Báo cáo Y học: Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues docx

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

´
noma de Madrid Spain;
2
Departamento de Neurobiologı
´
a del Desarrollo, Instituto Cajal, Consejo Superior de Investigaciones Cientı
´
ficas, Madrid, Spain;
3
Facultad de Ciencias del Medio Ambiente, Universidad de Castilla La Mancha, Toledo, Spain
Aralar1 and citrin are members of the subfamily of calcium-
binding mitochondrial carriers and correspond to two iso-
forms of the mitochondrial aspartate/glutamate carrier
(AGC). These proteins are activated by Ca
2+
acting on the
external side of the inner mitochondrial membrane.
Although it is known that aralar1 is expressed mainly in
skeletal muscle, heart and brain, whereas citrin is present in
liver, kidney and heart, the precise tissue distribution of the
two proteins in embryonic and adult tissues is largely
unknown. We investigated the pattern of expression of
aralar1 and citrin in murine embryonic and adult tissues at
the mRNA and protein levels. Insituhybridization analysis
indicates that both isoforms are expressed strongly in the
branchial arches, dermomyotome, limb and tail buds at early
embryonic stages. However, citrin was more abundant in the
ectodermal components of these structures whereas aralarl
had a predominantly mesenchymal localization. The strong
expression of citrin in the liver was acquired postnatally,

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

have the features of the MC superfamily and their N-terminal
extensions harbor EF-hand Ca
2+
-binding motifs [6].
Aralar1 and citrin, two members of the CaMC subfamily,
are nuclear-encoded proteins, with genes in human
chromosome 2 (SLC25A12 [9,10]) and 7 (SLC25A13
[8,11]), respectively. As recently demonstrated, aralar1 and
citrin are isoforms of the mitochondrial aspartate/glutamate
carrier (AGC) [12] which catalyzes a 1 : 1 exchange of
aspartate for glutamate and plays an important role in the
malate/aspartate shuttle, urea synthesis and gluconeogenesis
from lactate [13–15]. These two AGC isoforms are activated
by Ca
2+
on the external face of the inner mitochondrial
membrane [12].
Mutations in the human gene coding for citrin are
responsible for adult-onset type II citrullinemia (CTLN2:
603471) [8,16], an autosomal recessive disease caused by a
liver-specific deficiency in argininosuccinate synthetase
(ASS). In the liver, the AGC plays an important role in
the urea cycle by providing aspartate for incorporation into
argininosuccinate [17]. The mutations in the citrin gene in
patients affected by CTLN2 cause either truncation of the
protein or deletion of a loop between the transmembrane
spans [8,16], impairing the function of citrin as an AGC in
mitochondria. This impairment would presumably lead to a
failure in the supply of aspartate from mitochondria for
argininosuccinate synthesis, with consequent alterations in

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.

To address the first of these questions, we have now
studied the expression of aralar1 and citrin throughout
mouse development and in tissues of the adult rat with the
use of isoform-specific probes and antibodies. Our results
indicate that the two isoforms are widely expressed
throughout embryogenesis with a dynamic expression
pattern. The characteristic expression of aralar1 in skeletal
muscle and citrin in liver is only manifested at E18 or after
birth, respectively. Aralar1, but not citrin, is expressed early
(E11) in heart and it is preferentially localized to auricular
myocardium in late embryonic stages. Whereas citrin is
preferentially expressed in liver and kidney, the classical
gluconeogenic organs, a number of adult tissues and cell
types were found to express aralar1 preferentially over
citrin, including the adult lung and hematopoietic cells.
MATERIALS AND METHODS
Animals and tissues
mRNA expression was examined in Balb/c mice and Wistar
rats. Animals were kept in climate-controlled quarters under
a 12-h light cycle with free access to water and standard
chow diet. The animal facilities fulfilled the requirements of
the European laws, and the highest standards of animal care
were met in all experimental protocols.
Mouse embryos were collected from timed pregnant
Balb/c mice. The day of vaginal plug appearance was
considered embryonic day 0.5 (E0.5). The following tissues
and organs were dissected from 3-month-old rats: liver,
forebrain, cerebellum, heart, small intestine, stomach, lung,
kidney, testis, ovary, white adipose tissue, pancreas, bone
marrow, spleen and muscle. Bone marrow was obtained

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

antisense and sense cRNA probes. Whole-mount in situ
hybridizations were performed as described [21]. Briefly,
hybridizations were carried out at 65 °Cin50%formamide.
Post-hybridization washes were performed at the same
temperature and in the same buffer. Embryos staged at
embryonic day 11 (E11) were hybridized in toto.After
hybridization, embryos were photographed, dehydrated,
embedded in Paraplast, and sectioned with a microtome at
18 lm. For older embryos, E18.5, hybridizations were
carried out on tissue sections. Embryos were fixed in 4%
paraformaldehyde in 0.1
M
phosphate buffer, pH 7.3, at
4 °C overnight and then cryoprotected by immersion in
30% sucrose solution in phosphate buffer. Cryostat sections
16–20 lm thick were mounted on 2% 3-aminopropyltri-
ethoxysilane-coated slides, air-dried, and permeabilized with
proteinase K (10 lgÆmL
)1
in NaCl/P
i
containing 0.1%
Tween) for 5–10 min at room temperature. Sections were
then postfixed in 4% paraformaldehyde in phosphate
buffer, prehybridized for 1 h at 65 °C in 50% formamide,
and incubated with probes for 16 h at 65 °C. All the
staining patterns described below were obtained only with
antisense riboprobes and not with control sense riboprobes.
RNA analysis
Total RNA was extracted from rat tissues using the

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

mcKLH-conjugated peptides were injected into rabbits
using standard immunization procedures.
Westerns blots
Rat tissues were homogenized in 250 m
M
sucrose/10 m
M
Tris/HCl (pH 7.4)/protease inhibitors (1 m
M
iodoacetamide
and 1 m
M
phenylmethanesulfonyl fluoride) and centrifuged
at 750 g (10 min). The supernatant was then centrifuged at
10 000 g (15 min), and the pellets were collected to obtain
the crude mitochondrial fractions. Cells were scraped into
250 m
M
sucrose/20 m
M
Hepes/10 m
M
KCl/1.5 m
M
MgCl
2
/
1m
M
EDTA/1 m

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

4 °C for 24 h, and cryoprotected by immersion in 30%
sucrose. Free-floating cryostat 40-lm-thick sections were
first quenched with 3% H
2
O
2
in 10% methanol for 20 min in
potassium phosphate-buffered saline (NaCl/P
i
). After this
treatment, the sections were preincubated for 2–3 h in NaCl/
P
i
containing 5% horse serum and 0.25% Triton X-100 and
incubated overnight with antiaralar1 antibody at a dilution
of 1 : 100 in 1% horse serum and 0.25% Triton X-100 in
NaCl/P
i
. Secondary biotinylated antibody (goat anti-rabbit;
Vector; 1 : 150 dilution) was then incubated for 1–2 h,
followed by a 1-h reaction with avidin–biotin–peroxidase
complexes (regular ABC kit Vectastain; Vector). Sections
were developed using 0.05% 3,3-diaminobenzidine (Sigma)
in the presence of 0.03% H
2
O
2
in NaCl/P
i
for 1–2 min.

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

later (E18), the expression of aralar1 and, to a lesser extent,
citrin became clearly localized to selected brain regions such
as the cortex and hippocampus, the ventromedial thalamus,
the mitral cell layer of the olfactory bulb (Fig. 1L–M,l–m),
and the developing striatum (not shown). In the peripheral
nervous system, aralar1 and citrin mRNAs were detected, at
similar levels, in the trigeminal ganglia (Fig. 1L,l).
At E18, when organogenesis becomes a predominant
event in embryonic development, the mRNAs of the two
genes became differentially localized to particular organs
and tissues. Skeletal muscle showed high levels of aralar1
expression, whereas the detection of citrin was negligible in
this tissue (compare Fig. 1K with 1k). Similarly, aralar1 but
not citrin transcripts were present in the heart, mainly
confined to the auricular myocardium (compare Fig. 1I
with 1i). The gut endothelium expressed both citrin and
aralar1 (Fig. 1f–g,F–G), aralar1 transcripts being localized
to the basolateral region of the enterocytes (Fig. 1g). Gut
endothelium is a site where arginine biosynthesis occurs in
the suckling rat [22] and where the AGCs probably function
to provide aspartate for argininosuccinate synthesis. Both
genes were also expressed in the kidney but with a
differential distribution (Fig. 1H,h). In particular, only high
levels of citrin transcripts were found in the epithelium of the
tubules, whereas the expression of aralar1 was associated
with mesenchymal components (Fig. 1H,h).
In summary, the two AGC isoforms have a partially
overlapping expression pattern at early stages of embryo-
genesis. At later stages, the expression domain of the two
genes diverges, and aralar1 distribution becomes predom-

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,

aralar1 levels were higher in auricular than ventricular
Fig. 1. Comparison of expression pattern of citrin and aralar1 during murine embryonic development. Whole embryos from embryonic day (E) 11
(A–E, a–e), in toto E18 isolated organs (H–I, h–i), or transverse E18 cryostat tissue sections (F, G, L, M; f, g, l, m) were hybridized with digoxigenin-
labeled probes specific for the citrin or aralar1 genes. Images in (A–E) and (a–e) show E11 embryos, in toto (A, a) and in transverse paraffin sections
(B–E, b–e) taken from the embryos in (A and a) at the axial levels indicated by the dotted lines. Note the strong expression in the limb (B) and tail
buds (arrowhead in A), in the branchial arches (D) and dermomyotome (E), more strongly localized in the ectodermal component for citrin (A–E),
while to the mesenchyme for aralar1 (a–e). At E11, aralar1 (c) but not citrin (C) was expressed in the heart. Images in (F–M; f–m) illustrate the
comparative expression of citrin and aralar1 in the gut (F–G; f–g), kidney (H, h), heart (I, i), skeletal muscle (K, k), cortex (L, l), olfactory bulbs (M,
m) from E18 embryos. Note the stronger expression of aralar1 in skeletal muscle, heart and neural tissue. Note also the basolateral localization of
the in situ hybridization signal in the cells of the gut. Abbreviations: aer, apical ectodermal ridge; am, auricular myocardium; ba, branchial arch; cx,
cortex; dm, dermomyotome; h, heart, hc, hippocampus; lb, limb bud; mcl, mitral cell layer; smf, skeletal muscle fiber; tg, trigeminal ganglia; vmt;
ventromedial thalamus. Scale bars ¼ 500 lm (B, b; D, d; F, f; H, h; I, i; L, i); 250 lm (C, c; E, e; M, m); 100 lm(K,k);50lm(G,g).
3316 A. del Arco et al.(Eur. J. Biochem. 269) Ó FEBS 2002
myocardium (Fig. 2C). Lower levels of aralar1 were also
present in a wide range of tissues, including lung, kidney,
ovary, spleen, pancreas (particularly in b islets) and stomach
and to an even lower extent in mitochondrial extracts of
testis, intestine and liver.
Citrin was absent from the central nervous system,
skeletal muscle and lung but abundant in liver, kidney and
heart, in agreement with its mRNA distribution. Weaker
expression was also observed in ovary, testis, spleen,
stomach and pancreas, but not in b islets (Fig. 2B). Citrin
and aralar1 were hardly observed at all in the adult intestine
(Fig. 2B), where their expression was instead high at
embryonic day 18–19 (Fig. 1F–G,f–g and [18]). Neither
aralar1 nor citrin were detectable in either brown or white
adipose tissue (Fig. 2B). It is interesting to note that aralar1
protein is present in lung, where aralar1 mRNA was not
detectable [6,8,18]. Similarly, citrin and aralar1 proteins, but

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

P-labeled
DNA probe of human aralar1 under high-stringency conditions. The blot was subsequently stripped and reprobed under identical conditions with a
human citrin probe The size of the specific transcripts is indicated. Staining with ethidium bromide was carried out to verify the amount of RNA
loaded (lower panel). (B) Distribution of AGC isoforms in rat tissues; 20–30 lg mitochondrial protein was used for all tissues except for b islets
where 20 lg total protein extract obtained from 500 pancreatic islets was used. Blots for citrin and aralar1 were performed in parallel and
reincubated with anti-(b-F
1
ATPase). Aralar1 was detected with an antibody directed against its N-terminus, or against Aralar1 amino acids 507–
520 (results not shown). Citrin antibodies were against its N-terminus (upper panels) or against citrin amino acids 305–319 (lower panels), both at
1 : 2000 dilution. Bands correspond to 70 kDa (aralar1 and citrin) and 52 kDa for b-F
1
ATPase. (C) Western blot analysis of aralar1 and citrin in
auricular and ventricular myocardium. 20 lg of atria (A) and ventricle (V) mitochondrial extracts from two different animals were analysed. The
blot was incubated with anti-(aralar N-terminus) (1 : 5000) and anti-(b-F
1
ATPase) (1 : 5000), stripped and probed again with anti-(citrin
N-terminus) (1 : 2000). The amount of aralar1 (standardized to that of b-F
1
ATPase) was 1.5 and 0.9 in atria and ventricles, respectively. No
significant changes between atria and ventricle are observed for citrin levels (0.86 and 0.64 standardized values, in atria and ventricles, respectively).
(D) Immunoblotting of increasing amounts of recombinant citrin and aralar1. Known amounts (as indicated) of bacterially expressed aralar1 and
citrin N-terminal regions were loaded on to gels and blotted and processed in parallel with an identical dilution (1 : 2000) of their respective specific
antiserum antibodies. Note the different amounts of recombinant aralar1 and citrin used.
Ó FEBS 2002 Development of aspartate/glutamate mitochondrial carriers (Eur. J. Biochem. 269) 3317
CTLN2 [8,23], indicating that aralar1 does not compensate
for the loss of citrin in liver. Surprisingly, however, we
detected a low but unequivocal presence of aralar1 protein
in the adult liver (Fig. 2B, see also Fig. 3C). Furthermore,
even though aralar1 mRNA was not detected by Northern
blots of whole liver (Fig. 2A), similarly to that reported by

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-

poietic organs in prenatal mammalian development [25]. We
detected by Western blotting aralar1 and citrin in liver
mitochondrial extracts from rat embryos (E18), neonates
(1–6 h after birth, P0) and adult animals (3 months old, A).
Figure 3C shows that aralar1 levels decreased dramatically
after birth, from about 3.9 at E18 to 2.3 in P0 and 0.9 in
adults (numbers correspond to the aralar1 signal standard-
ized to that of b-F
1
ATPase; mean of two experiments). This
decrease matches the gradual loss of liver hematopoiesis at
the end of fetal life, when spleen and bone marrow become
the major hematopoietic organs [24]. In contrast, citrin
levels in liver mitochondrial extracts increased markedly
during postnatal development (Fig. 3C). Indeed, Iijima
et al. [18] found that citrin expression increases in liver just
before birth, in parallel to that of ASS and carbamoyl
phosphate synthetase, to provide full development of the
urea cycle early in postnatal life [26].
DISCUSSION
This study shows the mitochondrial expression levels of
aralar1 and citrin proteins in a large number of tissues and
organs. The distribution of aralar1 and citrin mRNAs has
been previously compared in different tissues and during
postnatal development [8,18]. Although the protein levels in
some tissues are consistent with their mRNA data, we have
obtained new information on the tissue distribution of the
AGCs which is of great interest in the search for specific
functions for these proteins. In particular, aralar1 is present
in many more tissues than suggested by its mRNA

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-

old rats (A). The blots were processed as described in the legend to Fig. 2B
5
.
3318 A. del Arco et al.(Eur. J. Biochem. 269) Ó FEBS 2002
two isoforms during early embryogenesis. At early stages of
embryonic development, the mRNAs of both isoforms were
localized particularly in actively growing structures (limb
and tail buds, apical ectodermal ridge, etc.) but with
different tissue distributions. Later, the two isoforms show a
widespread and dynamic expression pattern that does not
always reflect their final distribution in adult tissues. For
example, both aralar1 and citrin mRNAs are expressed in
the dermamyotome, from which skeletal muscle will
originate, but there is progressive loss of citrin expression
throughout embryogenesis (compare Fig. 1K with 1k), and
it is finally absent from adult skeletal muscle. Similarly, both
aralar1 and citrin are expressed in the central and peripheral
nervous system at E18, but only aralar1 is observed in the
adult brain. In contrast, only aralar1 was expressed in
the developing heart, but both AGC isoforms are present in
the adult rat tissue (Fig. 2). Interestingly, however, the two
isoforms are differentially distributed, and aralar1 is more
abundant in the atrial myocardium.
Overall, the substantial overlap in the distribution of the
two AGC isoforms in early embryos suggests a redundancy
of function and may explain why CTLN2 patients with non-
functional citrin protein do not suffer from major develop-
mental symptoms. The finding that most adult tissues
express aralar1, with the notable exception of liver hepato-
cytes, contrasts with previous indications that aralar1

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

´
utica Bayer, S.A., and by an institutional
grant from the Fundacio
´
nRamo
´
n Areces to the Centro de Biologı
´
a
Molecular ÔSevero OchoaÕ. We thank Dr Isabel Valverde for providing
the extracts of b islets and Professor J. M. Cuezva for the gift of
antibodies to b-F
1
ATPase. We also thank Dr Alberto Martı
´
nez-
Serrano for critical reading of the manuscript.
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