Characterization of a dCTP Transport Activity Reconstituted from
Human Mitochondria*
Edward G.
Bridges
,
Zaoli
Jiang
, and
Yung-chi
Cheng§
From the Department of Pharmacology, Yale University School of
Medicine, New Haven, Connecticut 06520
 |
ABSTRACT |
A protein fraction of mitochondria from human
acute lymphocytic leukemia cells, which could be reconstituted into
proteoliposomes to have dCTP transport activity, has been partially
purified by hydroxyapatite and blue Sepharose chromatography. The dCTP
transport activity in proteoliposomes was time-dependent
and could be activated by Ca2+ and to a lesser extent
by Mg2+. None of the other divalent cations tested could
activate the transport activity. The Km value of
dCTP in the presence of Ca2+ was shown to be 3 µM. dCDP but not dCMP or dCyd could inhibit the transport
activity. Other deoxynucleoside triphosphates could also inhibit the
uptake of dCTP with the potency dGTP = dATP > TTP. Although
ATP could competitively inhibit dCTP uptake with a
Ki value of 8 µM, the reconstituted
dCTP uptake activity was not sensitive to the ATP/ADP carrier inhibitor
atractyloside or the sulfhydryl reagent N-ethylmaleimide.
This suggests that the dCTP transport system studied is not the same as
the ATP/ADP carrier. In conclusion, these studies describe the first
functionally reconstituted mitochondrial carrier that displays an
efficient transport activity for dCTP.
 |
INTRODUCTION |
The inner membrane of mitochondria normally possesses very low
electrophoretic permeability to most molecules. However, through the
action of specific transport systems, the inner mitochondrial membrane
is selectively permeable to a number of co-factors, metabolites, and
nucleotides (1, 2). The AAC1
is the only mitochondrial nucleotide carrier successfully isolated and
reconstituted in an active state (3). The AAC is highly selective for
ADP and ATP, mediating the import of ADP from the cytoplasm and export
of matrix ATP. The ATP-Mg/Pi carrier is another nucleotide
transport activity that has been described in isolated mitochondria (4)
but has not been successfully isolated. Another member of the family of
anion transport proteins that reside within the mitochondrial inner
membrane is the phosphate carrier that catalyzes transport of inorganic
phosphate into the mitochondrial matrix, where the phosphate is
utilized for phosphorylating ADP to ATP (5).
The existence of a mechanism for mitochondrial dNTP uptake has been
suggested by DNA synthesis experiments using isolated mitochondria
(6-8). These studies indicated that exogenous dNTPs could be utilized
by isolated mitochondria to synthesize mitochondrial DNA. In addition,
based on previous studies from our laboratory, antiviral nucleoside
analog triphosphates in mitochondria appear to originate from the
cytoplasm (9). In these studies, the absence of
2',3'-dideoxycytidine-induced mitochondrial toxicity in cytoplasmic
dCyd kinase-deficient cells suggested that the cytoplasmic-formed
metabolite 2',3'-dideoxycytidine triphosphate is the nucleotide source
in the inhibition of mitochondrial DNA synthesis. Thus, the
2',3'-dideoxycytidine triphosphate metabolites inside mitochondria
appear to originate from the cytoplasm. However, the experimental
evidence supporting the existence of a dNTP carrier in intact
mitochondria is only circumstantial and not definitive as for other
mitochondrial metabolite carriers.
The final proof for the existence of a carrier protein is its isolation
and functional reconstitution. To that end, we demonstrate that upon
incorporation into lipid vesicles, partially purified mitochondrial
protein catalyzes dCTP uptake. This transport activity displays unique
substrate specificity and inhibitor sensitivity from that previously
described for mitochondrial anion transporters. To our knowledge, this
is the first report of a substantially purified preparation of this
class of mitochondrial carrier activity in functional form.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
Hydroxyapatite (Bio-Gel HPT) was purchased from
Bio-Rad. Blue Sepharose and Sephadex G-50 were from Amersham
Pharmacia Biotech.
-32P-Labeled nucleotides were
from Amersham Pharmacia Biotech. Soybean asolectin
(L-
-phosphatidylcholine), cholesterol, Triton X-114, Dowex 1 × 4 (100-200 mesh, chloride form), atractyloside, and nucleotides were from Sigma.
Preparation of [
-32P]dCTP--
Nucleoside
diphosphate kinase (from Baker's yeast; Sigma) was used to prepare
[
-32P]dCTP from dCDP as follows. A 1-ml mixture
containing 10 units of nucleoside diphosphate kinase, 2 mM
[
-32P]GTP (100 µCi), and 0.5 mM dCDP in
150 mM Tris acetate, pH 7.5, and 10 mM
MgCl2 was incubated at 37 °C for 10 min. The reaction was stopped by the addition of 3 volumes of ice-cold methanol and
incubated on ice for 15 min. After centrifugation at 14,000 × g for 10 min, the supernatant containing
[
32P]dCTP was evaporated to dryness and resuspended in
500 µl of water. The mixture containing dCDP, GDP,
[
-32P]GTP, and [
-32P]dCTP was
chromatographed by ion exchange HPLC using a Whatman Partisil-SAX
column (4.6 mm × 25 cm) at a flow rate of 1 ml/min. The
nucleotides were resolved with a gradient consisting of water to 30 mM buffer (potassium phosphate, pH 6.7) from 0 to 10 min, 30-150 mM buffer from 10 to 15 min, 150 mM
buffer from 15 to 50 min, and 300 mM buffer from 50 to 80 min. Peaks were identified by authentic standards, and the peak
corresponding to [
-32P]dCTP was collected. The
purified [
-32P]dCTP was diluted 5-fold with water,
placed over a DEAE-Sephadex A-25 column (2-ml bed volume), and washed
with 20 ml of water to remove the phosphate buffer, and the
[
-32P]dCTP was eluted with 10 ml of ammonium formate
(500 mM). The eluted sample was freeze-dried to remove the
ammonium formate and resuspended to a volume of 2 ml in water. The
purity of [
32P]dCTP was verified by ion exchange HPLC
as described above, and the radiospecificity was determined to be 90 µCi/mmol.
Preparation of Mitochondria and Partial Purification of Proteins
with dCTP Transport Activity--
Mitochondria were isolated from
acute lymphocytic leukemia cells after leukophoresis of a patient in
blast crisis and further purified on a discontinuous sucrose gradient
as described previously (9). Frozen mitochondria (100-150 mg) were
solubilized for 10 min on ice in 10 ml of a buffer containing 20 mM Hepes (pH 7.0), 1% Triton X-114, 150 mM
Na2SO4, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine. The detergent was separated
from the aqueous phase by centrifugation at 280,000 × g for 1 h at 4 °C. The upper aqueous phase was
decanted, and the detergent phase was resuspended to 10 ml in 20 mM Hepes (pH 7.0), 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine (buffer B). The detergent
extract was applied to an HPT column (1 g of dry HPT/20 mg of protein) connected in tandem with a blue Sepharose column (1 ml of Blue Sepharose/2 mg of protein) equilibrated with buffer B. The unbound protein-containing fraction was eluted from the tandem columns with
buffer B.
Preparation of Proteoliposomes--
Asolectin was further
purified as described (10). For use in transport studies, cholesterol
(120 mg/ml final concentration) was added to the asolectin (200 mg/ml
final concentration). Cholesterol has been reported to prevent
protein-mediated leakage in reconstituted proteoliposomes (11). The
asolectin was stored in chloroform under nitrogen in a light proof
container at
20 °C. Immediately before use, an aliquot of
asolectin was dried under a stream of nitrogen. Following the addition
of 2 ml of 20 mM Hepes (pH 7.0), the lipid was
dispersed by sonication (Branson Sonifier 250; microtip output
control = 4; 80% duty cycle; on ice) until the mix appeared transparent.
Aliquots of freshly isolated protein fractions (typically 0.3 mg) were
mixed with lipid vesicles (55 mg) to a final volume of 1 ml. This
mixture was vortexed and then rapidly frozen in liquid nitrogen. For
ATP/ADP exchange reactions and dCTP efflux experiments, proteoliposomes
were prepared in the presence of 100 µM ADP and 20 µM [
-32P]dCTP, respectively. These
proteoliposomes could be stored at
80 °C up to 5 days without
appreciable loss of dCTP transport activity. Immediately prior to
assay, the samples were freeze-thawed twice (total of three cycles)
using liquid nitrogen and thawed a final time in an ice-water bath
followed by sonication (Branson Sonifier 250; microtip output
control = 2; 70% duty cycle; 30 burst; total sonication time = 25 s; on ice). For ATP/ADP exchange reactions and dCTP efflux
experiments, external ADP and [
-32P]dCTP,
respectively, were removed by passing the proteoliposomes over an ion
exchange column (Dowex; 1 × 3.5 cm) equlibrated with 20 mM Hepes (pH 7.0) at 4 °C. The first 1 ml of the turbid
eluate from the Dowex column was collected and then used for transport experiments. A heterogeneous population of large, primarily unilamellar proteoliposomes suitable for transport studies are generated by this
general freeze-thaw-sonicate procedure (12-15).
Assay Conditions for dCTP Uptake--
All measurements of
nucleotide uptake into reconstituted proteoliposomes were carried out
at 37 °C. Transport was initiated by the addition of 4 µl of
radiolabeled substrate (typically [
-32P]dCTP, 10 µM final concentration, 30 Ci/mmol) to 190 µl of
proteoliposomes in a final volume of 200 µl. For competition assays,
competitor was added to the desired concentration to the proteoliposome
mix before the radiolabeled substrate. After the desired incubation time, the reaction mixture was placed over a Dowex ion exchange column
(1 × 3.5 cm) equilibrated with 20 mM Hepes (pH 7.0)
at 4 °C in order to remove the external radioactivity not
transported into proteoliposomes. The liposomes were eluted with 4 ml
of ice-cold equilibration buffer, collected in 16 ml of scintillation
fluid, vortexed, and counted. Subtracting values obtained when a
100-fold molar excess of nonradiolabeled substrate was added 2 min
prior to the radiolabeled substrate made corrections for nonspecific uptake of radioactivity. For efflux experiments, external radioactivity was removed by passing the samples through a Dowex column and collecting and counting the liposomes as described above. The transport
activity was calculated by subtracting the experimental values from the
control values (efflux at 4 °C).
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RESULTS AND DISCUSSION |
Mitochondria possess a variety of specific carrier systems for the
transport of metabolites across the inner mitochondria membrane.
Because of the importance of mitochondrial toxicity in nucleoside
analog chemotherapy (for a review, see Ref. 16), we have sought to
extend our knowledge about dCTP uptake into mitochondria. Our procedure
for isolation and reconstitution of active dCTP transport from human
mitochondria consisted of three basic steps. First, sucrose gradient
isolated mitochondria were extracted with Triton X-114. Second,
hydrophobic membrane proteins in the detergent were separated from the
aqueous phase. Finally, the detergent phase containing solubilized
membrane proteins was chromatographed on HPT and blue Sepharose. When
applying these conditions to mitochondria from acute lymphocytic
leukemia cells, the activity of dCTP transport was significantly
enriched, but it remains a heterogeneous mixture of proteins.
In Table I the activity of the protein
obtained by blue Sepharose chromatography relative to that of the HPT
chromatography is reported. For comparison, we report also the data on
the total mitochondrial membrane extract. However, reconstitution of a
total membrane extract in liposomes and its comparison with the data obtained from a smaller number of proteins after chromatography should
be done with caution and may not be fully reliable because of
differences in protein-protein interactions, liposome size, and passive
permeabilities. Uptake of [
-32P]dCTP could be detected
in proteoliposomes reconstituted using crude mitochondrial protein
extracts and detergent phase membrane proteins with a specific activity
of 2.8 and 18 pmol/min/mg protein, respectively. However, only minimal
dCTP transport activity could be detected in proteoliposomes using
aqueous phase proteins after detergent phase separation. Approximately
180- and 90-fold increases in the specific activity of dCTP uptake in
proteoliposomes compared with crude extracts were obtained after HPT
and blue Sepharose chromatography, respectively. The decrease in
specific activity after blue Sepharose chromatography compared with HPT
elutes may indicate the presence of multiple dCTP transport activities
resolved by blue Sepharose. Alternatively, this result may suggest the removal of a protein(s) component of a multisubunit complex responsible for dCTP transport. Furthermore, there is a large increase in the total
activity recovered in the HPT eluate (i.e. 965%) relative to the starting material. The recovery of activity greater than the
starting material may reflect the removal of an inhibitor or
interfering activity. Further studies will be required to define the
role of these factors in the above observations.
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Table I
Partial purification of the dCTP carrier activity from human acute
lymphocytic leukemia mitochondria
Sucrose gradient isolated mitochondria were used as a starting
material. Protein fractions were incorporated into phospholipid
vesicles as described under "Experimental Procedures." Uptake was
started by the addition of 10 µM [ -32P]dCTP
to proteoliposomes in a buffer containing 20 mM Hepes (pH
7.0), 0.2 mM EDTA, and 2 mM CaCl2. The
dCTP uptake activity was measured for 1 min at 37 °C.
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Hydroxyapatite chromatography is important because it offers a large
single purification step common to mitochondrial anion transporters
(17-23). Under the conditions described under "Experimental Procedures," the reconstituted protein eluate after HPT
chromatography contained atractyloside-sensitive AAC activity in
addition to dCTP transport activity (data not shown). To improve the
purification of the dCTP carrier activity, the protein eluate after HPT
chromatography was applied to a blue Sepharose column to remove the AAC
activity (24). Table II shows that after
HPT and blue Sepharose chromatography the internalized ADP as a
counterion did not stimulate ATP uptake in reconstituted
proteoliposomes. This is in contrast to HPT protein eluates
reconstituted into proteoliposomes, where internalized ADP dramatically
increased ATP uptake (data not shown). Furthermore, ATP uptake in
proteoliposomes was observed after HPT and blue Sepharose
chromatography but was not sensitive to the AAC inhibitor atractyloside. These results demonstrate that the reconstituted protein
eluate after HPT and blue Sepharose chromatography is capable of ATP
transport, but this activity is not associated with AAC activity.
Further studies will be required to determine whether one or more
proteins are present that play a role in dCTP and ATP transport.
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Table II
ATP uptake in proteoliposomes
The protein eluate after HPT and blue Sepharose chromatography was
obtained and reconstituted as described under "Experimental
Procedures." For ATP/ADP exchange reactions, proteins were
reconstituted in the presence of 100 µM ADP. Uptake
reactions were initiated by the addition of 10 µM
[ -32P]ATP. dCTP was added 2 min prior to the addition of
radiolabeled substrate as indicated. Means and S.D. values from three
separate experiments are presented.
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The unbound protein fraction from HPT and blue Sepharose chromatography
was functionally characterized for dCTP transport activity after
incorporation into liposomes. To obtain a base line of transport
activity in the absence of divalent cations, the effect of EDTA on
transport using 10 µM [32P]dCTP in
reconstituted proteoliposomes was measured (Fig.
1A). A significant
dose-dependent decrease in dCTP uptake was observed with a
maximum effect at 0.2 mM EDTA. We next measured the effect of 1 mM divalent cations on dCTP uptake in the presence of
0.2 mM EDTA (Fig. 1B). Mn2+ and
Zn2+ had a negligible effect, while Cu2+ and
Co2+ inhibited dCTP uptake. However, Ca2+
dramatically increased dCTP uptake, whereas Mg2+ and
Ni2+ moderately increased uptake. In addition, NaCl and KCl
were observed to have no effect on dCTP uptake (data not shown). The
effects of Ca2+ and Mg2+ were further
investigated in Fig. 1C. A dose-dependent
increase in dCTP uptake was noted with Ca2+ that was
saturable at about 2 mM (an 8-fold increase), whereas the
effect of Mg2+ was less dramatic (a 2.5-fold increase).

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Fig. 1.
Optimization of dCTP uptake.
Proteoliposomes were prepared from the protein eluate after HPT and
blue Sepharose chromatography as described under "Experimental
Procedures." Uptake was initiated by the addition of 10 µM [ -32P]dCTP for 1 min. A
depicts the effect of EDTA on the uptake of dCTP by proteoliposomes in
the presence of 20 mM Hepes (pH 7.0). B
illustrates the effect of divalent cations on the dCTP carrier activity
in 20 mM Hepes (pH 7.0) and 0.2 mM EDTA.
Vesicles were incubated for 1 min with the indicated additions at a
concentration of 2 mM before initiating uptake. The control
value of dCTP uptake in the absence of divalent cations was 23 pmol/min/mg of protein. C illustrates the effect of
Mg2+ and Ca2+ on dCTP uptake. Vesicles were
incubated for 1 min with 20 mM Hepes (pH 7.0), 0.2 mM EDTA, and MgCl2 ( ) or CaCl2
( ) as indicated.
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What effect is Ca2+ having to stimulate dCTP transport
activity? The transport assay described in this study consisted of
several components that, in principle, could all be targets for the
cations: negatively charged phospholipids, the negatively charged
substrate dCTP, and the inserted carrier protein. Interaction of
Ca2+ with the anionic substrate dCTP is questionable
because of the large discrepancy in the actual concentrations used.
Interestingly, the affinity of Mg2+ for ATP has been
reported to be about 10 times higher than that of Ca2+
(25). If Mg2+ interacts with the triphosphate moiety of
dCTP similar to that of ATP to stimulate dCTP transport in
reconstituted proteoliposomes, the activation effect of
Mg2+ should be seen at lower concentrations. Second, the
results presented in this paper cannot simply be due to surface charge
screening of the phospholipid vesicles, since there are significant
differences with respect to the stimulation power between the various
cations tested. Finally, although the protein may be the most
reasonable choice as the target of the activation by Ca2+,
further studies will be required to address this issue.
The effect of protein concentration on the reconstitution of the dCTP
transport activity was investigated by progressively increasing the
protein concentration in the reconstitution mix while keeping the lipid
concentration constant (55 mg/ml). Increasing the protein concentration
from 0.05 to 0.4 mg/ml resulted in a parallel increase in the
transporter-mediated uptake of [32P]dCTP (Fig.
2A). There also was a slight
increase in the background rate of uptake with increasing protein
concentration (<2 pmol/min at 0.4 mg/ml protein), but this increase
was much less pronounced than the increase in the rate of
protein-mediated uptake. From these experiments, we selected 0.3 mg/ml
protein as an optimum condition to characterize dCTP uptake.

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Fig. 2.
Characterization of dCTP uptake.
Proteoliposomes were prepared from the protein eluate after HPT and
blue Sepharose chromatography as described under "Experimental
Procedures." Vesicles were incubated with [32P]dCTP in
the presence of 20 mM Hepes (pH 7.0), 2 mM
CaCl2, and 0.2 mM EDTA. A
illustrates the effect of protein concentration on the uptake of dCTP
by proteoliposomes. Uptake was initiated by the addition of
10 µM [ -32P]dCTP for 1 min. The protein
concentration of the initial reconstitution mixture was varied from
0.05 to 0.4 mg/ml. B illustrates the time- and
temperature-dependent uptake of [ -32P]dCTP
by proteoliposomes reconstituted with 0.3 mg/ml protein. Uptake was
initiated by the addition of 10 µM [ -32P]dCTP.
Vesicles were incubated for the indicated times at 37 °C ( ) or
4 °C ( ). Proteoliposomal uptake of [ -32P]dCTP is
plotted as pmol of [ -32P]dCTP accumulated per
milligram of proteoliposomal protein. C illustrates
the time-dependent efflux of dCTP in proteoliposomes
(reconstituted with 0.3 mg/ml protein) loaded with 20 µM
[ -32P]dCTP as described under "Experimental
Procedures." Transport was initiated by incubation at 37 °C.
D illustrates the time- and
temperature-dependent uptake of [ -32P]dCTP
by proteoliposomes reconstituted with 0.3 mg/ml protein. Uptake was
initiated by the addition of 20 µM
[ -32P]dCTP and the vesicles incubated for the
indicated times at 37 ( ) or 4 °C ( ).
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In Fig. 2B the dCTP uptake catalyzed by mitochondrial
protein reconstituted into liposomes has been plotted as a function of
time. There is an apparent initial rapid component occurring within 1 min and a slower uptake component that reached a steady state within
5-10 min. A temperature dependence in dCTP uptake is also observed
with negligible [32P]dCTP uptake when the proteoliposomes
are incubated at 4oC. Subsequent experiments were conducted
at 1 min to assess the initial uptake of dCTP in reconstituted
proteoliposomes. Because the insertion of protein in the lipid vesicle
is in a random orientation, unidirectional transport in both directions
may explain the time-dependent change in the transport
rate. To measure the efflux of dCTP, proteoliposomes were preloaded
with [
-32P]dCTP. Fig. 2C illustrates a
time-dependent efflux of [
-32P]dCTP from
proteoliposomes similar to that observed for influx. To directly
address if dCTP is transported without prior breakdown, dCTP was
radiolabeled with 32P in the
-position only and used for
uptake studies. Fig. 2D illustrates the time- and
temperature-dependent uptake of
[
-32P]dCTP. Under these conditions,
[
-32P]dCTP uptake decreases in the presence of excess
nonradiolabeled dCTP.2
However, a 10-fold molar excess of inorganic phosphate has no effect on
[
-32P]dCTP uptake, suggesting that protein-mediated
uptake of 32P-labeled inorganic phosphate (e.g.
the phosphate transporter) plays no role in the observed transport
activity.2 Thus, it is unlikely than dCTP is broken down
before uptake into proteoliposomes. In addition, the exchange of a
counterion (including dCDP or ADP) for dCTP in reconstituted
proteoliposomes, similar to that for ATP and ADP in the AAC nucleotide
transport system, has not been observed.2
We have thus far demonstrated the transport activity of reconstituted
mitochondrial protein by measuring the uptake of radioactive substrate
into proteoliposomes. With this assay method, the amount of substrate
taken up can be measured, but it is not possible to obtain precise
initial kinetic parameters because the radioactive substrate remaining
outside the proteoliposomes has to be removed before the radioactive
substrate transported inside the vesicles can be measured. Further
studies were performed to elucidate a dose-response curve for dCTP
uptake in proteoliposomes. The value obtained from a Lineweaver-Burk
plot (Fig. 3) indicated an apparent Km of 3 µM. The inhibition of dCTP
uptake in proteoliposomes by ATP was also studied. Fig. 3 demonstrates
that ATP competitively inhibits dCTP uptake. The Ki
value was estimated to be 8 µM by a replot of the slopes
versus the ATP concentration. Numerous studies in cell
culture, particularly that of the T-lymphoblastic cell line CEM, have
demonstrated dCTP intracellular pool sizes ranging from 8 to 38 µM (26-30). Whereas CEM cells are rapidly dividing cells
in culture, another study using peripheral blood lymphocytes reported a
dCTP pool size of 1.5 µM in quiescent cells and 18 µM in phytohemagglutinin-activated lymphocytes (31). Thus, the mitochondrial dCTP transport activity reported herein may be
significant within the range of these reported intracellular dCTP
concentrations.

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Fig. 3.
Interaction of dCTP with reconstituted
proteoliposomes and inhibition by ATP. Proteoliposomes (0.3 mg/ml
protein) were prepared after HPT and blue Sepharose chromatography as
described under "Experimental Procedures." Transport was initiated
by the addition of [ -32P]dCTP at the indicated
concentrations for 1 min in the presence of 20 mM Hepes (pH
7.0), 2 mM CaCl2, and 0.2 mM EDTA.
The data are presented as a double-reciprocal plot of dCTP uptake.
Velocity is expressed as pmol of dCTP uptake/min/mg of protein. The
figure illustrates the dependence of the rate of dCTP uptake
in proteoliposomes on substrate concentration and the inhibition of
dCTP uptake in reconstituted proteoliposomes by ATP. Vesicles were
incubated with [ -32P]dCTP in the presence of ATP.
Velocity is expressed as pmol of dCTP uptake/min/mg of protein.
Concentrations of ATP were 0 ( ), 5 ( ), 10 ( ), and 20 ( )
µM. The Ki value for ATP was
determined by replot of the slopes as shown in the inset.
These data represent the means and S.D. values of at least three
experiments in duplicate.
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The substrate specificity of this novel mitochondrial carrier activity
was examined by investigating the ability of dCTP and its derivatives
to inhibit uptake of the unidirectional, inward flux of 10 µM [32P]dCTP (Table
III). If [32P]dCTP were
broken down to [32P]dCDP or [32P]dCMP for
transport, we would expect excess dCDP or dCMP to decrease uptake by
dilution of the radiospecificity of [32P]dCDP or
[32P]dCMP derived from the action of phosphatase present
in the preparation. Excess unlabeled dCTP is included as a positive
control. Uptake of [32P]dCTP was most affected by the
triphosphate of deoxycytidine (dCTP), while the diphosphate form (dCDP)
exhibited a moderate effect. In contrast, the nucleoside deoxycytidine
and its monophosphate derivative (dCMP) had little effect on
[32P]dCTP uptake (<10% inhibition). Thus, the
reconstituted transporter discriminates between different phosphate
forms of deoxycytidine. The uptake of
- and
-labeled
[32P]dCTP into proteoliposomes and the effect of excess
dCDP and dCMP on uptake suggests that dCTP is the primary substrate
used for transport.
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Table III
Effect of competitors and inhibitors on the uptake of
[32P]dCTP by proteoliposomes
Competitors were added immediately before the radiolabeled substrate.
Atractyloside and NEM were added at a concentration of 1 mM
3 min before the labeled substrate. Proteoliposomes were prepared after
HPT and blue Sepharose chromatography as described under
"Experimental Procedures." Transport was started by the addition of
10 µM [ -32P]dCTP to proteoliposomes in a
buffer containing 20 mM Hepes (pH 7.0), 0.2 mM
EDTA, and 2 mM CaCl2. The concentration of
competitors was 100 µM. Phosphate was added at a
concentration of 5 mM. Means and S.D. values from three
experiments in duplicate are presented.
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The effects of other nucleoside triphosphates on
[32P]dCTP uptake are shown in Table
IV. At equimolar concentrations, the
purines GTP, ATP, dATP, and dGTP moderately reduced
[32P]dCTP uptake in proteoliposomes with more potent
inhibition with a 5-fold excess of competitor. Interestingly, these
purines were more effective than TTP or UTP in competing for
[32P]dCTP uptake in proteoliposomes. These results raise
the possibility that the observed transport activity may not be
specific for dCTP only but may reflect the activity of a general dNTP
carrier. Further studies will be required to assess if the competition
represents uptake of other dNTPs.
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Table IV
Effect of nucleoside triphosphates on dCTP transport activity
The protein eluate from HPT and blue Sepharose chromatography was
reconstituted into phospholipid vesicles as described under
"Experimental Procedures." Transport was initiated by the addition
of 10 µM [ -32P]dCTP and one of the competing
nucleotides. Means and S.D. values from three separate experiments are
presented. The control value of dCTP uptake was 189 pmol/min/mg
protein.
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The sensitivity of the reconstituted carrier to externally added
inhibitors was investigated using the sulfhydryl reagent N-ethylmaleimide (a potent inhibitor of the phosphate
carrier) (32), atractyloside (a specific inhibitor of the AAC) (33), and phosphate. N-Ethylmaleimide, atractyloside, and
phosphate had no significant inhibitory effect on dCTP transport (Table III). However, a 50% increase in dCTP uptake occurs in the presence of
atractyloside. The mechanism by which atractyloside increases dCTP
uptake is unclear. These data indicate that the phosphate carrier and
the AAC do not play a role in the dCTP uptake activity observed in this
study. We have not assessed the uptake of other dNTPs, although it is
possible that there are other carrier activities for these metabolites.
To our knowledge, this is the first report of a preparation of a
mitochondrial dCTP carrier activity in functional form from any source.
This transport activity suggests an important role of bioactive
endogenous dNTP uptake in mitochondria. Thus, cytoplasmic pools of
dNTPs may be available to mitochondria, which are continually being
damaged, and for replication of their DNA. The transport activity
reported in this study may also be important in mediating the
mitochondrial accumulation of cytotoxic nucleoside analogs used in
chemotherapy. Clinical and laboratory findings have focused attention
on damage to mitochondrial function as a mechanism of toxicity of
various drugs. Indeed, available evidence supports the hypothesis that
the myopathy induced by anti-human immunodeficiency virus nucleoside
analogs is due to inhibition of mitochondrial DNA synthesis (34,
35). The existence of the mitochondrial dCTP carrier activity described
in this study may provide insight into the molecular mechanism
underlying nucleoside analog-induced mitochondrial toxicity and protection.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants F32GM17474 (to E. G. B.) and AI-38204 (to Y. C. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
These two authors contributed equally to this work.
§
To whom correspondence should be addressed: Dept. of Pharmacology,
Yale University School of Medicine, P.O. Box 802066, New Haven, CT
06520. Tel.: 203-785-7119; Fax: 203-785-7129; E-mail: cheng.lab{at}yale.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
AAC, ATP/ADP
carrier;
HPT, hydroxyapatite.
2
E. G. Bridges, Z. L. Jiang, and
Y. C. Cheng, unpublished observations.
 |
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