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Plant Physiol. (1999) 120: 841-848
Purification and Characterization of the Reconstitutively Active
Citrate Carrier from Maize Mitochondria1
Giuseppe Genchi,
Anna Spagnoletta,
Aurelio De Santis,
Luisa Stefanizzi, and
Ferdinando Palmieri*
Department of Pharmaco-Biology, Laboratory of Biochemistry and
Molecular Biology, University of Bari and Consiglio Nazionale delle
Ricerche Unit for the Study of Mitochondria and Bioenergetics, 70125 Bari, Italy (G.G., A.S., L.S., F.P.); Department of
Pharmaco-Biology, University of Calabria, 87030 Cosenza, Italy (G.G.); and Department of Evolutionistic and Experimental Biology, University
of Bologna, 40126 Bologna, Italy (A.D.S.)
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ABSTRACT |
The citrate carrier from maize
(Zea mays) shoot mitochondria was solubilized with
Triton X-100 and purified by sequential chromatography on
hydroxyapatite and hydroxyapatite/celite in the presence of
cardiolipin. SDS-gel electrophoresis of the purified fraction showed a
single polypeptide band with an apparent molecular mass of 31 kD. When
reconstituted into liposomes, the citrate carrier catalyzed a pyridoxal
5 -P-sensitive citrate/citrate exchange. It was purified 224-fold with
a recovery of 50% and a protein yield of 0.22% with respect to the
mitochondrial extract. In the reconstituted system the purified citrate
carrier catalyzed a first-order reaction of citrate/citrate (0.065 min 1) or citrate/malate exchange (0.075 min1). Among the various substrates and inhibitors
tested, the reconstituted protein transported citrate,
cis-aconitate, isocitrate, L-malate, succinate, malonate, glutarate,  -ketoglutarate, oxaloacetate, and
-ketoadipate and was inhibited by pyridoxal 5-P,
phenylisothiocyanate, mersalyl, and
p-hydroxymercuribenzoate (but not
N-ethylmaleimide), 1,2,3-benzentricarboxylate,
benzylmalonate, and butylmalonate. The activation energy of the
citrate/citrate exchange was 66.5 kJ/mol between 10°C and 35°C; the
half-saturation constant (Km) for citrate
was 0.65 ± 0.05 mM and the maximal rate
(Vmax) of the citrate/citrate exchange was
13.0 ± 1.0 µmol min1 mg1 protein at
25°C.
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INTRODUCTION |
Metabolite transport occurs in mitochondria via a series of
carrier proteins spanning the inner membrane (LaNoue and Schoolwerth, 1979 ; Day and Wiskich, 1984; Hanson, 1985; Heldt and Flügge, 1987; Douce and Neuburger, 1989). The main properties of all these carriers have been studied in intact mitochondria. However, essential for the identification of a transport protein and for its detailed functional and structural characterization are the purification and the
reconstitution of the purified protein in artificial membranes. To
date, six of the plant mitochondrial metabolite carriers have been
partially purified, reconstituted into liposomes, and kinetically studied, namely the dicarboxylate (Vivekananda et al., 1988), Glu/Asp
(Vivekananda and Oliver, 1989), monocarboxylate (Vivekananda and
Oliver, 1990), tricarboxylate (McIntosh and Oliver, 1992), and
phosphate (McIntosh and Oliver, 1994) carriers from pea seedlings and
the -ketoglutarate from maize (Zea mays) shoots (Genchi
et al., 1991). Only the ADP/ATP translocator has been purified to homogeneity from maize shoot mitochondria, characterized, and partially
sequenced (Genchi et al., 1996).
The citrate carrier, also known as the tricarboxylate carrier, is an
intrinsic protein of the inner mitochondrial membrane, which exchanges
cytoplasmic malate for citrate synthesized inside the mitochondrion.
The exported citrate is an important source of C skeleton for synthetic
processes, especially for Glu biosynthesis that takes place mainly in
the chloroplast compartment (Hanning and Heldt, 1993).
In this paper we describe the purification of the citrate carrier from
maize cv B 73 shoot mitochondria using functional reconstitution as a
monitor of carrier activity during isolation. Upon SDS-PAGE the
purified citrate transport protein appears to be a single polypeptide
with an apparent molecular mass of 31 kD. The functional properties, as
well as the basic kinetic data of the purified carrier incorporated
into liposomes, are also described.
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MATERIALS AND METHODS |
Plant Material and Chemicals
Maize (Zea mays L. cv B 73) kernels, obtained from
Maisadour (Mont De Marsan, France), were surface-sterilized for 5 min
in 1% (w/v) sodium hypochlorite and rinsed in distilled water. Seeds were allowed to imbibe in water at 25°C overnight, then they were sown on a layer of hydrophilic cotton in plastic boxes and covered by a
sheet of thin, wet paper. Seedlings were grown for 4 to 5 d in a
dark-controlled environmental chamber at 30°C and 95% RH before
harvesting. Hydroxyapatite (Bio-gel HTP) was obtained from Bio-Rad;
Triton X-100, celite 535, acrylamide, and
N,N-methylenebisacrylamide were obtained from
Serva; Dowex AG1-X8 (100-200 mesh), egg-yolk phospholipids (lecithin
from eggs), and Amberlite XAD-2 were obtained from Fluka;
[1,5-14C]citrate was obtained from Amersham;
cardiolipin was obtained from Avanti-Polar Lipids (Alabaster, AL); and
Sephadex G-75 was obtained from Pharmacia. All other reagents were of
the highest purity commercially available.
Isolation and Purification of Maize Mitochondria
Maize shoots were disrupted with a Braun mixer in 3 volumes of
ice-cold 0.4 M Suc, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1% (w/v) BSA, and 0.05% (w/v) Cys three times
for approximately 1 min each. The homogenate was filtered through a
layer of nylon sheet (80-µm pores; Saacilene 150T, Gaudenzi Tecnica
Industriale, Padova, Italy) and centrifuged for 20 min at
10,000g. The pellet was resuspended in a washing medium
containing 0.3 M Suc, 5 mM Tris-HCl, pH 7.2, and centrifuged for 5 min at 1,000g. The
decanted supernatant was layered onto a discontinuous Suc gradient, and purification of the mitochondria was carried out according to the
method of Douce et al. (1972), except that 5 mM
Tris-HCl (pH 7.2) was used instead of 10 mM
phosphate buffer in all of the purification steps. Purified
mitochondria were suspended at a concentration of 15 to 16 mg protein
mL1 washing medium (pH 7.2), frozen in liquid
N2, and stored at  80°C.
Purification of the Citrate Carrier
Maize shoot mitochondria were solubilized in 3% Triton X-100
(w/v), 20 mM
Na2SO4, 1 mM
EDTA, and 10 mM Pipes (1,4-piperazinediethanesulphonic acid), pH 7.0 (buffer A), at a final concentration of 15 mg protein mL1 buffer. After 10 min at 0°C, the mixture
was centrifuged at 105,000g per 15 min. The citrate carrier
was purified by hydroxyapatite and hydroxyapatite/celite chromatography
as follows: 225 µL of ultracentrifuged supernatant (Triton extract)
supplemented with cardiolipin (0.5 mg in 25 µL of buffer A) was
applied to a hydroxyapatite column (0.8 cm in diameter, containing
1.0 g of dry material) and eluted with 0.1% Triton X-100 and 10 mM Pipes, pH 7.0 (buffer B). The first 500 µL
was collected and 300 µL of this hydroxyapatite eluate was applied to
a hydroxyapatite:celite column (7:1; Pasteur pipettes with 300 mg of
dry material). The first 300 µL was collected eluting with buffer B. All of the operations were performed in a cold room at 4°C.
Reconstitution of the Citrate Carrier into Liposomes
Liposomes were prepared as described previously (Bisaccia et al.,
1985) by sonication of 100 mg/mL egg yolk phospholipids in water for 60 min. Protein eluates were reconstituted by removing the detergent with
a hydrophobic ion-exchange column (Palmieri et al., 1995). In this
procedure the mixed micelles containing detergent, protein, and
phospholipids were repeatedly passed through the same Amberlite XAD-2
column. The composition of the reconstitution mixture was: 200 µL of
eluates from the different columns or 20 µL of the Triton extract
plus 180 µL of buffer A; 90 µL of egg yolk phospholipids in the
form of sonicated liposomes; 90 µL of 10% Triton X-114; 20 mM citrate or other substrates, as indicated in the legends
to the tables and figures; 150 µL of 100 mM Pipes (pH
7.0) in the presence of 20 mM KCl in a final volume of 700 µL. After the mixture was vortexed, it was passed 15 times through the Amberlite column (0.5 × 3.6 cm) preequilibrated with a buffer containing 10 mM Pipes, pH 7.0, and 20 mM
concentration of the substrate present in the starting mixture. All of
the operations were performed at 4°C, except the passage through the
column, which was carried out at room temperature.
Transport Measurements
The external substrate was removed by passing 650 µL of the
proteoliposomal suspension through a Sephadex G-75 column (0.7 × 15 cm) preequilibrated with 50 mM NaCl and 10 mM Pipes, pH 7.0. The first 600 µL of turbid
proteoliposomal eluate was collected and distributed in reaction
vessels (180 µL each), incubated at 25°C for 4 min, and used for
transport measurements by the inhibitor stop method (Palmieri and
Klingenberg, 1979). Transport was initiated by adding 10 µL of
[14C]citrate at the final concentrations
indicated in the legends to the tables and figures, and after the
desired time interval, transport was stopped by adding 10 µL of 350 mM pyridoxal 5-P. In control samples, the inhibitor was
added together with the labeled substrate at time 0. The external
radioactivity was removed by passing 180 µL of each sample through an
anion-exchange column (Dowex AG1-X8, chloride form, 0.5 × 5 cm).
The liposomes eluted with 1 mL of 50 mM NaCl were collected
in 4 mL of scintillation mixture, vortexed, and counted. Transport
activities were calculated from the experimental values minus the
controls. For kinetic measurements, initial transport rates were
obtained by measuring transport within 1.5 min.
Other Methods
Polyacrylamide slab-gel electrophoresis of acetone-precipitated
samples was performed in the presence of 0.1% SDS according to the
method of Laemmli (1970). A minigel system was used: gel size was 8 cm × 10 cm × 1.5 mm (thickness). The stacking gel contained 5% acrylamide, and the separation gel contained 17.5% acrylamide with
an acrylamide/bisacrylamide ratio of 30:0.8 to give a high resolution
of polypeptides with a molecular mass close to 30 kD. Staining was
performed by the silver nitrate method (Morrissey, 1981). Protein was
determined by the Lowry method modified for the presence of Triton
(Dulley and Grieve, 1975).
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RESULTS |
Purification of the Citrate Carrier
Maize shoot mitochondria were solubilized in Triton X-100 in the
presence of cardiolipin and subjected to chromatography on hydroxyapatite followed by a second chromatography on
hydroxyapatite/celite (Table I). The
passage of the mitochondrial extract through hydroxyapatite led to a
substantial purification of the citrate carrier. About 95% of the
proteins present in the extract were bound to this resin. In the
hydroxyapatite eluate 51% of the total activity of reconstituted
citrate transport was recovered and the specific activity was increased
16-fold. For further purification, the hydroxyapatite pass-through was
subjected to chromatography on hydroxyapatite/celite (see ``Materials and Methods''). By this purification step, the specific activity of
reconstituted citrate transport was increased 14- and 224-fold with
respect to that of hydroxyapatite eluate and of mitochondrial extract,
respectively. Approximately 50% of the total transport activity was
recovered with a protein yield of 0.22%.
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Table I.
Purification of the citrate carrier from maize
mitochondria
The proteoliposomes were loaded with 20 mM citrate and the
exchange was started by the addition of 0.1 mM external
[14C]citrate.
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Figure 1 shows a SDS-PAGE of
hydroxyapatite pass-through (lane 2) and hydroxyapatite/celite eluate
(lane 3) obtained from maize mitochondria solubilized with Triton
X-100. Under the conditions described in "Materials and Methods,"
the eluate from hydroxyapatite contained five to six protein bands in
the region of 29 to 36 kD and several transport activities
corresponding to the citrate carrier, the ADP/ATP carrier, the
-ketoglutarate carrier, and porin (voltage-dependent anion channel
of the outer mitochondrial membrane). Figure 1, lane 3, shows that a
single protein band with an apparent molecular mass of 31 kD was eluted
from the hydroxyapatite/celite column.
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| Figure 1.
Purification of the citrate carrier from maize
mitochondria. Results of SDS-gel electrophoresis of fractions obtained
by hydroxyapatite and hydroxyapatite/celite columns of maize
mitochondria solubilized with Triton X-100 are shown. Lane M, Protein
markers (from top to bottom: BSA, carbonic anhydrase, and Cyt
c); lane 1, Triton X-100 mitochondrial extract (180 µg
in 25 µL); lane 2, hydroxyapatite eluate (20 µg in 100 µL); lane
3, hydroxyapatite/celite eluate (2 µg in 160 µL).
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Properties of the Reconstituted Citrate Carrier
In all of the following experiments, the reconstituted system
consists of purified protein eluted from the hydroxyapatite/celite column (Fig. 1, lane 3) and incorporated into liposomes. In Figure 2, the time course of 0.1 mM
[14C]citrate uptake in proteoliposomes loaded
either with citrate or malate is reported. Citrate uptake increased
linearly with time for about 3 min in citrate-loaded liposomes and for
about 2 min in malate-loaded liposomes. Furthermore, the total amount of citrate per milligram of protein taken up into the proteoliposomes was different in the two types of vesicles; it was 30% lower with malate-loaded liposomes. These differences can easily be rationalized taking into account the difference in the affinities of citrate and
malate to the carrier (see below). There was no activity without incorporation of the carrier protein or with incorporation of heat-denatured carrier protein (2 min at 100°C) into the liposomes, or in the absence of internal citrate or malate.
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| Figure 2.
Time course of the citrate/citrate and
citrate/malate exchanges in reconstituted liposomes.
[14C]citrate (0.1 mM) was added at zero time
to reconstituted liposomes containing 20 mM citrate ( )
or 20 mM malate ( ). The insets represent the logarithmic
plots of ln citratemax
(Cm)/(citratemax citratet)
(Ct), where citratemax is the maximum citrate
exchange per mg protein and citratet is the citrate
exchange per mg protein at time t, according to the
relation ln citratemax/(citratemax citratet) = kt. The amount of citrate
taken up after reaching equilibrium was measured after 90 min; it was
13,920 and 10,310 nmol/mg protein for the citrate/citrate and
citrate/malate exchanges, respectively.
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The reaction order of the citrate/citrate and citrate/malate exchanges
was investigated by plotting the natural logarithm of the fraction of
equilibrium
citratemax/(citratemax
citratet) against time. As shown in the insets of
Figure 2, straight lines were obtained, demonstrating that the two
exchange reactions follow a first-order kinetic. First-order rate
constants, k, of 0.065 min1 and
t1/2 of 10.0 min for the citrate/citrate
exchange, and of 0.075 min1 and
t1/2 of 7.5 min for the citrate/malate
exchange were calculated. The initial rates of citrate uptake evaluated
by multiplying the first-order constants by the total amounts
transported at equilibrium were 904 nmol min1
mg1 protein (for the citrate/citrate exchange)
and 773 nmol min1 mg1
protein (for the citrate/malate exchange), respectively.
The rate of citrate/citrate exchange is temperature dependent. In
an Arrhenius plot, a straight line was obtained in the range from
10°C to 35°C (results not shown). The activation energy as derived
from the slope was 66.5 kJ/mol.
The substrate specificity of [14C]citrate
uptake with respect to intraliposomal counteranions was investigated in
proteoliposomes loaded with a variety of substrates. The intraliposomal
concentration of the anions used was 20 mM and the exchange
time was 10 min. The data reported in Table
II show that 0.1 mM
[14C]citrate could be taken up in exchange with
citrate, cis-aconitate, isocitrate,
L-malate, malonate, and succinate. Surprisingly,
labeled citrate could also be exchanged for oxaloacetate,
-ketoglutarate, glutarate, and, to a lower extent, -ketoadipate
and adipate. In contrast, [14C]citrate was not
significantly taken up in exchange with trans-aconitate, oxalate, pimelate, and -ketopimelate or with the substrates of other
mitochondrial carriers, such as PEP (McIntosh and Oliver, 1992), Asp,
Asn, Glu, Gln, Lys, phosphate, pyruvate, and ADP.
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Table II.
Dependence of citrate transport in reconstituted
liposomes on internal substrates
The proteoliposomes were loaded with the indicated substrates.
Transport was initiated by adding 0.1 mM
[14C]citrate. The results are the means of three
experiments.
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The sensitivity of the reconstituted citrate/citrate
exchange to externally added substrates and inhibitors was also
investigated (Tables III and IV). The citrate/citrate exchange was
strongly inhibited by citrate, cis-aconitate,
L-malate, succinate, malonate, oxaloacetate,
-ketoglutarate, and less efficiently by isocitrate, glutarate, and
-ketoadipate. In contrast, trans-aconitate,
PEP, pyruvate, Glu, phosphate, and ADP had no effect (Table
III). In addition, the citrate/citrate
exchange was inhibited by the sulfydryl reagents mersalyl and
p-hydroxymercuribenzoate (but not
N-ethylmaleimide), as well as by the lysyl-specific reagents
pyridoxal 5-P and phenylisothiocyanate. The substrate analog
1,2,3-benzenetricarboxylate and the dicarboxylate analogs
benzylmalonate and butylmalonate also inhibited the reconstituted citrate/citrate exchange, although less efficiently than in animal mitochondria, in agreement with previous results (Jung and Laties, 1979; Birnberg et al., 1982). In contrast, atractyloside,
phenylglyoxal, and 1,2,4-benzenetricarboxylate had no significant
effect (Table IV).
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Table III.
Sensitivity of citrate exchange in reconstituted
liposomes to externally added substrates
The proteoliposomes were loaded with 20 mM citrate and the
exchange was started by adding 0.1 mM
[14C]citrate. The external substrates were added together
with [14C]citrate. The data are the means of three
experiments. The control value of uninhibited citrate exchange was 6090 nmol 10 min1 mg1 protein.
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Table IV.
Sensitivity of citrate exchange in reconstituted
liposomes to inhibitors
The proteoliposomes were loaded with 20 mM citrate and the
exchange was started by adding 0.1 mM
[14C]citrate. The inhibitors were added together with
[14C]citrate except the SH reagents, which were added 2 min before the labeled substrate. The data are the means of three
experiments. The control value of uninhibited citrate exchange was 6127 nmol 10 min1 mg1 protein.
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In other experiments the ability of the citrate carrier to transport
-ketoglutarate and oxaloacetate in reconstituted liposomes was
further investigated. Figure 3A shows the
effect of adding 2.0 mM unlabeled -ketoglutarate to
liposomes that had been incubated with 0.1 mM
[14C]citrate in the presence of 10 mM internal cold citrate. The unlabeled -ketoglutarate
was added after a 90-min incubation, when the
[14C]citrate taken up by the proteoliposomes
had approached equilibrium. The addition of -ketoglutarate caused an
extensive efflux of the intraliposomal
[14C]citrate, indicating that the radioactive
citrate, taken up by the citrate/citrate homoexchange, is released by
exchange for externally added -ketoglutarate. Similar results were
obtained by using oxaloacetate instead of -ketoglutarate (Fig. 3B),
indicating that an exchange between citrate and oxaloacetate had
occurred. In contrast to -ketoglutarate and oxaloacetate, Glu and
Asp did not cause any efflux of intraliposomal
[14C]citrate from
[14C]citrate-loaded liposomes (Fig. 3, A and
B). As shown in Figure 3C, the addition of 2.0 mM citrate
or cis-aconitate to proteoliposomes incubated with 0.1 mM [14C]-ketoglutarate
(in the presence of 10 mM internal
-ketoglutarate) caused an extensive efflux of radiolabeled
-ketoglutarate. In contrast, trans-aconitate, i.e. a
tricarboxylate that is not transported by the reconstituted protein,
had virtually no effect on the intraliposomal [14C]-ketoglutarate content. That the
citrate/-ketoglutarate and the citrate/oxaloacetate exchanges were
mediated by the purified and reconstituted citrate carrier protein is
demonstrated by the inhibition of these exchanges by the same
inhibitors that inhibit the citrate/citrate exchange (data not shown).
Furthermore, there was no exchange between citrate and
-ketoglutarate (or oxaloacetate) when using proteoliposomes that had
been reconstituted with boiled citrate carrier protein (not shown).
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| Figure 3.
Substrate-induced efflux of
[14C]citrate or [14C]-ketoglutarate from
proteoliposomes prelabeled by carrier-mediated exchange.
[14C]citrate (0.1 mM) (A and B) or
[14C]-ketoglutarate (0.1 mM) (C) was added at time 0 to reconstituted liposomes containing 10 mM citrate (A and
B) or -ketoglutarate (C). Where indicated by the arrow, 2 mM nonradioactive -ketoglutarate or Glu (A), 2 mM nonradioactive oxaloacetate or Asp (B), or 2 mM nonradioactive citrate, cis-aconitate, or
trans-aconitate (C) was added. ( ), With no addition;
(), with -ketoglutarate; ( ), with Glu; ( ), with
oxaloacetate; ( ), with Asp; ( ), with citrate; ( ), with
cis-aconitate; (), with
trans-aconitate. Transport was stopped by adding
pyridoxal 5-P (see ``Materials and Methods'') after the indicated
time intervals.
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In additional experiments (not shown), we found that the purified
preparation of the 31-kD protein, as shown in Figure 1, lane 3, when
reconstituted into liposomes, did not catalyze the exchange reactions
ADP/ADP (adenine nucleotide carrier), malate/phosphate (dicarboxylate
carrier), Asp/Asp (Asp/Glu carrier), Glu/Glu (Glu carrier),
pyruvate/pyruvate (monocarboxylate carrier), and phosphate/phosphate (phosphate carrier). Thus, the purified citrate carrier is
obviously not contaminated by other mitochondrial carriers.
Km and Vmax Values
of Citrate Transport
To obtain the basic kinetic data of the citrate carrier from maize
shoot mitochondria the dependence of the exchange rate on substrate
concentration was studied by changing the concentration of externally
added [14C]citrate at a constant internal
concentration of 20 mM citrate. In 12 experiments of this
type an average of 0.65 ± 0.05 mM for the
Km and 13.0 ± 1.0 µmol
min1 mg1 protein for
the Vmax at 25°C were determined.
Inhibition by Substrates
The inhibition of the reconstituted citrate/citrate exchange by
various compounds was analyzed in the presence of different substrate
concentrations. -Ketoglutarate, cis-aconitate, succinate, and malate were all identified as competitive inhibitors, since they
were found to increase the apparent Km
without changing the Vmax of the citrate
exchange. The inhibition constants, Ki, are summarized in Table V.
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Table V.
Ki values for substrates competing with citrate for
the exchange reaction
The Ki values were calculated from double reciprocal plots of the rate
of citrate/citrate exchange versus substrate concentrations. 0.1 to 1.0 mM [14C]citrate was added to proteoliposomes
that contained 20 mM citrate and were incubated for 1 min
at 25°C. The competing anions were added simultaneously with
[14C]citrate at the appropriate concentrations.
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DISCUSSION |
The data presented in this study demonstrate that we were able to
isolate and purify a 31-kD protein from maize mitochondria that
catalyzes the transport of citrate. For purification we used a general
scheme applied in our laboratory for the isolation of other
mitochondrial carriers (Palmieri et al., 1995) with modifications of
several experimental conditions. The conclusion that the polypeptide of
31 kD that we have purified from maize mitochondria, is in fact the
citrate carrier is supported by the following evidence. Upon
reconstitution into liposomes, the purified carrier catalyzes a very
active [14C]citrate/citrate exchange.
Furthermore, the purified transporter exhibits a substrate specificity
(Tables II and III) and an inhibitor sensitivity (Table IV) that
partially resemble those observed for the citrate transport system in
animal and plant mitochondria (Bisaccia et al., 1989; Claeys and Azzi,
1989; McIntosh and Oliver, 1992). Besides citrate,
cis-aconitate, isocitrate, L-malate,
succinate, and malonate can be used as counter-anions. However, whereas
the citrate transporter partially purified from pea mitochondria
(McIntosh and Oliver, 1992) is insensitive to mersalyl, the maize
citrate carrier protein is inhibited by sulfydryl reagents (as is the mammalian carrier), suggesting that the latter contain an essential Cys residue. Furthermore, at variance with all previously characterized transport systems we found that the purified citrate antiporter from
maize mitochondria can also transport -ketoglutarate,
oxaloacetate, and -ketoadipate. These findings cannot be explained
by contamination of the citrate carrier with the -ketoglutarate
carrier (Genchi et al., 1991) and/or with the oxaloacetate carrier
(Ebbigausen et al., 1985) because the latter two transporters do not
catalyze citrate/-ketoglutarate, citrate/oxaloacetate, or
citrate/-ketoadipate exchanges (Ebbigausen et al., 1985; Genchi et
al., 1991). During the revision of this paper an interesting article
has appeared that describes an exchange of oxaloacetate with citrate,
malate, -ketoglutarate, succinate, and Asp in liposomes
reconstituted with Triton X-100-solubilized mitochondria from potato
(Hanning et al., 1999). In view of our results and the close similarity in the substrates transported (with the exception of Asp), it is likely
that the transport activities observed by Hanning et al. (1999) in
reconstituted mitochondrial extracts from potato are catalyzed by a
protein homologous to the citrate carrier that we purified from maize.
The citrate transporter protein may play an important role in etiolated
maize shoots under various physiological conditions. The citrate
exported from the mitochondria to the cytosol in exchange for
oxaloacetate can be cleaved by citrate lyase (Kaethner and ap Rees,
1985) to acetyl-CoA and oxaloacetate and used for fatty acid elongation
(Ohlrogge and Brause, 1995) and isoprenoid synthesis (McGarvey and
Croteau, 1995). The efflux of citrate, isocitrate, or -ketoglutarate
in exchange for malate or oxaloacetate may also be involved in other
metabolic processes, such as nitrate assimilation (Hanning and
Heldt, 1993) and amino acid biosynthesis, which require production of
-ketoglutarate in the cytosol. Thus, under these conditions citrate
and isocitrate exported from the mitochondria by the citrate
transporter can be converted to -ketoglutarate by the cytosolic
enzymes aconitase (Wendel et al., 1988) and isocitrate dehydrogenase,
and then to Glu (Chen and Gadal, 1990) by Gln synthetase and Glu
synthase system (Sukanya et al., 1994; Sakakibara et al., 1998). A
further significance for the citrate carrier protein from maize is the
possibility that it may transfer reducing equivalents from the
mitochondrial matrix to the cytosol by catalyzing a malate/oxaloacetate exchange. It should be noted that a malate-oxaloacetate shuttle has
been previously proposed in mammalian and plant mitochondria (Gimpel et
al., 1973; Passarella et al., 1977; Krömer and Heldt, 1991).
The purification and characterization of the reconstitutively active
citrate carrier from maize shoots represent important first steps
toward investigations of this carrier at a molecular level. No
N-terminal sequence was detected when samples of the intact carrier
protein were subjected to the Edman degradation (results not shown).
Therefore, it appears that the protein has a modified -amino group.
By using, in the first instance, complex mixtures of oligonucleotides
as primers with sequences based upon partial protein sequences of
fragments of the purified protein, we may be able to isolate clones
encoding the citrate carrier protein from a maize cDNA library.
Experiments in this respect are currently in progress in our
laboratory.
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FOOTNOTES |
1
This paper is dedicated to the memory of Prof.
Giacomino Randazzo.
*
Corresponding author; e-mail fpalm{at}farmbiol.uniba.it; fax
39-080-5442770.
Received December 9, 1998;
accepted March 28, 1999.
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Citrate transport in corn mitochondria.
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Abstract
Introduction