From the Department of Pharmaco-Biology, Laboratory
of Biochemistry and Molecular Biology, University of Bari, Via
Orabona 4, 70125 Bari, Italy, and the § Medical Research
Council Dunn Human Nutrition Unit, Hills Road,
Cambridge CB2 2, United Kingdom
Received for publication, May 19, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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The nuclear genome of Saccharomyces
cerevisiae encodes 35 members of a family of membrane proteins.
Known members transport substrates and products across the inner
membranes of mitochondria. We have localized two hitherto unidentified
family members, Odc1p and Odc2p, to the inner membranes of
mitochondria. They are isoforms with 61% sequence identity, and we
have shown in reconstituted liposomes that they transport the
oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a strict counter
exchange mechanism. Intraliposomal adipate and glutarate and to a
lesser extent malate and citrate supported
[14C]oxoglutarate uptake. The expression of Odc1p, the
more abundant isoform, made in the presence of nonfermentable carbon
sources, is repressed by glucose. The main physiological roles of Odc1p and Odc2p are probably to supply 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol where they are used in the
biosynthesis of lysine and glutamate, respectively, and in lysine catabolism.
A family of membrane proteins that transports metabolites involved
in oxidative phosphorylation and in other important functions in
mitochondria is found in the inner membranes of the organelle (1). The
sequences of members of this family are made of three related domains
of about 100 amino acids repeated in tandem, each probably being folded
into two transmembrane Yeast Strains and Growth Conditions--
Deletion of the yeast
nuclear genes ODC1 (ORF YPL134c) and ODC2 (ORF Y0R222w) was
accomplished by sequential homologous recombination of the auxotrophic
markers TRP1 and HIS3 at the respective loci of S. cerevisiae YPH499 strain (wild type: MATa ade2-101
his3- Subfractionation of Mitochondria and Quantitative
Immunoblotting--
Extractions of mitochondria with sodium carbonate
or with digitonin were performed as described previously (6). To
determine the amount of Odc1p and Odc2p in wild type mitochondria,
standard calibration curves were constructed using 10-500 ng of pure
recombinant ODC proteins as standards. After transfer of the proteins
to the same nitrocellulose membrane, the standards and the
mitochondrial samples were immunodecorated simultaneously. Once it had
been verified that the sample loading was within the linear range of the calibration curves, the densitometric signal intensity was used to
measure the amount of Odc1p and Odc2p.
Construction of the ODC Expression Plasmids--
The coding
sequences of ODC1 and ODC2 were amplified from S. cerevisiae
genomic DNA by polymerase chain reaction. Forward and reverse
oligonucleotide primers were synthesized corresponding to the
extremities of the ODC sequences with additional HindIII and
BamHI sites, respectively. The reverse primers also
contained 18 additional bases encoding a six-histidine tag immediately
before the translational termination codon. The products of polymerase chain reaction were cloned into the expression vector pYES2
(Invitrogen, Groningen, The Netherlands). The resulting expression
plasmids (pODC1 or pODC2) were introduced in the odc1 Overexpression in S. cerevisiae and Purification of the ODC
Proteins--
Mitochondria were isolated from odc1 Protein Chemical Characterization of Overexpressed ODC
Isoforms--
Proteins were analyzed by SDS-PAGE in 17.5% gels
(12) and either stained with Coomassie blue dye or transferred to
nitrocellulose membranes. The identities of purified Odc1p and Odc2p
were confirmed by matrix-assisted laser desorption ionization-time of
flight mass spectrometry of trypsin digests of the corresponding bands excised from a Coomassie-stained gel. Western blotting was carried out
with rabbit antibodies against the bacterially expressed ODC proteins.
The overproduction of ODC isoforms as inclusion bodies in the bacterial
cytosol and the purification of the inclusion bodies in host strain
Escherichia coli C0214(DE3) have been described previously
(9). The films were scanned with an LKB 2202 Ultroscan laser densitometer.
Reconstitution of the ODC Proteins into Liposomes--
Purified
ODC proteins were reconstituted by cyclic removal of the detergent with
a hydrophobic column (13). The composition of the initial mixture used
for reconstitution was: 200 µl of purified isoform (0.3-0.4 µg of
protein), 70 µl of 10% Triton X-114, 100 µl of 10% phospholipids
in the form of sonicated liposomes, 20 mM oxoglutarate
(except where otherwise indicated), 10 mM PIPES, pH 7.0, 0.7 mg of cardiolipin (Sigma), and water to a final volume of 700 µl.
These components were mixed throughly, and the mixture was recycled 13 times through an Amberlite column (Supelco) (3.0 × 0.5 cm)
pre-equilibrated with a buffer containing 10 mM PIPES, pH
7.0, and with the substrate at the same concentration as in the
starting mixture. All operations were performed at 4 °C, except the
passages through Amberlite, which were carried out at room temperature.
Transport Measurements--
External substrate was removed from
proteoliposomes on a Sephadex G-75 column preequilibrated with buffer F
(50 mM NaCl and 10 mM PIPES, pH 7.0). Transport
at 25 °C was started by adding [14C]oxoglutarate
(unless otherwise indicated) to the proteoliposomes and terminated by
addition of 30 mM pyridoxal 5'-phosphate and 10 mM bathophenanthroline (the "inhibitor stop" method
(13)). In controls, inhibitors were added with the labeled substrate. The external radioactivity was removed on Sephadex G-75, and the internal radioactivity was measured. The transport activity was the
difference between experimental and control values. The initial rate of
transport was calculated in mmol/min/g protein from the time course of
isotope equilibration (13). Various other transport activities were
also assayed by the inhibitor stop method. For efflux measurements, the
internal substrate pool of the proteoliposomes was made radioactive by
carrier-mediated exchange equilibration (13) with 0.1 mM
[14C]oxoglutarate added at high specific radioactivity.
After 60 min, the residual external radioactivity was removed by
passing the proteoliposomes again through a column of Sephadex G-75.
Efflux was started by adding unlabeled external substrate or buffer F alone and terminated by adding the inhibitors indicated above.
Subcellular Localization of the ODC Proteins--
Immunoreactive
bands on SDS-PAGE gels were detected with antibodies against Odc1p and
Odc2p. Bands with apparent molecular masses of about 36.5 and 35.0 kDa,
respectively, were detected in wild type mitochondria (Fig.
1, lane 1) but not in
mitochondria from the odc1
The submitochondrial location of Odc1p and Odc2p was examined by
separation of soluble and peripheral proteins from integral membrane
proteins of wild type mitochondria by carbonate treatment (Fig.
2). Both Odc1p and Odc2p remained in the
membrane protein fraction, as did the ADP/ATP carrier and (not shown)
Tom40p (marker proteins of inner and outer mitochondrial membranes,
respectively), but the matrix protein hsp70 and (not shown) the
intermembrane space protein cytochrome b2 were
in the soluble and peripheral protein fraction. In other experiments,
at 0.3% digitonin more than 80% of the outer membrane protein Tom40p,
and less than 10% of Odc1p, Odc2p and the ADP/ATP carrier, were
solubilized from wild type mitochondria. At higher levels of digitonin,
Odc1p and Odc2p were solubilized progressively in parallel with the
ADP/ATP carrier (data not shown). Therefore, Odc1p and Odc2p are
integral proteins of the inner mitochondrial membrane.
Expression in S. cerevisiae and Purification of the ODC
Proteins--
Odc1p and Odc2p were overexpressed at high levels in a
S. cerevisiae strain devoid of both corresponding genes
(odc1
The presence of the histidine tail at the C-terminal end of the
expressed ODC isoforms allowed their purification by a
Ni+-agarose affinity column (Fig. 3, lanes 5 and
6, and Fig. 1, lane 4). About 0.7 mg of Odc1p and
about 0.1 mg of Odc2p were obtained per liter of culture. The identity
of purified Odc1p and Odc2p was confirmed by matrix-assisted laser
desorption ionization-time of flight mass spectrometry.
Functional Characterization of Recombinant Odc1p and
Odc2p--
Proteoliposomes reconstituted with digitonin-solubilized
mitochondria isolated from odc1
The purified and reconstituted Odc1p and Odc2p catalyzed a very active
[14C]oxoglutarate/oxoglutarate exchange, which was
inhibited by a mixture of pyridoxal 5'-phosphate and
bathophenanthroline. No such activity was found with Odc1p and Odc2p
that had been boiled before incorporation into liposomes. Likewise, no
[14C]oxoglutarate uptake was observed into
proteoliposomes that did not contain internal oxoglutarate, indicating
that Odc1p and Odc2p do not catalyze a unidirectional transport
(uniport) of oxoglutarate but only the exchange reaction. To obtain
further information about the mechanism of transport catalyzed by Odc1p
and Odc2p, the efflux of [14C]oxoglutarate from
prelabeled active proteoliposomes was investigated because it provides
a more convenient assay for unidirectional transport (13). An
experiment performed with proteoliposomes reconstituted with Odc1p is
shown in Fig. 4. In the absence of external substrate, no efflux was observed even after incubation for
1 h. However, upon addition of external oxoglutarate, an extensive efflux of intraliposomal radioactivity occurred, and this efflux was
prevented completely by the presence of the inhibitors pyridoxal 5'-phosphate and bathophenanthroline (Fig. 4). Similar data were obtained using Odc2p instead of Odc1p. These results show clearly that
reconstituted Odc1p and Odc2p catalyze an obligatory exchange reaction
of internal oxoglutarate for external oxoglutarate. Furthermore, the
proteoliposomes did not catalyze homoexchanges for glutamate, aspartate, 2-oxoisocaproate, glutamine, ornithine, ADP, phosphate, sulfate, and carnitine (internal concentration, 10 mM;
external concentration, 1 mM).
The substrate specificity of purified Odc1p and Odc2p was investigated
further by measuring the uptake of [14C]oxoglutarate into
proteoliposomes that had been preloaded with various substrates. As
shown in Table II,
[14C]oxoglutarate was taken up efficiently by
proteoliposomes containing internal oxoglutarate, 2-oxoadipate,
2-oxopimelate, glutarate, adipate, pimelate, L-malate, and
D-malate. A much lower activity was observed in the
presence of internal oxaloacetate, succinate, citrate, and isocitrate.
A very low activity was found with internal malonate, suberate,
fumarate, and maleate, and (not shown) no exchange was found with
oxalate, aspartate, glutamate, 2-aminoadipate, 2-aminopimelate,
pyruvate, 2-oxobutyrate, 2-hydroxybutyrate, 2-oxovalerate, 2-oxoisocaproate, phosphate, sulfate, thiosulfate, ADP, ATP, ornithine, glutamine, and carnitine.
The [14C]oxoglutarate/oxoglutarate exchange reactions
catalyzed by reconstituted Odc1p and Odc2p were inhibited strongly by mercurials (mersalyl, p-chloromercuribenzene sulfonate, and
mercuric chloride), by pyridoxal 5'-phosphate, by bathophenanthroline, and by
In addition, the ability of nonradioactive potential substrates to
inhibit the oxoglutarate/oxoglutarate exchange was examined. The
effectiveness of dicarboxylates and 2-oxodicarboxylates with different
carbon chain length on the rate of oxoglutarate uptake are compared in
Fig. 5. With both reconstituted Odc1p and
(not shown) Odc2p, glutarate, adipate, and pimelate with 5-7 carbon atoms caused a significant inhibition of oxoglutarate uptake, whereas oxalate, malonate, succinate, and suberate had virtually no
effect. The presence of a carbonyl group on the dicarboxylate molecule
enhanced the inhibitory effect of the compounds with a maximum of
inhibition at six carbon atoms. In similar experiments (not shown) the
effect of other dicarboxylates on the rate of oxoglutarate uptake
catalyzed by the recombinant and reconstituted Odc1p and Odc2p was also
tested. The presence of a hydroxyl group on the C4
dicarboxylate molecule (as in malate) increased about 10-fold the
extent of the inhibition of oxoglutarate uptake with respect to the
corresponding dicarboxylate. Also, L- and
D-tartrate inhibited the uptake of oxoglutarate more
efficiently than succinate, their inhibitory effect being comparable
with that of malate. However, the C5 hydroxydicarboxylate,
hydroxyglutarate, was slightly less effective than glutarate, and the
C3 hydroxydicarboxylate (tartronate) was completely
ineffective like the corresponding dicarboxylate. Both the
cis- and trans-unsaturated dicarboxylates, fumarate and maleate, had no effect, nor had the aminodicarboxylates. D,L-Threo-hydroxyaspartate has been reported to
inhibit the uptake of oxoglutarate into intact yeast mitochondria (18),
but it had no effect on oxoglutarate transport catalyzed by
reconstituted Odc1p and Odc2p. In view of the inhibition of
oxoglutarate uptake by dicarboxylates carrying a carbonyl group, the
effect of oxomonocarboxylates on the rate of uptake of 0.4 mM oxoglutarate was also tested. Pyruvate, 2-oxovalerate
2-oxobutyrate, 2-oxoisocaproate, and 5-oxohexanoate (all at 8 mM concentration, i.e. 20 times greater than the
substrate) did not influence the rate of oxoglutarate uptake (data not
shown). Finally, several aminomonocarboxylates, (glycine, alanine,
valine, threonine, and serine), and other amino acids (glutamine,
asparagine, lysine, arginine, histidine, and ornithine) had no effect
on the oxoglutarate/oxoglutarate exchange (data not shown).
Kinetic Characteristics of Recombinant Odc1p and Odc2p--
The
kinetic constants of the recombinant purified Odc1p and Odc2p were
determined by measuring the initial transport rate at various external
[14C]oxoglutarate concentrations, in the presence of a
constant saturating internal concentration of 20 mM
oxoglutarate. The transport affinities (Km) and the
specific activities (Vmax) for the
oxoglutarate/oxoglutarate exchange at 25 °C, were 0.52 ± 0.08 mM and 252 ± 53 mmol/min/g protein for Odc1p (24 experiments) and 0.47 ± 0.07 mM and 73 ± 17 mmol/min/g protein for Odc2p (16 experiments). All of the compounds summarized in Table IV inhibited
oxoglutarate uptake by both isoforms competitively, because they were
found to increase the apparent Km without changing
the Vmax of oxoglutarate uptake (not shown). The
inhibition constants (Ki) of 2-oxoadipate are only
about 2-fold lower than the Km of oxoglutarate but
are about 5-7 times lower than the Ki values of
malate and 2-oxopimelate and more than 50 times lower than those of
oxaloacetate and succinate. The Ki values of malate
and citrate are similar to the Km values of the same
substrates for the reconstituted Odc1p, as determined from
Lineweaver-Burk plots of the rate of [14C]malate or
[14C]citrate uptake in the presence of a constant
internal oxoglutarate concentration of 20 mM. Under these
conditions the Km of malate was 1.3 ± 0.2 mM (seven experiments), and that of citrate was 5.7 ± 0.5 mM (three experiments). Taken together these results demonstrate that 2-oxoadipate and 2-oxoglutarate are the best substrates for reconstituted Odc1p and Odc2p.
Influence of the Carbon Source on the Expression of ODC
Proteins--
Because Odc1p and Odc2p appear to have virtually the
same transport properties, to shed light on the metabolic significance of the ODC isoforms the regulation of protein expression was examined. To quantify Odc1p and Odc2p, various amounts of mitochondrial samples
from yeast cells fed on glycerol were loaded onto the gel and
immunoblotted simultaneously with the appropriate range of bacterially
expressed Odc1p and Odc2p standards (see "Experimental Procedures"). In four determinations, the abundance of ODC proteins was 123 ± 30 pmol/mg of protein of Odc1p and 9 ± 2 pmol/mg
of protein of Odc2p.
The expression of Odc1p and Odc2p was investigated by immunoblot
analysis of mitochondria isolated from the wild type strain following
growth on different carbon sources. The expression of Odc1p is
repressed strongly by glucose, whereas Odc2p appears to be expressed at
comparatively higher levels on glucose and galactose medium than on
medium supplemented with nonfermentable carbon sources (Fig.
6).
Kinetic Properties of ODC Isoforms--
The transport
characteristics and kinetic parameters of the ODC proteins show that
they are isoforms of a novel mitochondrial transporter for C5-C7
oxodicarboxylates with greatest specificity for 2-oxoadipate and
2-oxoglutarate. ODC also transports the corresponding dicarboxylates
and to a lesser extent malate and citrate.
The substrate specificity of the yeast ODC isoforms is distinct from
that of any other previously characterized mitochondrial carrier. It
differs from that of the succinate-fumarate carrier (19), which is its
closest sequence homologue (5), because the former transports fumarate
and succinate with a very low efficiency (Km > 15 mM). ODC is also quite different from the mammalian oxoglutarate carrier. First, the yeast ODC isoforms and the bovine oxoglutarate carrier have sequence identities of 24 and 25%,
indicating that they are not orthologues. Second, the ODCs transport
C5-C7 oxodicarboxylates, whereas the mammalian oxoglutarate carrier transports C4 and C5 oxodicarboxylates (14, 20, 21). Third, the ODC
works best with C5-C7 dicarboxylates, whereas the mammalian oxoglutarate carrier displays optimal transport activity with C3 and C4
dicarboxylates (malonate, succinate, and maleate) (14, 20, 21). Fourth,
ODC appears to be less stereospecific than the mammalian oxoglutarate
carrier as it has equal affinity for L- and
D-malate, whereas the mammalian carrier has little or no affinity for the D-stereoisomer (14, 20, 21). Fifth, both isoforms of ODC accept the tricarboxylates citrate and isocitrate as
substrates, although with low affinity, whereas the specificity of the
mammalian oxoglutarate carrier is confined to dicarboxylates (14, 20,
21).
Regulation of Expression of the ODC Proteins--
Many yeast genes
involved in the tricarboxylic acid cycle, in oxidative phosphorylation
and in ATP generation are subject to repression by fermentable carbon
sources (catabolite repression). Under nonrepressed conditions in cells
fed on glycerol, Odc1p is about 15-fold more abundant than Odc2p.
However, ODC1 expression is strongly repressed by catabolites, whereas
ODC2 appears to be expressed at a higher level in the presence of
galactose and glucose (Fig. 6). This apparent increase could result
from the repression of most other mitochondrial proteins rather than
from induction of the ODC2 itself. An increase in ODC1 expression has been observed without significant change in ODC2 expression during the
diauxic shift when a culture of S. cerevisiae growing on
glucose in batch culture exhausted the glucose supply and began to
oxidize ethanol produced by fermentation (22). A temporal pattern of expression similar to that of ODC1 was observed for AAC1 and AAC2, the
gene for the major "aerobic" isoform of ADP/ATP translocase, whereas the transcript level of AAC3, which is induced under anaerobic conditions, remained constant (22). These considerations indicate that
Odc1p is the major ODC isoform under respiratory conditions and that
Odc2p is the prevailing isoform in the presence of glucose and possibly
in anaerobiosis. It should be noted that ODC1, AAC2 (encoding an
isoform of ADP/ATP translocase), and MIR1 (encoding the phosphate
carrier) are the only yeast mitochondrial carrier genes that increase
their expression following adaptive evolution in aerobic
glucose-limited conditions (23), indicating that they all have key
functions in cell metabolism.
Role of ODC in Lysine Metabolism--
In yeast, lysine is
synthesized via the
Reversal of the cytoplasmic part of the 2-aminoadipate pathway is used
in lysine catabolism in both S. cerevisiae and animals (24,
25). For this purpose, it is likely that 2-oxoadipate is imported by
ODC into mitochondria first to be converted into glutaryl-CoA by
2-oxoadipate dehydrogenase (a mitochondrial enzyme) and then
metabolized in a series of steps to acetyl-CoA. In mammals, cytosolic
2-oxoadipate is also produced by catabolism of tryptophan (26) and
possibly hydroxylysine. Therefore, it is likely that an ODC protein
exists in man and that defects in its activity could be linked to
2-ketoadipic acidemia. It has been suggested that this inborn error of
catabolism of lysine, tryptophan, and hydroxylysine (27) may be due to
2-oxoadipate dehydrogenase deficiency, but such a defect has not been
demonstrated directly.
The yeast strains used in the present investigation are devoid of the
LYS2 gene, encoding the major subunit of aminoadipate reductase
( Role of ODC in Nitrogen Assimilation--
In S. cerevisiae, nitrogen in the form of ammonium is assimilated either
by the action of isoforms 1 and 3 of glutamate dehydrogenase or by the
action of glutamine synthetase and glutamate synthase together (28).
These enzymes are cytoplasmic (29, 30), and nitrogen assimilation
requires the carbon skeleton of oxoglutarate. The major site of
oxoglutarate production is the mitochondrial matrix, and therefore,
another physiological role of ODC isoforms is probably to export it to
the cytoplasm. It should be stressed that it is unlikely that S. cerevisiae has an orthologue to the mammalian 2-oxoglutarate
carrier (6). The only yeast carriers to cluster on a phylogenetic tree
with the mammalian oxoglutarate carrier (5) have been identified as the
dicarboxylate and oxaloacetate-sulfate carriers (4, 6). Because the
odc1
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-helices joined by an extensive hydrophilic
sequence. The three repeats are linked by shorter hydrophilic
sequences. The repeats in the various family members are all related,
and various sequence features are conserved (2, 3). The nuclear genome
of Saccharomyces cerevisiae encodes 35 members of this
family (4). They include three isoforms of the ADP/ATP translocase and
the carriers for phosphate, citrate, dicarboxylate, ornithine,
succinate-fumarate, oxaloacetate-sulfate, and carnitine (see Ref. 5 for
a review; Refs. 6 and 7). Hitherto, the functions of other
family members have been unknown. Two of them, Odc1p and
Odc2p1 are 61% identical in
sequence. As described here, they have been overexpressed in S. cerevisiae and shown to be isoforms in the inner mitochondrial
membrane where they transport C5-C7 oxodicarboxylic acids, including
2-oxoadipate and 2-oxoglutarate, by a counter exchange mechanism. The
main physiological roles of these novel transporters are likely to be
in cytoplasmic biosynthesis of lysine and glutamate by supplying
2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix and in
lysine catabolism.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
200 leu2-
1 ura3-52 trp1-
63 lys2-801). Deletants were
verified by polymerase chain reaction and Western blot analysis.
Yeast cells were precultured on synthetic complete medium (8)
supplemented with 3% glycerol and 0.1% glucose. Tryptophan,
histidine, and uracil were omitted where the genotype permitted. For
growth studies, exponentially growing cells were harvested by
centrifugation, washed with growth medium and diluted with the same
medium until a final optical density of 5 × 10
3 at
600 nm was reached. For the preparation of mitochondria, precultures were diluted 35-fold in YP medium (1% yeast extract, 2%
bacto-peptone, pH adjusted to 4.8 with HCl) and grown in the presence
of the same carbon sources to mid exponential phase. Galactose (0.45%) was added 6 h before harvesting. For the estimation of the
expression of ODCs, yeast cells were grown at 30 °C to mid-log phase
in YP medium supplemented with either 2% glucose, 2% galactose, 3%
glycerol, 2% ethanol, or 3% lactate and then harvested by
centrifugation (3000 × g, 5 min).
odc2
double
mutant, and transformants (odc1
odc2
/pODC1 or odc1
odc2
/pODC2
cells) were selected for uracil auxotrophy. Other experimental
conditions have been described before (9).
odc2
/pODC1 or
odc1
odc2
/pODC2 cells according to standard procedures (10) and
solubilized in buffer A (500 mM NaCl, 10 mM
PIPES, pH 7.0)2 containing
0.8% digitonin (w/v) and 0.1 mM phenylmethylsulfonyl fluoride at a final concentration of 0.2-0.4 mg protein/ml. After incubation for 20 min at 4 °C, the mixture was centrifuged
(138,000 × g, 20 min). The supernatant (1.1 ml) was
mixed for 1 h at 4 °C with 0.45 ml of
nickel-nitrilotriacetic-agarose (Qiagen, Hilden, Germany) previously
equilibrated with buffer A. Then the resin was packed into a column
(0.5-cm internal diameter) and washed extensively with the following
buffers: B, 500 mM NaCl, 0.8% digitonin, 10 mM
imidazole, 0.5% Triton X-100, 7.5% glycerol, 10 mM PIPES, pH 7.5 (2 ml); C, 300 mM NaCl, 0.8% digitonin, 10 mM imidazole, 0.1% Triton X-100, 5% glycerol, 10 mM PIPES, pH 7.5 (2 ml); D, 100 mM NaCl, 0.6%
digitonin, 10 mM imidazole, 0.05% Triton X-100, 1%
glycerol, 10 mM PIPES, pH 7.5 (1 ml); and E, 50 mM NaCl, 0.3% digitonin, 10 mM imidazole,
glycerol 0.5%, 10 mM PIPES, pH 7.0 (1 ml). Finally pure
ODC proteins were eluted with a buffer containing 50 mM
NaCl, 0.1% digitonin, 80 mM imidazole, and 10 mM PIPES, pH 7.0. Protein concentrations were determined by
the Lowry method modified for the presence of detergent (11) or by
laser densitometry (9).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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odc2
double mutant (Fig. 1, lane
2). The antibody against Odc2p cross-reacted with Odc1p (upper
band), and both antibodies reacted with an unidentified band of about
33.0 kDa that was also present in the odc1
odc2
mitochondria. The
contents of the ADP/ATP carrier and (not shown) the phosphate,
succinate-fumarate and dicarboxylate carriers detected with specific
antibodies were essentially the same in both wild type and
odc1
odc2
mitochondria. Therefore, the absence of both ODC
proteins from the double mutant does not affect the expression of other
mitochondrial carriers. Furthermore, the phenotype of the
odc1
odc2
strain was studied by comparison of the growth of the
mutant cell with the parental strain in shake flask cultures on
different media. Both the wild type and the deletion strain yeast
exhibited substantial and similar growth on either rich medium (YP) or
synthetic complete medium supplemented with either 2% glucose, 2%
galactose, 3% glycerol, 2% ethanol or 3% lactate, indicating that
the absence of the ODC proteins does not impair the respiratory
function of mitochondria.
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Fig. 1.
Immunoblot analysis of ODC proteins in yeast
mitochondria. 25 µg of mitochondrial protein from wild type
(lane 1) and odc1 odc2
cells (lane 2) were
separated by SDS-PAGE, transferred to nitrocellulose, and
immunodecorated with antibodies directed against Odc1p, Odc2p and the
ADP/ATP carrier (Aac2p). Lane 3, 10 and 1 µg of
mitochondrial protein from odc1
odc2
/pODC1 and
odc1
odc2
/pODC2 cells, respectively. Lane 4, 350 ng
(Odc1p) and 5 ng (Odc2p) of recombinant ODC transport proteins purified
from mitochondria in lane 3.
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Fig. 2.
Submitochondrial localization of Odc1p and
Odc2p. Analysis by SDS-PAGE and Western blotting of soluble and
peripheral proteins (S) and intrinsic membrane proteins
(P) from yeast mitochondria (5 µg of protein in each slot)
blotted with antisera directed against Odc1p, Odc2p, the ADP/ATP
carrier (Aac2p; inner membrane component), and the mitochondrial hsp70
(mt-hsp70; matrix protein). In the top two panels the
reaction was terminated once Odc1p and Odc2p became visible.
odc2
strain) (Fig. 3,
lanes 3 and 4). Their apparent molecular masses were about 38 and 37 kDa (the calculated values including the initiator
methionine and the histidine tail were 35,006 and 34,807 Da,
respectively). The successful overexpression and targeting of the
episomal Odc1p and Odc2p to mitochondria was confirmed by Western
blotting of isolated mitochondria from the odc1
odc2
/p0DC strains
(Fig. 1, lane 3), because the amount of mitochondrial protein applied in lane 3 of Fig. 1 was 2.5 (from
odc1
odc2
/p0DC1 cells) and 25 (from odc1
odc2
/p0DC2 cells)
times less than the amount of wild type mitochondrial protein applied
in lane 1 of Fig. 1. The differences in the molecular mass
of immunodecorated bands in lane 3 (odc1
odc2
/p0DC
mitochondria) and lane 1 (wild type mitochondria) are a
reflection of the presence of a C-terminal histidine tag in the
recombinant proteins.
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Fig. 3.
Purification of the overexpressed ODC
proteins. Proteins were separated by SDS-PAGE and stained
with Coomassie blue dye. Lanes M, markers (bovine serum
albumin, carbonic anhydrase and cytochrome c). Lanes
1-4, mitochondrial protein (100 µg) from wild type (lane
1), odc1 odc2
mutant (lane 2),
odc1
odc2
/pODC1 (lane 3), and odc1
odc2
/pODC2
(lane 4) strains. Cells were harvested 6 h after
addition of galactose. Lanes 5 and 6, 2 µg of
Odc1p (lane 5) and 4 µg of Odc2p (lane 6)
purified from mitochondria in lanes 3 and 4,
respectively.
odc2
/p0DC1 or odc1
odc2
/p0DC2
strains were able to catalyze an active
[14C]oxoglutarate/oxoglutarate homoexchange (Table
I). A lower oxoglutarate transport was
observed upon reconstitution of the digitonin extract from wild type
mitochondria, whereas liposomes reconstituted with the extract from
odc1
odc2
mitochondria showed a very low but reproducible
oxoglutarate exchange. Furthermore, oxoglutarate transport measured
upon reconstitution of the mitochondrial extract isolated from the
double deletion strain transformed with the pYES2 vector harboring the
sequence encoding the yeast oxaloacetate carrier (odc1
odc2
/pOAC1
strain) (6), the yeast carnitine carrier (odc1
odc2
/pCRC1 strain)
(7), or with the empty pYES2 vector (not shown) was not significantly
increased.
Oxoglutarate homoexchange in liposomes reconstituted with mitochondrial
extracts from various yeast strains
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Fig. 4.
Efflux of [14C]oxoglutarate
from proteoliposomes. Proteoliposomes were reconstituted with
recombinant Odc1p in the presence of 20 mM oxoglutarate,
and then the internal substrate pool was labeled by carrier-mediated
exchange equilibration. After removal of external substrate by Sephadex
G-75 chromatography, the efflux of [14C]oxoglutarate was
started by adding buffer F alone ( ) or 10 mM
oxoglutarate (
), or 10 mM oxoglutarate, 30 mM pyridoxal 5'-phosphate, and 10 mM
bathophenanthroline (
) in the same buffer.
Dependence on internal substrate of the transport properties of
proteoliposomes reconstituted with recombinant Odc1p or Odc2p
-cyanocinnamate (Table III).
Also, both Odc1p and Odc2p were inhibited considerably by
N-ethylmaleimide. The impermeable dicarboxylate analogues
butylmalonate and phenylsuccinate, which are known to be powerful
inhibitors of the oxoglutarate and dicarboxylate carriers (14, 15),
decreased the reconstituted transport activities rather poorly. Also,
the tricarboxylate analogue 1,2,3-benzenetricarboxylate, a very
efficient inhibitor of the citrate carrier (16), had a rather mild
inhibitory effect, and carboxyatractyloside, a powerful inhibitor of
the ADP/ATP carrier (17), had little or no effect on the activities of
Odc1p and Odc2p.
Effect of inhibitors on the [14C]oxoglutarate/oxoglutarate
exchange by proteoliposomes reconstituted with Odc1p and Odc2p
-cyanocinnamate were added 2 min
before the labeled substrate; the other inhibitors and external
substrates were added together with [14C]oxoglutarate. The
final concentration of the inhibitors was 4 mM, except for
mercurials (10 µM), carboxyatractyloside (0.1 mM), and N-ethylmaleimide and
-cyanocinnamate
(2 mM). Similar results were obtained in three independent
experiments in duplicate.
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Fig. 5.
Effect of dicarboxylates and
2-oxodicarboxylates with different carbon chain length on the rate of
[14C]oxoglutarate uptake into proteoliposomes
reconstituted with recombinant Odc1p. Proteoliposomes were
preloaded with 20 mM oxoglutarate and reconstituted with
recombinant Odc1p. Transport was initiated by the addition of 0.4 mM [14C]oxoglutarate and terminated after
45 s. Oxalate, malonate, succinate, glutarate, adipate, pimelate,
and suberate ( ) and the corresponding 2-oxoacids (
) were added
simultaneously with [14C]oxoglutarate at 4.0 mM concentration. The control value for uninhibited
2-oxoglutarate uptake was 108 mmol/min/g protein. The results are given
as a percentage of inhibition of the control. Similar results were
obtained in three separate experiments in duplicate.
Competition with [14C]oxoglutarate uptake in proteoliposomes
containing recombinant yeast Odc1p and Odc2p
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Fig. 6.
Comparison of the expression of Odc1p and
Odc2p on various carbon sources. Cells were harvested from
exponentially growing cells on YP medium supplemented with the
indicated carbon sources. Amounts of ODC proteins were estimated by
densitometry upon immunodecoration of mitochondrial proteins with
specific antisera. Similar results were obtained in three
independent experiments in duplicate. The amount of Odc1p and Odc2p
present in mitochondria from glycerol-fed cells was taken as
100%.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminoadipate pathway, whereby 2-oxoadipate is
produced in the mitochondrial matrix and 2-aminoadipate is converted
into lysine in the cytoplasm (24). The results reported here suggest
that 2-oxoadipate is exported by ODC from the mitochondrial matrix to
the cytoplasm where it is transaminated to 2-aminoadipate (Fig.
7). Because ODC functions by a strict
exchange mechanism, the carrier-mediated efflux of 2-oxoadipate
requires uptake of a counter-substrate. On the basis of transport
measurements (Table II), 2-oxoglutarate, malate, or another transported
Krebs cycle intermediate (according to the metabolic conditions) can
fulfill this role and satisfy an important anaplerotic role by
compensating the Krebs cycle for the 2-oxoadipateglutarate withdrawn
for 2-oxoadipate synthesis.
View larger version (26K):
[in a new window]
Fig. 7.
Compartmentalization of selected enzymes
involved in lysine biosynthesis in S. cerevisiae and
the role of the mitochondrial oxodicarboxylate carrier. The
dashed lines indicate the entry of nitrogen into the
glutamate molecule.
-aminoadipate-semialdehyde dehydrogenase), and so confirmation of
the proposed involvement of the ODC proteins in lysine metabolism
requires construction of appropriate strains with a functional LYS2.
odc2
strain grew on different fermentable and nonfermentable
carbon sources at rates similar to the parental strain, the
mitochondrial ODC proteins are not indispensable for respiration, but
this does not imply that the ODC is not involved in cytosolic glutamate
formation. The synthetic media used in this study contained
glutamate, which may suffice to sustain the growth of mutant cells.
Thus, more stringent growth conditions may be required to observe a
phenotype. Also, yeast has alternative mechanisms for generating
cytosolic 2-oxoglutarate to support nitrogen assimilation. It is
possible that impairment of oxoglutarate export from mitochondria may
be circumvented by the cytosolic NADP-dependent isocitrate
dehydrogenase, Idp2p, which is sufficient for growth of S. cerevisiae without glutamate in the absence of the mitochondrial
isozymes when oxoglutarate cannot be generated in the matrix (31).
Another possibility is that yeast mitochondria contain a second
unidentified oxoglutarate transporter.
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FOOTNOTES |
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* This work was supported in part by the Consiglio Nazionale delle Richerche Target Project on Biotechnology, by the Ministero dell'Università e della Ricerca Scientifica e Tecnologica, by the Piano Biomedicina, Progetto 1 Cluster C04 legge 488/92, and by the European Social Fund.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.
¶ To whom correspondence should be addressed. E-mail: walker @mrc-dunn.cam.ac.uk.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M004332200
1 The names ODC1 and ODC2 (corresponding to ORF YPL134c and ORF YOR222w, respectively) have been reserved for the genes encoding the two isoforms of the yeast oxodicarboxylate carrier.
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ABBREVIATIONS |
---|
The abbreviations used are: PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); PAGE, polyacrylamide gel electrophoresis; ODC, oxodicarboxylate carrier.
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REFERENCES |
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