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Structural and Functional Characteristics of Two Sodium-coupled
Dicarboxylate Transporters (ceNaDC1 and ceNaDC2) from
Caenorhabditis elegans and Their Relevance to Life Span*
Received for publication, August 27, 2002, and in revised form, December 10, 2002
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M208763200
You-Jun Fei‡§, Katsuhisa Inoue‡, and Vadivel Ganapathy‡
¶
From the Departments of ‡Biochemistry and Molecular Biology, and
¶
Obstetrics and Gynecology,
Medical College of Georgia, Augusta, Georgia 30912
We have cloned and functionally characterized two
Na
ⴙ
-coupled dicarboxylate transporters, namely ceN
-
aDC1 and ceNaDC2, from Caenorhabditis elegans. These
two transporters show significant sequence homology
with the product of the Indy gene identified in Drosoph-
ila melanogaster and with the Na
ⴙ
-coupled dicarboxy
-
late transporters NaDC1 and NaDC3 identified in mam-
mals. In a mammalian cell heterologous expression
system, the cloned ceNaDC1 and ceNaDC2 mediate Na
ⴙ
-
coupled transport of various dicarboxylates. With succi-
nate as the substrate, ceNaDC1 exhibits much lower
affinity compared with ceNaDC2. Thus, ceNaDC1 and
ceNaDC2 correspond at the functional level to the mam-
malian NaDC1 and NaDC3, respectively. The nadc1 and
nadc2 genes are not expressed at the embryonic stage,
but the expression is detectable all through the early
larva stage to the adult stage. Tissue-specific expression
pattern studies using a reporter gene fusion approach in
transgenic C. elegans show that both genes are coex-
pressed in the intestinal tract, an organ responsible for
not only the digestion and absorption of nutrients but
also for the storage of energy in this organism. Inde-
pendent knockdown of the function of these two trans-
porters in C. elegans using the strategy of RNA interfer-
ence suggests that NaDC1 is not associated with the
regulation of average life span in this organism,
whereas the knockdown of NaDC2 function leads to a
significant increase in the average life span. Disruption
of the function of the high affinity Na
ⴙ
-coupled dicar
-
boxylate transporter NaDC2 in C. elegans may lead to
decreased availability of dicarboxylates for cellular pro-
duction of metabolic energy, thus creating a biological
state similar to that of caloric restriction, and conse-
quently leading to life span extension.
Two different Na
⫹
-coupled dicarboxylate transporters
(NaDC)
1
have been identified in mammalian tissues (1–7).
These are NaDC1 and NaDC3. NaDC1 is Na
⫹
-coupled, electro
-
genic, and exhibits low affinity for its dicarboxylate substrates.
The K
t
value (Michaelis-Menten constant) is in the range of
0.1– 4.0 m
M (1–4). This isoform is expressed primarily in the
brush border membrane of intestinal and renal epithelial cells.
The physiological function of NaDC1 is to absorb the interme-
diates of the citric acid cycle, such as citrate, succinate,
␣
-ke-
toglutarate, fumarate, and malate, in the intestine and kidney.
NaDC3 is also a Na
⫹
-coupled and electrogenic dicarboxylate
transporter, but it exhibits relatively higher affinity for its
substrates compared with NaDC1 (5–7). The K
t
value is in
micromolar range. The NaDC3 is expressed primarily in the
basolateral membrane of intestinal and renal epithelial cells.
However, it is also found in tissues such as liver, placenta, and
brain. NaDC3 in the kidney is involved in generating the driv-
ing force for the organic anion transporter OAT1 to facilitate
the active entry of organic anions into the tubular cells across
the basolateral membrane (8). In the brain, NaDC3 mediates
the cellular uptake of N-acetylaspartate, a process closely
linked to myelination (9). Therefore, the physiological functions
of the NaDCs may extend beyond the mediation of cellular
entry of citric acid cycle intermediates. Recently, we reported
on the molecular identification of the third member of this
family in mammals (10, 11). This transporter, known as Na
⫹
-
coupled citrate transporter (NaCT), mediates the cellular up-
take of citrate in a Na
⫹
-coupled manner.
In a recent study by Rogina et al. (12), a NaDC-like trans-
porter, coded by the Indy (for I am not dead yet) gene, has been
implicated in the regulation of life span in Drosophila. The
investigators of this study suggested that defects in one copy of
the Indy gene (heterozygosity) can lead to less efficient produc-
tion of cellular energy and that, as a consequence, the meta-
bolic profile of the fruit fly changes resulting in life span ex-
tension. The eating behavior of the organism is not altered,
however. The decreased generation of cellular energy due to
the heterozygous mutation in the Indy gene creates a biological
situation resembling that of caloric restriction, which in other
animal models leads to an extension of life span (13). Recently,
we have identified (14) the transport function of Drosophila
INDY. This transporter mediates the cellular uptake of a broad
spectrum of citric acid cycle intermediates in a Na
⫹
-independ
-
ent manner. These characteristics of drINDY have now been
confirmed independently by Knauf et al. (15).
Studies of life span extension are difficult, if not impossible,
to conduct in mammals, particularly in humans. But it is
relatively a simple task to monitor the mean and maximum life
span in other animal models such as Caenorhabditis elegans.A
number of features make C. elegans especially suitable for
studies of life span extension. This organism has a short life
span with a mean life span of ⬃15 days. In addition, there are
* This work was supported by National Institutes of Health Grants
DA10065 and HD33347 (to V. G.) and by an intramural grant from the
Medical College of Georgia Research Institute (to Y. J. F.). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. E-mail: yjfei@mail.
mcg.edu.
1
The abbreviations used are: NaDC, Na
⫹
-coupled dicarboxylate
transporters; NaCT, Na
⫹
-coupled citrate transporter; ceNaDC, C. el
-
egans NaDC; hNaDC, human NaDC; hNaCT, human NaCT; drINDY,
Drosophila INDY; RT, reverse transcriptase; GFP, green fluorescent
protein; HRPE, human retinal pigment epithelial; NMDG, N-methyl-
D-glucamine; dsRNA, double-stranded RNA; YFP, yellow fluorescent
protein; CFP, cyan fluorescent protein; RNAi, RNA interference.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 8, Issue of February 21, pp. 6136 –6144, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
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techniques available to silence genes in this organism as a
means of assessing the role of specific genes in the maintenance
of life span. Therefore, with an aim to investigate the potential
role of NaDC family in life span, we set out to clone the
C. elegans counterparts of mammalian NaDCs and subse-
quently to monitor the influence of these transporters on life
span by using the RNAi technique to down-regulate their func-
tion. These studies have successfully led to the molecular and
functional identification of two different Na
⫹
-coupled dicar
-
boxylate transporters (ceNaDC1 and ceNaDC2) analogous to
the mammalian NaDC1 and NaDC3. In addition, studies of the
influence of these two transporters on life span have shown
that disruption of the function of the high affinity transporter
ceNaDC2, but not that of the low affinity transporter ceNaDC1,
leads to a significant extension of the average life span in
C. elegans.
EXPERIMENTAL PROCEDURES
Nematode Culture— A wild type nematode strain, C. elegans N2
(Bristol-Myers Squibb Co.), was obtained from the Caenorhabditis Ge-
netics Center (St. Paul, MN). Nematode culture was carried out using a
standard procedure with a large scale liquid cultivation protocol (16–
19). The nematodes were cleaned by sedimentation through 15% (w/v)
Ficoll 400 in 0.1 M NaCl. The pellet was then used for total RNA
preparation.
Extraction and Purification of Poly(A)
⫹
RNA—Total RNA was iso
-
lated using the TRIzol reagent (Invitrogen). Poly(A)
⫹
mRNA was puri
-
fied by affinity chromatography using oligo(dT)-cellulose.
Reverse Transcription (RT)-PCR and Hybridization Probe Prepara-
tion—A pair of PCR primers specific for the putative C. elegans nadc1
gene was designed based on the sequence of the cosmid F31F6.6 (Gen-
Bank
TM
accession number Z69884), 5⬘-GCC TCC AAG CAA AAT GTC
TC-3⬘ (forward primer) and 5⬘-CTA ACG CAA ATC CAC CTC C-3⬘
(reverse primer). A second pair of PCR primers specific for putative C.
elegans nadc2 gene was designed based on the sequence of the cosmid
K08E5.2 (GenBank
TM
accession number Z30974), 5⬘-TCA TCC TTC
CAA CAC CAT CC-3⬘ (forward primer) and 5⬘-ACC ATT CCA CTT CCA
AAC AC-3⬘ (reverse primer). Poly(A)
⫹
RNA (⬃0.5
g) isolated from
mixed stage C. elegans was taken as template to perform RT-PCR using
an RT-PCR kit from PerkinElmer Life Sciences. A single RT-PCR
product was obtained with an estimated size of ⬃1.0 and ⬃0.9 kb for the
cenadc1 and the cenadc2 genes, respectively, as predicted by the dis-
tance between the two primers in each pair. The RT-PCR products were
gel-purified and subcloned into pGEM-T Easy Vector (Promega, Madi-
son, WI). The molecular identity of the inserts was established by
sequencing. These cDNA fragments were used as probes to screen a
C. elegans cDNA library.
Construction of a Directional C. elegans cDNA Library—The Super-
Script Plasmid System from Invitrogen was used to establish the cDNA
library using the poly(A)
⫹
RNA from C. elegans. The transformation of
the ligated cDNA into Escherichia coli was performed by electropora-
tion using ElectroMAX DH10B competent cells. The bacteria plating,
the filter lifting, the DNA fragment labeling, and the hybridization
methods followed the routine procedure (20). The DNA sequencing of
the full-length ceNaDC1 cDNA and ceNaDC2 cDNA clones was per-
formed using an automated PerkinElmer Life Sciences Applied Biosys-
tems 377 Prism DNA sequencer and the Taq DyeDeoxy terminator
cycle sequencing protocol.
Vaccinia/T7 Expression System—Functional expression of the ceN-
aDC cDNAs in mammalian cells was done in human retinal pigment
epithelial (HRPE) cells using the vaccinia virus expression system as
described previously (5, 7, 9). HRPE cells grown in 24-well plates were
infected with a recombinant vaccinia virus (VTF
7–3
) at a multiplicity of
10 plaque-forming units/cell. The virus was allowed to adsorb for 30 min
at 37 °C with gentle shaking of the plate. Cells were then transfected
with the plasmid DNA (empty vector pSPORT or ceNaDC1 cDNA or
ceNaDC2 cDNA constructs) using the lipofection procedure (Invitro-
gen). The cells were incubated at 37 °C for 12 h and then used for
determination of transport activity. Cells transfected with pSPORT
alone without the cDNA insert were used as the control to determine
endogenous transport activity in these cells. [
3
H]Succinate uptake was
determined at 37 °C as described previously (5, 7, 9). In most experi-
ments, the uptake medium was 25 m
M Hepes/Tris (pH 7.5), containing
140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl
2
, 0.8 mM MgSO
4
,and5mM
glucose. In experiments in which the cation and anion dependence of
the transport process was investigated, NaCl was replaced iso-osmoti-
cally by LiCl, KCl, sodium gluconate, or N-methyl-
D-glucamine
(NMDG) chloride. The transport activity in cDNA-transfected cells was
adjusted for the endogenous activity to calculate the ceNaDC
cDNA-specific activity. The endogenous succinate transport activity in
vector-transfected cells was always less than 10% compared with the
succinate transport activity measured in cells transfected with either
ceNaDC1 cDNA or ceNaDC2 cDNA. Experiments were done in tripli-
cate, and each experiment was repeated at least three times. Results
are presented as means ⫾ S.E.
Semi-quantitative RT-PCR—An RT-PCR assay with the cenadc1-or
cenadc2-specific primers described above was used to study the devel-
opmental stage-specific expression pattern of ceNaDC1 mRNA and
ceNaDC2 mRNA. A Quantum RNA 18 S internal standard (Ambion,
Austin, TX) was used for the semi-quantitative RT-PCR. Total RNA
(⬃1.0
g) isolated from different developmental stages of C. elegans
(embryo, early larva, late larva, and adult) was taken as template to
perform reverse transcription using an RT-PCR kit from PerkinElmer
Life Sciences. The reverse transcription was initiated with random
hexamers and carried out in a DNA thermal cycler (GeneAmp PCR
System 9600) and thin-walled reaction tubes (PerkinElmer Life Sci-
ences) at 42 °C for 60 min, followed by incubation at 99 °C for 5 min to
inactivate the reverse transcriptase (Maloney murine leukemia virus-
reverse transcriptase). Reverse transcription was followed by PCR in a
multiplex format, in which the gene-specific primers and the primers
for the internal control (18 S rRNA) with their competimers were com-
bined at a predefined ratio. The PCR cycle number was titrated accord-
ing to the manufacturer’s protocol to ensure that the reaction was
within the linear range. A competimer was included to prevent the
highly abundant rRNA from being overwhelmingly amplified during
the reaction, and an optimal 18 S primer:competimer ratio was also
pre-established by trial and error. The resultant multiplex PCR prod-
ucts were resolved in an 1.0% agarose gel, and the intensity of the
gene-specific and the 18 S rRNA-specific bands was determined using
SpectraImager 5000 Imaging system and AlphaEase 32-bit software
(Alpha Innotech, San Leandro, CA). The steady state levels of ceNaDC1
mRNA and ceNaDC2 mRNA at different developmental stages were
assessed from the relative ratios of the intensity of the ceNaDC1-
specific RT-PCR product or ceNaDC2-specific RT-PCR product to the
intensity of the 18 S rRNA-specific RT-PCR product at each of these
stages.
Analysis of Tissue-specific Expression Pattern of cenadc1 and cen-
adc2—To study the tissue-specific expression pattern of the nadc1 and
nadc2 genes in C. elegans, transcriptional cenadc1::gfp and
cenadc2::gfp fusion genes were constructed, and transgenic animals
expressing these transgenes were developed. The expression pattern of
the cenadc1 and cenadc2 genes was investigated in live transgenic
animals based on the expression pattern of the GFP reporter. A pair of
primers for construction of a transcriptional cenadc1::gfp fusion gene
was designed to amplify the cenadc1 promoter. The forward primer,
5⬘-CGC GTC GAC GCT TAC ATC ATT CTT GTA TTT TTC-3⬘, corre-
sponds to the nucleotide positions 28,630 –28,659 of the cosmid F31F6
(GenBank
TM
accession number Z69884). The primer contains an incor
-
porated SalI restriction site at its 5⬘ end. The reverse primer, 5⬘-ATA
GGA TCC ATG ATT GGA GGC TCT GCA ATA CTA-3⬘, corresponds to
the nucleotide positions 29,797–29,772 in the same cosmid. A BamHI
site was incorporated in this primer at the 5⬘ end. The SalI and BamHI
sites were introduced into these primers for subsequent cloning into a
GFP expression vector. An ⬃1.2-kb DNA fragment of the cenadc1
promoter was amplified using the cosmid F31F6 DNA as template.
Similarly, a pair of primers for construction of a transcriptional
cenadc2::gfp fusion gene was also designed. The forward primer, 5⬘-
GTC GAC AAA ATA TGT ATT AGC CAC ATA AAA CCC-3⬘, corre-
sponds to the nucleotide positions 13,966–13,998 of the cosmid K08E5
(GenBank
TM
accession number Z30974). The reverse primer, 5⬘-GGA
TCC ATT TTC CGC ACA TGC CGA ATT TGC AT-3⬘, corresponds to the
nucleotide positions 15,461–15,432 in the same cosmid. A SalI site and
a BamHI site were incorporated in these primers for subsequent sub-
cloning purposes. A ⬃1.5-kb DNA fragment of the cenadc2 promoter
was amplified using the cosmid K08E5 as template. The PCR products
were digested with SalI and BamHI and inserted into a GFP expression
vector pPD117.01 (a generous gift from Dr. A. Fire, Carnegie Institu-
tion, Baltimore, MD) at a SalI/BamHI site. In these minigene con-
structs, a built-in mec7 promoter (⬃0.9 kb) in the expression vector was
replaced by the cenadc1 and cenadc2 promoter fragments in such a way
that the GFP transcription is under control of the putative promoters of
the cenadc1 and cenadc2 genes, respectively. The minigene fusion con-
structs were verified by sequence analysis. Transgenic lines were es-
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tablished using a standard germ line transformation protocol (17, 18).
Syncytial gonad injection was carried out according to a standard pro-
cedure (18). For microinjection, a computerized injection system, Trans-
jector 5246 and Micromanipulator 5171 from Eppendorf (Hamburg,
Germany), and a Nikon Eclipse TE 300 inverted microscope with No-
marski differential interference contrast optics were used. A cloned
mutant collagen gene containing the rol-6 (plasmid pRF4, kindly pro-
vided by Dr. M. Koelle, Yale University School of Medicine, New Haven,
CT) was used as a dominant genetic marker for DNA transformation.
Coinjection of this dominant marker with the GFP fusion constructs
allowed progeny selection of the transformed animals by their “roller”
phenotype. The F1 rollers were picked up according to their character-
istic rolling behavior and cultured individually to establish transformed
lines. F2 rollers with extrachromosome arrays were selected for fluo-
rescence microscopy to determine the GFP expression pattern. Stable
transgenic lines were established by the
␥
-irradiation method from the
F2 rollers, and the background was cleaned up by several times of
outcross (17, 21).
Double Labeling Fluorescent Protein Expression System—Two mod-
ified versions of the Aequora victoria green fluorescent protein (GFP),
designated as CFP and YFP with cyan-shifted and yellow-shifted spec-
tra (22), respectively, were used to simultaneously follow the expression
patterns of ceNaDC1 and ceNaDC2 in C. elegans. For the construction
of the cenadc1 promoter-driven CFP and the cenadc2 promoter-driven
YFP expression vectors, the GFP coding region in the GFP-expression
vector cenadc1::gfp (pPD117) and cenadc2::gfp (pPD117) was substi-
tuted by the CFP and YFP coding regions, respectively. The CFP and
YFP coding regions (⬃950 bp) were obtained by an EcoRI/KpnI diges-
tion of the vectors L4666 (pPD133.58) and L4664 (pPD133.51), respec-
tively (kindly provided by Dr. A. Fire, Carnegie Institution, Baltimore,
MD). The cenadc1::cfp and cenadc2::yfp expression vectors were linear-
ized by SalI digestion and coinjected into the distal arms of the
C. elegans syncytial gonads as described earlier. The extrachromosome
arrays were used for fluorescence microscopy to compare the expression
pattern of CFP and YFP in the same transgenic animal. Epi-fluores-
cence microscopic analysis of the expression of CFP and YFP was
performed using an Axiophot microscope (Carl Zeiss, Thornwood, NY).
Excitation and emission filter settings are as follows: for CFP exami-
nation, excitation at 436 ⫾ 20 nm, dichroic 455 nm LP, and emission at
480 ⫾ 40 nm; for YFP examination, excitation at 500 ⫾ 20 nm, dichroic
515 nm LP, and emission at 535 ⫾ 30 nm LP (22). The filter sets were
purchased from Chroma Technology (Brattleboro, VT). The SPOT-
cooled CCD color digital camera (Diagnostic Instruments Inc., St. Ster-
ling Heights, MI) and its associated data acquisition software were used
to record the fluorescence images.
Bacteria-mediated RNA Interference (RNAi)— A fragment of the
coding region of ceNaDC1 cDNA was generated by PCR and subcloned
into a pGEM-T easy vector (Promega, Madison, WI). The DNA fragment
was released by EcoRI digestion and inserted into a “double T7” plasmid
(pPD129.36, a generous gift from Dr. A. Fire, Carnegie Institution,
Baltimore, MD) at an EcoRI site within the multiple cloning site. A host
strain DH5
␣
was used for the first transformation. Competent host
bacteria HT115 (DE3) (kindly provided by the Caenorhabditis Genetics
Center, St. Paul, MN)) expressing T7-RNA polymerase from an induc-
ible promoter was prepared using a standard CaCl
2
method (20). The
double T7 promoter-containing plasmid with the cenadc1 gene-specific
DNA fragment inserted between the two T7 promoter regions was
transformed into the competent HT115 (DE3) cells and plated onto
standard LB ⫹ tetracycline (12.5
g/ml) ⫹ ampicillin (100
g/ml)
plates. HT115 cells harboring the double-T7 plasmid were cultured and
induced to express dsRNA using 0.4 mM isopropyl-

-D-1-thiogalactopy-
ranoside at 37 °C for 4 h. The experimental worms were transferred
onto isopropyl-

-D-1-thiogalactopyranoside-containing nematode
growth medium plates with the induced bacteria HT115 lawn for bac-
teria feeding experiments. The empty vector pPD129 was processed
similarly for use as a negative control, and the bacteria harboring this
plasmid were used to feed the control worms. Sufficient quantities of
bacteria HT115 were seeded on the testing plates for the worms to
consume to prevent the worms from starving and to ensure that dsRNA
was always present in the testing plates during the entire experimental
period for the experimental worms. A similar experimental strategy
was used for ceNaDC2. To serve as a positive control for the bacteria-
mediated RNAi in the assessment of the influence of ceNaDC1 and
ceNaDC2 on life span, we monitored the influence of DAF-2 on life span
using an identical experimental approach. Homozygous daf-2
⫺/⫺
knockout in C. elegans is known to enhance the life span dramatically
(⬃2-fold) (23, 24). Therefore, if the knockdown of DAF-2 function by
bacteria-mediated RNAi in C. elegans doubles the life span, this can be
taken as a positive control for the validity of the experimental approach
to assess the role of ceNaDC1 and ceNaDC2 in life span. For this
purpose, we obtained an ⬃0.8-kb DNA fragment specific for C. elegans
DAF-2 by RT-PCR using the following primer pairs: 5⬘-CGAACAAAA-
CACATCACAGAC-3⬘ (forward primer) and 5⬘-TCCATCATTTCCATCA-
CAACC-3⬘ (reverse primer) using the nematode total RNA as the tem-
plate. This fragment was then subcloned into pGEM-T easy vector. The
fragment was then released from the vector by EcoRI digestion, and the
released insert was cloned into the double T7 plasmid pPD129.36 at the
EcoRI site at the multiple cloning region. Shuttling of this plasmid into
HT115 (DE3) bacteria, induction of double-stranded RNA, and feeding
of the worms with the bacteria were carried out as described earlier.
Life Span Measurement— Life span of age-synchronous nematodes
was determined at 20 °C. Eggs obtained from gravid hermaphrodites
using an alkaline hypochlorite treatment procedure (18) were dispensed
on nematode growth medium Petri dishes with bacteria lawn and
allowed to hatch. Worms were inspected every day until death and were
scored as dead when they were no longer able to move even in response
to prodding with a platinum-wire pick. Each day, dead worms were
removed from plates and the deaths were recorded. Experiments were
started with 60 worms for each RNAi treatment (10 per plate). The
worms were transferred to a new plate every day during the reproduc-
tive period and every 3 days afterward to avoid contamination by their
offspring. Worms that died from matricidal hatching (the bag-of-worms
phenotype) and the worms that crawled off the plates or burrowed into
the agar were replaced by spare worms. A backup reservoir plate of ⬃30
spare worms was started at the same time as the experimental worms
and was identically treated for this purpose. To avoid the influence of
any potential subjective judgment of the experimenter in identifying
the dead worms on the experimental outcome, the life span measure-
ment studies were repeated with the experimenter blinded with regard
to the identity of the individual experimental groups. Statistical anal-
ysis was performed using the Microsoft EXCEL 2000 analysis ToolPak.
Mean life spans from different groups were compared using the Stu-
dent’s t test assuming unequal population variances.
RESULTS
Molecular Cloning and Structural Characterization of ceN-
aDC1 and ceNaDC2—The cloned ceNaDC1 cDNA is 1,989 bp
long and contains a poly(A) tail (GenBank
TM
accession number
AY090484). The 5⬘- and 3⬘-untranslated regions of this cDNA
are 12 and 173 bp long, respectively. The ceNaDC1 protein,
deduced from the coding region of the cDNA, contains 582
amino acids (Fig. 1) with a predicted molecular mass of 64 kDa
and an isoelectric point of 6.64. The ceNaDC2 cDNA is 2,250 bp
long and contains a poly(A) tail (GenBank
TM
accession number
AY090485). The 5⬘- and 3⬘-untranslated regions of this cDNA
are 50 and 500 bp long, respectively. The ceNaDC2 protein,
deduced from the coding region of the cDNA, contains 566
amino acids (Fig. 1) with a predicted molecular mass of 62 kDa
and an isoelectric point of 7.69. According to the Kyte-Doolittle
plot with a 21-amino acid window size, ceNaDC1 as well as
ceNaDC2 possess 12 putative transmembrane domains.
Following a multiple protein sequence alignment of the two
ceNaDCs, the three members of the human NaDC family, and
the drINDY using the PILEUP and in combination with the
MOTIFS program in the GCG package, a sodium symporter
family signature motif was identified within these transporter
proteins (Fig. 1). A consensus pattern established for the sig-
nature sequence is as follows: (S)SX(2)FX(2)P(V)(G)X(3)NX-
(I)V, where the X denotes the flexible amino acid residues
preceding the number in parentheses, and the numerical value
indicates the permitted number of the flexible amino acid res-
idues in the consensus. This sodium symporter family is a
group of integral membrane proteins that mediate the cellular
uptake of a wide variety of molecules including di- or tricar-
boxylates and sulfate by a transport mechanism involving so-
dium cotransport (sodium symporters). They are grouped into a
single gene family on the basis of sequence and functional
similarities. This group consists of the following proteins: the
sodium/sulfate cotransporters and sodium/dicarboxylate co-
transporters identified in yeast, C. elegans, Drosophila, and
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mammals; the putative sulfur deprivation response regulator
(SAC1) from Chlamydomonas reinhardtii; and the hypothet-
ical protein YfbS from E. coli (25). These transporter proteins
usually consist of 430 – 620 amino acids. They are highly
hydrophobic and contain 11 or 12 putative transmembrane
regions. The highly conserved sodium symporter signature
motif is located in or near the penultimate transmembrane
domain.
The molecular identity of mammalian or C. elegans func-
tional counterpart of drINDY is not known at present. There-
fore, we compared the primary structure of ceNaDC1 and ce-
NaDC2 with that of drINDY and mammalian NaDC1, NaDC3,
and NaCT. With a pairwise comparison analysis, ceNaDC1 is
more closely related to drINDY (51% similarity and 37% iden-
tity) than ceNaDC2 (46% similarity and 35% identity). Simi-
larly, hNaDC1 and hNaCT are more closely related to drINDY
(52% similarity and 40% identity) than hNaDC3 (50% similar-
ity and 37% identity). However, the differences are small, and
it is difficult to conclude whether ceNaDC1 or ceNaDC2 is the
C. elegans functional counterpart of drINDY based on the
structural comparison. Similarly, this structural analysis does
not allow definitive conclusion with regard to the question of
whether NaDC1or NaDC3 is the mammalian functional coun-
terpart of drINDY. Structural comparison reveals that both
ceNaDCs have similar sequence homology with hNaDC1,
hNaDC3, and hNaCT. Thus, the sequence data have failed to
provide any useful hint with respect to the functional identity
of the two ceNaDCs.
The cenadc1 and cenadc2 genes are located on chromosomes
X and III, respectively. Both genes, excluding the unidentified
promoter region in respective genes, are ⬃3.5 kbp in size (C.
elegans data base, ACeDB, data version WS57). The presence of
10 exons in the cenadc1 gene and 8 exons in cenadc2 gene was
deduced by a comparison between the sequences of the cloned
cDNAs and the respective genes in the GenBank
TM
data base
(F31F6.6 and K08E5.2) from the nematode genome sequence
project. The structural organization of the cenadc1 and cenadc2
genes is shown in Fig. 2.
Functional Characterization of ceNaDC1 and ceNaDC2 Us-
ing a Heterologous Expression System—The functional analysis
of the cloned ceNaDCs was carried out by heterologous expres-
sion of the cDNAs in HRPE cells using the vaccinia virus
expression system (5, 7, 9). Cells transfected with vector alone
served as the control. The transport function was monitored by
the uptake of [
3
H]succinate. Initial studies on the time course
of uptake indicated that the uptake was linear at least up to 5
min. All subsequent studies were therefore carried out with a
2-min incubation. With a succinate concentration of 10
M and
in the presence of Na
⫹
, the uptake of succinate increased
12-fold in cells expressing ceNaDC1 compared with control
cells (Fig. 3A). Under similar conditions, the increase in succi-
nate uptake was 24-fold in the case of ceNaDC2. Thus, both
FIG.1. A multiple protein sequence comparison of the representative members in the NaDC superfamily. The software PILEUP
(version 10.2) in the GCG package (from the Genetic Computer Group Inc., Madison, WI) was used to establish this multiple sequence alignment.
Gaps are introduced to make an optimum alignment and are indicated by dots/dashes. The names of the transporters are indicated at the
beginning of the protein sequence. The sodium/symporter family signature sequence is highlighted by a box.
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ceNaDC1 and ceNaDC2 mediate the uptake of succinate in the
presence of Na
⫹
. The uptake via these two transporters was,
however, obligatorily dependent on the presence of Na
⫹
be
-
cause substitution of Na
⫹
with Li
⫹
,K
⫹
, or NMDG abolished
completely the cDNA-induced increase in succinate uptake.
There was no involvement of anions in the uptake process as
indicated by comparable uptake activities for both transporters
in the presence of NaCl or sodium gluconate (data not shown).
The substrate selectivity of the uptake process mediated by
ceNaDCs was then studied by competition analysis by moni-
toring the ability of various monocarboxylates and dicarboxy-
lates (5 m
M) to compete with succinate for the uptake process
(Fig. 3B). Uptake measurements were made in parallel in
vector-transfected cells and in cDNA-transfected cells, and
then the cDNA-specific uptake was calculated by subtracting
the uptake in vector-transfected cells from the uptake in
cDNA-transfected cells. Only the cDNA-specific uptake was
used in the analysis. Among the various dicarboxylates tested,
the ceNaDC1-mediated succinate uptake was inhibited mark-
edly by fumarate, malate,
␣
-ketoglutarate, dimethylsuccinate,
and N-acetylaspartate. In contrast to fumarate, its stereoiso-
mer maleate failed to compete with succinate for transport via
ceNaDC1. Malonate, a structural homolog of succinate, not
only failed to inhibit the uptake of succinate but actually
caused a significant stimulation of succinate uptake. The
monocarboxylates pyruvate, lactate, and

-hydroxybutyrate
caused only a minimal inhibition of succinate uptake.
The substrate selectivity of ceNaDC2 was more or less sim-
ilar to that of ceNaDC1. The uptake of succinate mediated by
ceNaDC2 was inhibited significantly by fumarate, malate,
␣
-ketoglutarate, dimethylsuccinate, and N-acetylaspartate,
whereas the monocarboxylates had only a minimal effect. How-
ever, there were some notable differences between ceNaDC1
and ceNaDC2. Maleate was able to inhibit ceNaDC2-mediated
succinate uptake, whereas ceNaDC1-mediated succinate up-
take was not affected. Malonate, which caused a significant
stimulation of succinate uptake via ceNaDC1, had minimal
effect on succinate uptake via ceNaDC2. Another notable fea-
ture was that fumarate and malate were much more potent in
inhibiting ceNaDC2-mediated succinate uptake than in inhib-
iting ceNaDC1-mediated succinate uptake, suggesting that
there may be significant differences in substrate affinities be-
tween the two transporters. But, interestingly the trend in the
inhibitory potency was opposite for dimethylsuccinate and N-
methylaspartate. These two dicarboxylate derivatives were
more potent in inhibiting ceNaDC1-mediated succinate uptake
than in inhibiting ceNaDC2-mediated succinate uptake.
The cDNA-specific succinate uptake was saturable for ceN-
aDC1 as well as for ceNaDC2, and the data conformed to the
Michaelis-Menten equation describing a single saturable sys-
tem (data not shown). The Michaelis-Menten constant (K
t
) was
0.73 ⫾ 0.05 m
M for ceNaDC1 and 60 ⫾ 9
M for ceNaDC2. Thus,
with succinate as the substrate, ceNaDC2 exhibits a 10-fold
greater affinity than ceINDY1. The competition studies sug-
gest that the same may be true for other dicarboxylate sub-
strates such as fumarate and malate. These data show that
FIG.2.Structure of cenadc1 and ce-
nadc2 genes. Exons are indicated by
filled boxes and numbered accordingly; in-
trons are shown by solid lines. The un-
translated regions in exons are indicated
by open boxes. The consensus polyadenyl-
ation signal AATAAA is also shown. Sizes
and positions of the exons and introns are
drawn to the exact scale.
FIG.3. A, ion dependence of ceNaDC-mediated succinate uptake in
HRPE cells. Uptake of 10
M succinate was measured in buffers con-
taining 140 m
M concentrations of sodium, lithium, potassium, or
NMDG (as chloride salts). Values represent means ⫾ S.E. for four
determinations. Uptake of 10
M succinate measured in the vector
(pSPORT)-transfected cells served as a control for endogenous uptake
activity. The uptake in cDNA-transfected cells is given as percent of
uptake in vector-transfected cells. B, substrate specificity of the ceN-
aDC-mediated uptake. Uptake of 10
M [
3
H]succinate was measured in
the absence or presence of potential inhibitors (5 m
M) in cells trans-
fected with vector alone, ceNaDC1 cDNA, or ceNaDC2 cDNA. The
cDNA-specific uptake was calculated by adjusting for the uptake in
vector-transfected cells. The cDNA-specific uptake in the absence of
inhibitors was taken as the control (100%), and the uptake in the
presence of inhibitors is given as percent of this control value.
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ceNaDC1 is a low affinity Na
⫹
/succinate cotransporter, and
ceNaDC2 is a high affinity Na
⫹
/succinate cotransporter. There
-
fore, these two C. elegans dicarboxylate transporters corre-
spond at the functional level to mammalian NaDC1 and
NaDC3, respectively. NaCT shows very little ability to trans-
port succinate and thus is not related to either ceNaDC1 or
ceNaDC2 in terms of transport function. Drosophila INDY does
have the ability to transport various dicarboxylate intermedi-
ates of citric acid cycle (14, 15). But the transport function is
not Na
⫹
-dependent. Furthermore, Drosophila INDY has a
much higher affinity for citrate, a tricarboxylate, than for di-
carboxylates (14).
The effect of Na
⫹
on the uptake of succinate was then inves
-
tigated by measuring the uptake in the presence of varying
concentrations of extracellular Na
⫹
in cells transfected with
either ceNaDC1 cDNA or ceNaDC2 cDNA. Again, the uptake
values were adjusted for the endogenous uptake activity meas-
ured under identical conditions in cells transfected with vector
alone. The concentration of Na
⫹
in the uptake medium was
varied from 0 to 140 m
M. The osmolality of the medium was
maintained by adding appropriate concentrations of NMDG
chloride as a substitute for NaCl. The relationship between the
cDNA-specific uptake and Na
⫹
concentration was sigmoidal for
both ceNaDC1 and ceNaDC2, suggesting the involvement of
multiple Na
⫹
ions per succinate molecule transported. The
uptake rates failed to reach saturation within the concentra-
tion range of Na
⫹
employed in these studies.
Developmental Stage-specific Expression Pattern of ceNaDC1
mRNA and ceNaDC2 mRNA—To monitor the relative expres-
sion levels of ceNaDC1 mRNA and ceNaDC2 mRNA during
different stages of C. elegans development, synchronized cul-
tures were obtained, and total RNA was isolated at each of the
following four stages of development: embryo, early larva (lar-
va stages 1 and 2), late larva (larva stages 3 and 4), and adult.
The steady state levels of mRNAs for ceNaDC1 and ceNaDC2
were then determined by semi-quantitative RT-PCR with 18 S
rRNA as an internal control for variations in RNA input into
RT-PCRs. The levels of ceNaDC1 mRNA and ceNaDC2 mRNA
were compared at different developmental stages based on
relative intensities of ceNaDC-specific RT-PCR products com-
pared with that of 18 S rRNA-specific RT-PCR product (Fig. 4).
ceNaDC1 mRNA expression was not detectable at the embryo
stage. Abundant expression of this mRNA was evident, how-
ever, at the early larva stage. There was a transient decrease in
ceNaDC1 mRNA levels at the late larva stage, but the levels
increased again during subsequent development into the adult
stage. The levels of ceNaDC1 mRNA as assessed by the relative
band intensities of RT-PCR products for ceNaDC1 and 18 S
rRNA at these four stages, namely embryo, early larva, late
larva, and adult, were 0, 0.87, 0.24, and 0.77. In the case of
ceNaDC2, the mRNA was below detectable levels in the em-
bryo, but the expression was easily detectable at the early larva
stage. The levels of mRNA reached the maximum at the late
larva stage. The relative mRNA levels for ceNaDC2 at the four
stages (embryo, early larva, late larva, and adult) were 0, 2.5,
4.2, and 3.7.
Tissue-specific Expression Pattern of cenadc1 and cenadc2
Genes—We first studied the tissue expression pattern of cen-
adc1 and cenadc2 genes in C. elegans using the transgenic GFP
fusion technique in which the transgene consisted of the cen-
FIG.4. Developmental stage-spe-
cific expression of ceNaDC1 and ceN-
aDC2. Following RT-PCR, 10
lofthe
products were separated in a 1.2% (w/v)
agarose gel to show the size and intensity
of the cenadc gene-specific and the 18 S
rRNA-specific fragments. A 1.0-kb DNA
marker (Invitrogen) was used as a molec-
ular mass standard. The upper bands
(⬃850 bp long) were derived from the ce-
nadc1 (upper panel) and cenadc2 (lower
panel) transcripts; the lower bands (⬃480
bp) were amplified from 18 S rRNA and
served as an internal control. The RNA
samples that served as templates were
prepared from different developmental
stages of C. elegans: embryos (Emb), early
larva stage (L1&2), late larva stage
(L3&4), and adults. In addition, RNA pre-
pared from a mixture of C. elegans at dif-
ferent developmental stages was also
used (Mix).
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adc1 promoter fused with GFP cDNA or the cenadc2 promoter
fused with GFP cDNA. In both cases, the expression of GFP is
controlled by the respective promoter. Thus, the expression
pattern of GFP would match the expression pattern of the
cenadc1 and cenadc2 genes because of the control of the expres-
sion of the GFP reporter by the respective gene-specific pro-
moters. With this technique, we found that GFP expression is
restricted to the intestinal tract whether the expression of GFP
is driven by the ceNaDC1 promoter or by the ceNaDC2 pro-
moter (Fig. 5, A and B), indicating that both cenadc1 and
cenadc2 genes are expressed in the intestinal tract. The expres-
sion pattern is evident from the early larva stage through the
adult stage (data not shown). The GFP fluorescence is detect-
able throughout the intestinal tract, starting from the pharynx
all the way through the anus. In the case of both promoters, the
expression level of GFP is significantly greater in the anterior
half of the intestine than in the posterior half.
Because both cenadc1 and cenadc2 are expressed in the same
tissue, we employed a double-labeling approach to verify the
coexpression pattern of the two genes. In this approach, we
used two different fluorescent protein reporters, each driven
independently by either cenadc1 promoter (CFP) or cenadc2
promoter (YFP). Transgenic animals were developed that ex-
pressed both of the reporter constructs. The expression of CFP
as well as YFP was then examined in the same transgenic
animal under a fluorescence microscope with different excita-
tion and emission filter settings. These experiments showed
that the cenadc2 promoter-controlled YFP and the cenadc1
promoter-controlled CFP were coexpressed in the intestinal
tract (Fig. 5, C and D). This expression pattern was confirmed
with at least 10 transgenic animals.
Influence of RNAi-mediated Knockdown of the Function of
ceNaDC1 and ceNaDC2 on Average Life Span—The knock-
down of the function of ceNaDC1 by feeding the wild type N2
worms on bacteria expressing the ceNaDC1-specific dsRNA did
not show any significant influence on average life span nor on
the maximal life span (Fig. 6). The average life span of these
worms was same as that of the worms fed on bacteria harboring
the empty vector pPD129 (pPD129 control, 15.3 days; ceNaDC1
knockdown, 14.8 days, p ⬎ 0.05). In contrast, the knockdown of
the function of ceNaDC2 by feeding the wild type N2 worms on
bacteria expressing the ceNaDC2-specific dsRNA enhanced
significantly (p ⬍ 0.0001) the average life span of the worms
(pPD129 control, 15.3 days; ceNaDC2 knockdown, 17.6 days).
The increase in average life span induced by ceNaDC2 knock-
down was 15%. We used DAF-2 knockdown as a positive control
in these experiments. Worms feeding on bacteria expressing
DAF-2-specific dsRNA exhibited an average life span of 30
days, showing that the knockdown of the function of DAF-2
doubles the average life span. This influence of DAF-2 knock-
down on life span is similar to the influence of homozygous
knockout of daf-2 gene function on life span (23, 24). This
attests to the validity of the experimental approach indicating
that the bacteria-mediated RNAi strategy is as effective as
homozygous knockout strategy.
DISCUSSION
We have described in this paper the cloning and functional
characterization of two transporters in C. elegans that mediate
the transport of several intermediates of the citric acid cycle.
Both transporters are Na
⫹
-coupled and exhibit broad substrate
specificity for dicarboxylates. They do not interact with mono-
carboxylates. At the functional level, these two transporters,
named ceNaDC1 and ceNaDC2, resemble the mammalian
Na
⫹
-coupled dicarboxylate transporters NaDC1 and NaDC3,
respectively.
Even though ceNaDC1 and ceNaDC2 generally resemble
NaDC1 and NaDC3, respectively, in terms of functional char-
acteristics, there is one important difference. This difference
relates to the interaction of these transporters with certain
derivatives of succinate such as dimethylsuccinate and
N-acetylaspartate. Dimethylsuccinate is considered to be a spe-
cific substrate for mammalian high affinity transporter NaDC3
(26, 27). The low affinity transporter NaDC1 does not tolerate
substitutions in the carbon backbone of succinate. Thus, di-
methylsuccinate and dimercaptosuccinate are recognized pref-
erentially by NaDC3. In contrast to the mammalian counter-
parts, it is ceNaDC1, the low affinity transporter in C. elegans,
that interacts with dimethylsuccinate with much higher affin-
ity compared with ceNaDC2. Interaction with N-acetylaspar-
FIG.5.Tissue-specific expression pattern of cenadc1 and cenadc2 genes. Expression of GFP was driven by the cenadc1 (A) and cenadc2
(B) gene promoters in stably transformed transgenic C. elegans. The insets A1 and B1 are the bright field images (low magnification) of the animals
for A and B, respectively. C, (YFP expression driven by the cenadc2 promoter) and D (CFP expression driven by the cenadc1 promoter) show the
expression pattern of YFP and CFP in the same animal. The inset C1/D1 is the bright field image of the same worm for both C and D.
Sodium-coupled Dicarboxylate Transporters in C. elegans6142
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tate also follows a similar pattern. In mammals, NaDC3 shows
high affinity for this succinate analog (9). In contrast, it is
ceNaDC1, not ceNaDC2, that shows high affinity for this
compound.
Na
⫹
-activation kinetics of succinate uptake mediated by ce
-
NaDC1 and ceNaDC2 shows that multiple Na
⫹
ions are in
-
volved in the transport mechanism. Succinate exists as a diva-
lent anion under the experimental conditions (i.e. pH 7.5), and
therefore the number of Na
⫹
ions involved per transport cycle
will determine whether or not the transport process is influ-
enced by membrane potential. However, the exact number of
Na
⫹
ions transported with succinate per transport cycle could
not be determined in the present studies because the activation
of succinate uptake by Na
⫹
did not saturate within the range of
Na
⫹
concentrations employed in the study. We tried to express
ceNaDC1 and ceNaDC2 in Xenopus laevis oocytes to evaluate
the electrogenic nature of these two transporters by using the
two-microelectrode voltage clamp method, but the transporters
were not functionally expressed in this heterologous system.
We do not know the reasons for the lack of expression. We are
currently trying different expression vectors for this purpose.
Successful expression of these transporters in X. laevis oocytes
may become essential to demonstrate unequivocally whether or
not ceNaDCs are electrogenic.
In mammals, the expression of NaDC1 is restricted primar-
ily to the intestine and kidney, whereas the expression of
NaDC3 is evident not only in the intestine and kidney but also
in the liver, brain, and placenta (26). Furthermore, NaDC1 and
NaDC3 exhibit differential distribution in the apical versus
basolateral membrane of the polarized cells in the intestine,
kidney, liver, and placenta. NaDC1 is localized to the apical
membrane of the intestinal and renal tubular cells. In contrast,
NaDC3 is localized to the basolateral membrane of the renal
tubular cells, sinusoidal membrane of the hepatocytes, and the
brush border membrane of the placental syncytiotrophoblast
(26). The physiological function of NaDC1 in the intestine and
kidney is to facilitate the absorption of exogenous dicarboxy-
lates in the intestine and the reabsorption of endogenous di-
carboxylates in the kidney. In the liver and placenta, NaDC3
may play a role in the cellular entry of circulating dicarboxy-
lates for subsequent metabolic utilization. Because these dicar-
boxylates are present in the circulation only in micromolar
concentrations, the high affinity transporter NaDC3 has obvi-
ous advantages over the low affinity transporter NaDC1 to
perform this function. In C. elegans, the low affinity trans-
porter NaDC1 as well as the high affinity transporter NaDC2
are expressed predominantly in the intestinal tract. The C.
elegans intestinal tract is a tubular structure made up of a
single layer of 20 donut-shaped cells (28). Unlike in mammals,
the intestinal tract in C. elegans performs a variety of functions
in addition to the digestion and absorption of dietary nutrients.
It is a primary site of synthesis and storage of fat as the energy
source, a function similar to that of liver and adipose tissue in
mammals. The cells of the intestinal tract in C. elegans are
polarized, with numerous microvilli on the luminal surface
analogous to the apical membrane of the enterocytes in mam-
mals. The basolateral membrane of the intestinal cells is in
contact with the pseudocoelomic space that is filled with fluid
that supplies nutrients to the rest of the cells in the body. In
this respect, there is a lot of similarity between the intestinal
tract in C. elegans and the liver and adipose tissue in mam-
mals. We have provided evidence in this paper in support of
coexpression of NaDC1 and NaDC2 in the cells of the intestinal
tract in C. elegans. It is not known, however, whether these two
transporters are distributed differentially in the apical versus
basolateral membrane of the intestinal cells.
The physiological functions of NaDC1 and NaDC2 in C.
elegans intestinal tract are not known. We used the RNAi
technique to silence the function of these two transporters to
evaluate their influence on life span in this organism. This
technique is very effective in silencing the function of any
specific protein as evidenced by the doubling of the average life
span by silencing the function of DAF-2. Homozygous muta-
tions in daf-2 gene lead to doubling of life span in C. elegans
(23, 24). Since RNAi-mediated targeting of daf-2 also doubles
the life span, we conclude that this technique is very effective
in silencing the function of any targeted gene. RNAi-mediated
interference of NaDC1 function does not have any noticeable
effect on the average life span as well as on the maximal life
span, whereas targeting NaDC2 by this approach results in a
significant increase in the average and maximal life span. We
speculate that NaDC2 is localized to the basolateral membrane
of the intestinal cells where it functions in the cellular entry of
endogenous dicarboxylates for subsequent metabolic utiliza-
tion and energy production. Interference with this function
leads to a metabolic state analogous to that of caloric restric-
tion, thus resulting in life span extension. It has been well
established in C. elegans that caloric restriction (29) or sup-
pression of metabolic energy production within the mitochon-
dria (30) is associated with a significant increase in life span.
FIG.6.Influence of the knockdown
of ceNaDC1 and ceNaDC2 on life
span in C. elegans. The knockdown of
ceNaDC1 and ceNaDC2 was carried out
by feeding the worms with bacteria pro-
ducing ceNaDC1- or ceNaDC2-specific
dsRNA. The knockdown of DAF-2 was
used as a positive control. The survival
curves were plotted according to the
Kaplan-Meier algorithm using Sigma Plot
(version 6.0, SPSS Inc., Chicago). These
curves show the survival probability of
the wild type animals at a given day after
hatching under the influence of the gene-
specific dsRNAs. Each group was from a
total of four experiments. The total num-
ber of worms in each group at the begin-
ning of the life span experiment was 240.
Sodium-coupled Dicarboxylate Transporters in C. elegans 6143
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It is interesting to note that Indy gene is expressed in Dro-
sophila in tissues such as the fat body, midgut, and oenocytes
(12, 15). The fat body in this organism is involved in the
metabolism and storage of major energy sources (fat, glycogen,
and protein). The metabolic functions of this organ are similar
to those of liver in mammals. The same is true with the intes-
tinal tract in C. elegans where NaDC2 expression is seen.
However, the transport characteristics of ceNaDC2 are very
different from those of Drosophila INDY even though disrup-
tion of NaDC2 function enhances life span in C. elegans as
disruption of INDY does in Drosophila. In addition to NaDC1
and NaDC2 reported in this paper, a recent search of the C.
elegans data base has revealed that there are three other genes
coding for putative transporters with structural similarity to
Drosophila INDY. Cloning and functional characterization of
these putative transporters will be required to establish the
molecular identity of the gene that is the C. elegans functional
counterpart of Drosophila INDY. The present studies have
clearly shown that NaDC2 is involved in the regulation of life
span in C. elegans, but it is likely that additional transporters
with NaDC2-like transport function may exist in this organism
and function in the regulation of life span.
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Sodium-coupled Dicarboxylate Transporters in C. elegans6144
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Ganapathy
You-Jun Fei, Katsuhisa Inoue and Vadivel
Relevance to Life Span
and TheirCaenorhabditis elegansfrom
Transporters (ceNaDC1 and ceNaDC2)
of Two Sodium-coupled Dicarboxylate
Structural and Functional Characteristics
FUNCTION AND BIOGENESIS:
MEMBRANE TRANSPORT STRUCTURE
doi: 10.1074/jbc.M208763200 originally published online December 11, 2002
2003, 278:6136-6144.J. Biol. Chem.
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