CaMCs) with new
structural characteristics [6–8]. The CaMC subfamily mem-
bers have a bipartite structure. Their C-terminal domains

the stability/activity of liver ASS, one of the symptoms of
CTLN2.
Citrin is strongly expressed in both liver and kidney
[6–8,18]. However, CTLN2 is a liver-specific metabolic
Correspondence to J. Satru´ stegui, Departamento de Biologı
´
a
Molecular, Centro de Biologı
´
a Molecular Severo Ochoa,
Universidad Auto
´
noma de Madrid, 28049-Madrid, Spain.
Fax: 00 34 91 3974799, Tel.: 00 34 91 3974872,
E-mail:
Abbreviations: AGC, aspartate/glutamate carrier; MC, mitochondrial
carrier; CaMC, calcium-binding mitochondrial carrier; CTLN2,
adult-onset type II citrullinemia; ASS, argininosuccinate synthetase.
(Received 14 February 2002, revised 19 April 2002,
accepted 23 May 2002)
Eur. J. Biochem. 269, 3313–3320 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03018.x
deficiency, and ASS levels are normal in other tissues such
as kidney [8,19]. This difference can be explained by the
observation that aralar1, the second human AGC isoform,
is also expressed in kidney [6,18] and human kidney cell lines
[12,18], therefore it may compensate for the loss of citrin in
the kidney of patients with CTLN2. This raises a general
question of whether the two isoforms are expressed in the
same tissues and cell types and whether these isoforms play
thesamerole.

from the tibia bone. Rat pancreatic b islets were isolated by
collagenase digestion and standard procedures [20]. Brown
adipose tissue was collected from 1-day-old pups. Rat
fetuses staged at embryonic day 18 (E18) delivered by
cesarian section and newborn pups (1–6 h after sponta-
neous delivery) were used to study postnatal development of
the liver.
Cell lines
HEK-293T cells were cultured in Dulbecco’s modified
Eagle’s medium containing 5% fetal bovine serum (Gibco-
BRL) at 37 °Cina7%CO
2
atmosphere. RAW 264 and
Jurkat cells were grown in RPMI 1640 medium with 5%
fetal bovine serum under identical conditions.
Probes
The mouse aralar1 probe used was a 381-bp PstIfragment
obtained from the mouse EST clone W82002 (ATCC). A
probe specific for mouse citrin was generated by RT-PCR
using 2 lg total RNA obtained from adult mouse liver as
template. The oligonucleotides used, ara2-rat5 (5¢-AT
CTGTCCTGTGTGCTCCGG-3¢) and ara2-mouse3
(5¢-TCCATGGGTGTAACCTGACC-3¢), were designed
from mouse citrin cDNA sequence [11]. The amplified
fragment was subcloned into the blunted pSTBlue-1
(Novagen) and verified by sequencing.
In situ
hybridization
The 381-bp and 557-bp fragments of aralar1 and citrin
cDNA were transcribed to generate digoxigenin-labeled

guanidine isothiocyanate method. Northern blot analysis
was carried out using 20 lg total RNA from different rat
tissues as previously described [7]. As human and rat
nucleotide sequences are highly homologous ( 90%
identity), we used fragments of human citrin and aralar1
cDNAsasprobes.
1
The blot was stripped on 0.1% SDS at
100 °C for 30 min, and reprobed under identical conditions.
Antibodies
An antibody to the N-terminal half of aralar1 (amino acids
12–343) was described previously [6]. A citrin-specific
antibody was generated to amino acids 9–278 of the
N-terminal half of citrin expressed in bacteria. The construct
for bacterial expression has been previously described [7]. In
addition, selected regions of human citrin (amino acids 305–
319) and human aralar1 (amino acids 507–520), with
Jameson and Wolf antigenic indexes of  1.7, as predicted
by the peptidestructure program from the CGC (Genetic
Computer Group, Madison, WI, USA) package, were used
to generate epitope-specific antibodies. These regions of
human aralar1 and citrin are conserved in the mouse
proteins. The citrin 305–319 peptide was conjugated with
mcKLH (mariculture keyhole limpet hemocyanin) using an
Imject Immunogen EDC conjugation kit (Pierce). The
3314 A. del Arco et al.(Eur. J. Biochem. 269) Ó FEBS 2002
aralar1 507–520 peptide was conjugated with maleimide-
activated mcKLH (Pierce) through a cysteine added at the
N-terminus of the peptide, as recommended by the supplier.
The purified citrin protein (amino acids 9–278) and

M
EGTA/1 m
M
dithiothreitol/protease
inhibitors, pH 7.4, homogenized and subjected to differen-
tial centrifugations as described above.
Mitochondrial fractions were analysed by Western blot-
ting using an Enhanced Chemiluminiscence (ECL) kit
(Amersham). Antibody to the N-terminus of aralar1 was
used at a dilution of 1 : 5000, and antibodies to the
N-terminus of citrin, citrin 305–319 and aralar1 507–520
were used at a dilution of 1 : 2000. To control for the amount
of mitochondrial protein loaded, blots were stripped and
incubated with an antibody to the mitochondrial protein
b-F
1
ATPase (a gift from J. M. Cuezva, Centro de Biologia
Molecular devero Ochoa, JAM, Spain)
2
at a dilution of
1 : 5000. The densities of the bands were evaluated with a
Bio-Rad GS-710 calibrated imaging densitometer.
Immunocytochemistry
The animals were anesthetized with sodium pentobarbital,
and perfused through the cardiac ventricle, first with 50 mL
0.9% NaCl followed by 250 mL fixative solution containing
4% paraformaldehyde in 0.1
M
phosphate buffer, pH 7.4, at
room temperature. The tissues were removed, postfixed at

Sections were mounted on to polylysine-coated slides,
dehydrated, delipidated, and mounted in DPX (BDH).
RESULTS
Expression of
aralar1
and
citrin
during embryonic
mouse development
The expression of aralar1 and citrin was studied by using
in situ hybridizations in toto or on cryostat tissue sections,
depending on the stage of the embryos. The data obtained
partially confirmed and further extended those reported by
Sinasac et al. [11] on embryonic expression of citrin in
mouse.
At E11, the earliest stage analyzed, both aralar1 and citrin
were expressed throughout the developing embryo, with
stronger expression in the branchial arches, the developing
dermomyotome, the limb and the tail buds (Fig. 1A–E,a–e).
In spite of the apparent similarities in distribution, citrin
expression was predominantly, although not uniquely,
associated with the ectodermal, whereas aralar1 expression
was more abundant in the mesenchymal components of
these structures (Fig. 1B–E,b–e). In particular, citrin but not
aralar1 was strongly expressed in the apical ectodermal
ridge of the limb and on the tip of the tail bud (Fig. 1A,B).
As an additional difference between the two genes, aralar1
but not citrin transcripts were found in the heart (Fig. 1C,c).
At later stages of development (E13–E15), the mRNAs of
the two genes were also detected in neural tissue. A few days

inant in brain, heart and skeletal muscle, whereas citrin
expression only predominates in kidney.
Distribution of AGC isoforms in tissues
from the adult rat
In adult rat tissues, the distribution of aralar1 and citrin
transcripts was analysed by Northern blot. Two aralar1
transcripts of  2.7 and 3.8 kb were detected in all positive
tissues (Fig. 2A) as in humans [6]. The hybridization signal
was higher for the 2.7-kb than the 3.8-kb mRNA. Expres-
sion was stronger in heart and skeletal muscle, followed by
brain, and lower in kidney. No aralar1 mRNA was detected
in liver. On the other hand, the rat citrin gene presented a
Ó FEBS 2002 Development of aspartate/glutamate mitochondrial carriers (Eur. J. Biochem. 269) 3315
single transcript of about 3 kb, consistent with the size of
the mouse citrin cDNA and with the data reported for
human [7,11,18]. Citrin mRNA was abundant in liver,
kidney and heart but was notably absent from brain or
skeletal muscle. Therefore, the expression pattern of both
citrin and aralar1 in rat is consistent with that described for
human and mouse tissues [6,8,18].
The data on the mRNA distribution of the AGC
isoforms were complemented by the analysis of the content
of the respective proteins in mitochondria-enriched extracts
using Western blots with isoform-specific antibodies. As
shown in Fig. 2B, aralar1 levels were highest in heart,
forebrain, cerebellum and skeletal muscle, in agreement
with its mRNA distribution. Mitochondrial extracts from
two types of skeletal muscles, the fast-twitch glycolytic
extensor digitorum longus and the slow-twitch oxidative
soleus, showed similar levels of aralar1 protein. In heart,

not their corresponding mRNAs [18], were detected in
spleen and testis. Altogether, these results show that mRNA
levels are poor indicators of the levels of the AGC proteins.
To assess the relative expression of aralar1 and citrin,
mitochondrial fractions from a few representative tissues
were processed in parallel with either known amounts of
recombinant citrin [7] and aralar1 [6] or mitochondrial
fractions from HEK-293T cells overexpressing either
aralar1 or citrin, or control HEK-293T cells, which have
citrin/aralar1 ratios of about 0.5, 12, and 2.4, respectively
[12]. Serial dilution of the recombinant proteins (Fig. 2D) or
mitochondrial extracts from HEK-293T cells overexpress-
ing aralar1 or citrin (not shown) revealed a linear relation
between the amounts of the protein and the densities of the
immunoreactive bands. The analysis indicated that spleen,
heart (particularly the ventricle), ovary, and stomach have
similar levels of citrin and aralar1. Liver, kidney, whole
pancreas and testis clearly have higher levels of citrin than
aralar1. In contrast, central nervous system tissue, skeletal
muscle, lung and possibly auricular myocardium (Fig. 2C)
predominantly have aralar1.
Expression of
aralar1
in cells from the hematopoietic
system
Citrin is expressed at high levels in the liver. This molecule is
the liver-specific AGC isoform, as citrin deficiency causes
Fig. 2. Pattern of expression of aralar1 and citrin in adult rat tissues. (A) Northern blot analysis of tissue-specific expression patterns of AGC
isoforms. Northern blots with 20 lg total RNA from adult rat heart, kidney, brain, liver and skeletal muscle were hybridized with a
32

Iijima et al. [18] for different liver cell types (hepatocytes,
stellate, endothelial and Kupffer cells), rat aralar1 cDNA
was readily amplified by RT-PCR from liver mRNA (A. del
Arco et al., unpublished data), indicating that aralar1
transcripts are present in this tissue, albeit at very low levels.
To determine the cellular source of aralar1, sections from
adult rat liver were immunostained with specific antibodies.
As observed in Fig. 3A, aralar1 was not localized to the
parenchymal hepatocytes, but to sparse spindle-shaped cells
which, by morphological criteria and position, probably
correspond to Kupffer cells, the liver resident macrophages
[24]. The fact that Iijima et al. [18] did not detect aralar1
mRNA in isolated Kupffer cells in Northern blots probably
reflects either changes in aralar1 mRNA during isolation
and plating of the cells or the lack of correspondence
between the AGC mRNA and protein levels, a situation
also found for both aralar1 and citrin in lung, spleen and
testis, as mentioned above. The presence of aralar1 in
Kupffer cells is further supported by the observation that
aralar1 is expressed by other cells of the hematopoietic
system. Thus, it is present in mitochondrial extracts
obtained from a murine macrophage-like cell line, the
RAW 264 cells (Fig. 3B). Aralar1 mRNA and protein were
also detected in Jurkat cells and human T-lymphocytes,
respectively (Fig. 3B and data not shown) and in bone
marrow (Fig. 3B). In contrast, citrin was not detected in
RAW 264 cells (Fig. 3B), and citrin mRNA and protein
were absent from human T-lymphocytes and Jurkat cells [7]
(Fig. 3B).
Fetal liver together with the yolk sac are the hemato-

distribution [6–8,18]. Thus, although aralar1 mRNA is
highly represented in brain, skeletal muscle and heart,
aralar1 protein is not restricted to these excitable tissues but
it is also found in lung, stomach, pancreas (particularly
b cells), kidney, and ovary, and it is the main isoform
present in hematopoietic tissues. On the other hand, citrin
was expressed not only in kidney and liver, the classic
gluconeogenic organs, but was present at significant levels in
heart, stomach, pancreas and testis. The absence of
detectable aralar1 mRNA in tissues where aralar1 protein
is readily observed suggests that its expression may be
regulated at post-transcriptional levels, as is known for
other mitochondrial proteins involved in bioenergetic func-
tions [27].
The distribution of citrin mRNA in mouse embryos has
been studied previously [11]. However, this is the first time
that aralar1 expression has been studied in mouse embryos
by in situ hybridization and compared with that of citrin.In
contrast with the situation in the adult animal, this study
shows that there is a wide overlap in the expression of the
Fig. 3. Expression of aralar1 in cells of the immune system. (A) Immunohistochemical detection of aralar1 in liver rat sections. Aralar1 positive cells
are indicated by arrows. No signal is observed in hepatocytes. Those sections in which either the primary or secondary antibodies or the ABC
reagent were omitted were negative. Scale bar ¼ 75 lm. (B) Western blot analysis of aralar1 and citrin in cells from the immune system and
hematopoietic tissues. Mitochondrial extracts (20 lg) obtained from human Jurkat cells (a T-cell leukemia cell line) and the mouse macrophage cell
line RAW 264, as well as the hematopoietic tissues, bone marrow and spleen, were loaded on to gels. Mitochondrial extracts from HEK-293T cells
with a known aralar1/citrin ratio were included as an internal control [12]. The membranes were processed as described in Fig. 2B
4
with antibodies to
aralar1 N-terminus (1 : 5000) and citrin N-terminus (1 : 2000). (C) Western blot analysis of AGC isoforms during rat liver development. The
mitochondrial extracts (20 lg per lane) were obtained from fetuses at embryonic day 18 (E18), from pups 1–6 h after birth (P0), and from 3-month-

distribution was restricted to excitable tissues [6–8] and
may explain why citrin deficiency only affects the liver.
Indeed, CTLN2 patients have normal levels of ASS in
tissues other than the liver [8,18,19,28], suggesting that
the function provided by citrin, i.e, the efflux of aspartate
from mitochondria as substrate of ASS, can also be
accomplished by aralar1, a protein more widely expressed
than previously believed. This argues against major func-
tional differences between the two isoforms, and is consis-
tent with the results obtained with the recombinant proteins
reconstituted in proteoliposomes, and expressed in human
cells [12]. On the other hand, the presence of a single major
AGC isoform, aralar1, in skeletal muscle, central nervous
system, and cells from the hematopoietic system suggests
that mutations in aralar1 would have a preferential impact
in these tissues.
ACKNOWLEDGEMENTS
This work was supported by grants from the Spanish Direccion
General de Investigacio
´
nCientı
´
ficayTe
´
cnica, Comunidad Auto
´
noma
de Madrid, Quı
´
mica Farmace

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