ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
The Transport
180,
160-168
of Oxaloacetate
S. PASSARELLA, Department
F. PALMIERI, of Biochemistry, Received
(1977)
in Isolated AND University
July
Mitochondria
E. QUAGLIARIELLO of Bari,
Italy
6, 1976
The mechanism of mitochondrial oxaloacetate transport has been investigated by measuring the rate and the extent of exchange reactions between intramitochondrial anions and added oxaloacetate. The exchange between oxaloacetate and intramitochondrial oxoglutarate is insensitive to mersalyl at a concentration which completely inhibits the dicarboxylate carrier. Oxaloacetate causes efflux of intramitochondrial Pi, malonate, and malate. Mersalyl inhibits completely the oxaloacetate/Pi exchange, but only partially the oxaloacetate/malonat and the oxaloacetate/malate exchanges. The inhibition of the last two reactions decreases on increasing the time of incubation. Butylmalonate inhibits more than phenylsuccinate the exchange oxaloacetate&“P,,. , whereas phenylsuccinate is a more effective inhibitor than butylmalonate of the oxaloacetat~,,/[‘%!]oxoglutarat+~ exchange. The apparent K, values ranged from 0.6 to 1.2 mM for the oxaloacetate/oxoglutarate exchange and from 6.5 to 10 mM for the oxaloacetat.e/Pi exchange. The inhibition of oxoglutarate uptake by oxaloacetate is competitive. Oxaloacetate inhibits the malonate/Pi exchange competitively and it is a noncompetitive inhibitor of the Pi/P, exchange. It is concluded that oxaloacetate may be transported across the mitochondrial membrane by the oxoglutarate carrier and, much less effectively, by the dicarboxylate carrier. The implications of these findings are discussed. acid (sodium salt), [U-‘4Clsucrose, and 3H,0 were obtained from the Radiochemical Center (Amersham, England); rotenone was from F. P. Penick and Co. (New York); N-ethylmaleimide, oligomycin, and mersalyl were from Sigma; 2-phenylsuccinic acid was from K & K Laboratories, Inc. (Plainview, New York); oxaloacetate and glutamate dehydrogenase were from Boehringer; benzene-1,2,3-tricarboxylic acid and 2-butylmalonic acid were from Aldrich (Milwaukee, Wisconsin). Loading of mitochondria with labeled metabolites. Rat-liver or rat-heart mitochondria were isolated as previously described (10, 111, using a medium consisting of 0.25 M sucrose, 1 mM EGTA,’ and 20 mM Tris-HCl, pH 7.2. The mitochondria (40-50 mg of protein) were incubated at 20°C in 10 ml of medium consisting of 100 rnM KCl, 1 rnM EGTA, 20 mM Tris-HCl, pH 6.8, and one of the following metabolites (2 mM): Pi, malonate, malate, or 2-oxoglutarate. In this incubation medium, 1 pg/ml of rotenone was also present to block NADH dehydrogenase and therefore inhibit the oxidation of NAD+dependent substrates. When mitochondria were loaded with P,, the pH was 6.4 and 5 *g/ml of oligomycin was added to inhibit the incorporation of P,
Although several investigations in the last decade have indicated that oxaloacetate can cross the inner mitochondrial membrane (l-91, the mechanism by which the 0x0 acid is transported is still a matter of debate. Gimpel et al. (7) concluded that, in rat-liver mitochondria, oxaloacetate is transported only by the dicarboxylate carrier. However, in a study on the kinetics and the specificity of the oxoglutarate carrier, it was suggested that oxaloacetate is a substrate of the oxoglutarate carrier (8, 9). In this paper, the problem of the permeability of mitochondria to oxaloacetate is reinvestigated by measuring the exchange between intra- and extramitochondrial anions and by testing their sensitivity to inhibitors. The kinetics of oxaloacetate exchange and the competition with other anions are analyzed for possible carrier mechanisms. MATERIALS [32PlPhosphoric C4]malonic acid
acid, (sodium
AND
METHODS
~-lU-~~C]malic acid, salt), 2-ox0-[5-‘~Clglutaric
’ Abbreviations used: EGTA, ethylenenitrilolltetraacetic acid: droxyethyl)-1-piperazineethanesulfonic
ll-
lethylene-bis(oxyHEPES, 4-(2-hyacid.
160 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
MITOCHONDRIAL
OXALOACETATE
into ATP. When 2-oxoglutarate was used, 2 mM arsenite was added to inhibit oxoglutarate dehydrogenase. After 2 min, the mitochondria were washed in the medium without the metabolite and then suspended (40-50 nig of mitochondrial protein/ml). The intramitochondrial metabolite was labeled by adding to the mitochondrial suspension carrier-free labeled metabolite (approximately 1 &i/ml of mitochondrial suspension, i.e., about 0.02 &i/nmol and about 1 nmol/mg of mitochondrial protein, except for ,,Pi, whose activity was about lo” times higher). The mitochondria, loaded in this way and suspended in 1 ml of reaction mixture in the absence of counteranion for 2 min at 8”C, contained labeled metabolites at a concentration ranging from 12 to 24 mM, whereas the extramitochondrial concentration was less than 20 WM. In some experiments, the mitochondria were labeled with both [‘YJloxoglutarate and 32Pi. This was achieved by loading the mitochondria with 2-oxoglutarate, as described above, and by introducing to the mitochondrial suspension carrier-free [“Cloxoglutarate and carrier-free 3zPi, in the amount of 1 &i/ ml each. This is justified, since oxoglutarate does not exchange with Pi and the intramitochondrial content of Pi is still high in oxoglutarate-loaded mitochondria. Equilibration of the radioisotope between the extramitochondrial and intramitochondrial pools of oxoglutarate and Pi is obtained after 10 min at 0°C. Arsenite and oligomycin were present all through the entire procedure. Measurement of the exchange between intra- and extramitochondrial metabolites. Mitochondria loaded with labeled metabolite (about 2 mg of protein) were incubated at 8°C in 1.0 ml of medium consisting of 100 mM KCl, 1 mM EGTA, 20 mM 4-(2hydroxyethyll-l-piperazineethanesulfonic acid-Tris (HEPES-Tris), pH 7.0, in the presence of 2 pg of rotenone and 5 c(g of oligomycin. In some reactions, the medium included the inhibitors arsenite, Nethylmaleimide, mersalyl, phenylsuccinate, butylmalonate, or benzene-1,2,3-tricarboxylate, as indicated in the figure legends. After 1 min, the exchange was initiated by addition of the unlabeled metabolite and terminated 2 min later (unless otherwise specified) by centrifugation in an Eppendorf microcentrifuge. At 2 min, the exchange is at or near the equilibrium. The percentage exchange was calculated according to the equation : percentage exchange = 100 (Ca - Cp)lCa, where Cp and Ca represent the radioactivity measured in the presence and absence of external anions, respectively. Ca was approximately 2 x 10” cpm/mitochondrial pellet. In the absence of external anion, the mitechondria lose less than 10% of their labeled intramitochondrial metabolite in 2 min at 8°C. The results were corrected for this small time-dependent spontaneous leakage of radioactivity. Measurement of the kinetics of metabolite er-
TRANSPORT
161
change. The kinetics of the exchange were studied by using the inhibitor stop method, essentially according to the procedure described previously (12). Mitochondria loaded with the indicated metabolite were incubated at 8°C in 1.0 ml of medium consisting of 0.2 M sucrose, 10 mM KCl, 20 mM HEPESTris, pH 7.0, 1 IIIM MgCIP, 2 mM arsenite, 1 pg of rotenone, and 3 pg of oligomycin. When phosphateloaded mitochondria were used, the suspension also contained 1 mM N-ethylmaleimide. After 1 min, the assay was started by addition of unlabeled (for efflux) or labeled (for uptake) substrate and terminated 4-6 s later by rapid addition of an inhibitor. During this period, the rate of exchange was constant within the limits of experimental error. Butylmalonate (20 mM) and 20 mM phenylsuccinate were used to inhibit the dicarboxylate and the oxoglutarate carrier, respectively. Other methods. After rapidly centrifuging the mitochondria in an Eppendorf microcentrifuge for 1 min at O”C, the radioactivity in the pellets and supernatants was measured as described previously (13). The mitochondrial protein was determined by a modified biuret method (14). In some experiments, oxoglutarate was assayed enzymatically with glutamate dehydrogenase (15). Inorganic phosphate was estimated by the method of Beremblum and Chain (16). For measuring the amount of metabolite in the matrix, the space available to 3H,0 and [‘Vlsucrose was determined, in parallel experiments, as previously described (13). RESULTS
Exchange of Oxaloacetate with Intramitochondrial Oxoglutarate or Pi In Fig. la, the ability of oxaloacetate to exchange with [14C]oxoglutarate in oxoglutarate-loaded mitochondria is shown. Arsenite and rotenone were present to inhibit the oxidation of oxoglutarate. In agreement with previous results (8, 91, externally added oxaloacetate causes a significant efflux of intramitochondrial [14C]oxoglutarate, half maximal exchange being reached at 0.5 mM oxaloacetate. Analyses of supernatants and pellets for oxoglutarate, assayed enzymatically with glutamate dehydrogenase, were in good agreement with the radioactive data. Figure lb shows that oxaloacetate is also able to induce the efflux of Pi from Pi-loaded mitochondria [N-ethylmaleimide was present to inhibit the Pi carrier (17, 18) specifically]. However, the amount of oxaloacetate required for half maximal ex-
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PASSARELLA,
PALMIERI,
AND QUAGLIARIELLO
FIG. 1. (a) Exchange of intramitochondrial oxoglutarate with added oxaloacetate. [‘4C10xoglutarate-loaded mitochondria were incubated in the presence of oxaloacetate and 2 mM arsenite for 2 min at 8°C (see Materials and Methods). Mitochondrial protein was 1.7 mg. 0, without mersalyl; H, with 60 PM mersalyl; x , with 20 mM phenylsuccinate. In the absence of counteranion, the addition of 20 mM phenylsuccinate has no effect. (b) Exchange of intramitochondrial Pi with added oxaloacetate. 32P,-loaded mitochondria were incubated in the presence of oxaloacetate and 1 mM N-ethylmaleimide for 2 min at 8°C (see Materials and Methods). Mitochondrial protein was 1.9 mg. 0, without mersalyl; 0, with 60 PM mersalyl.
change with Pi is much higher. In the control experiment, the extent of the Pi/Pi exchange was larger and saturated early (not shown). To examine the contribution of the dicarboxylate carrier to the oxaloacetate/Pi and oxaloacetate/oxoglutarate exchanges, these reactions were tested for sensitivity to mersalyl (Fig. 1). While the oxaloacetate/Pi exchange is completely abolished by mersalyl, the oxaloacetateloxoglutarate exchange is unaffected. Since mersalyl at the concentrations used (20-40 nmol/mg of protein) completely inhibits the dicarboxylate carrier without affecting the oxoglutarate carrier (17-191, these results indicate that the dicarboxylate carrier is involved in the oxaloacetate/Pi exchange, but not in the oxaloacetate/oxoglutarate exchange. Figure 1 also shows that the exchange is oxaloacetateloxoglutarate strongly inhibited by phenylsuccinate, a rather powerful inhibitor of the oxoglutarate carrier (8). Exchange of Oxaloacetate with Intramitochondrial Dicarboxylates
In Figs. 2 and 3, the ability of oxaloacetate to promote efflux of dicarboxylates was tested using malonateor malateloaded mitochondria. Malonate and mal-
ate are transported by both the dicarboxylate and the oxoglutarate carriers, whereas malate, but not malonate, is also a substrate for the tricarboxylate carrier (see 20, 21). Figure 2 reports the exchange of intramitochondrial malonate with added Pi, oxaloacetate, or malonate and the sensitivity to mersalyl. All three externally added substrates cause malonate efflux, although Pi and malonate are more active than oxaloacetate: at 1 mM concentration, about 40% exchange is found with oxaloacetate compared to 80% with Pi or malonate. Furthermore, these exchange reactions show different sensitivities to mersalyl. While the PJmalonate exchange is completely abolished by mersalyl, only partial inhibition is seen on the oxaloacetate/malonate and malonate/malonate exchanges. Since the dicarboxylate carrier is completely inhibited by mersalyl, the mersalyl-insensitive part of the oxaloacetate/ malonate and malonatelmalonate exchanges points to the existence of a transport mechanism different from the dicarboxylate carrier. Figure 3 shows that the oxaloacetatel malate exchange is also partially inhibited by mersalyl at a concentration which inhibits completely the PJmalate exchange, i.e., the dicarboxylate carrier. In the same
MITOCHONDRIAL
OXALOACETATE
>---y 0
163
TRANSPORT
'---t
-e
. . . . . . . . . . w-------t ~-
25
5 ISUt3SIRAlEl
-
-----_. 75
A 10
imM)
FIG. 3. Exchange of intramitochondrial malate with added oxaloacetate, oxoglutarate. or P,, and its sensitivity to the inhibitors mersalyl, benzene-1,2,3tricarboxylate, and phenylsuccinate. \‘4C]Malateloaded mitochondria were incubated in the presence of 1 mM N-ethylmaleimide and oxaloacetate (O), oxoglutarate CO), or Pi (A) for 2 min at 8°C (see Materials and Methods). l , oxoglutarate plus 60 FM mersalyl; A, P, plus 60 FM mersalyl; n , oxaloacetate plus 60 PM mersalyl; x, oxaloacetate plus 5 mM benzene-1,2,3tricarboxylate; 0, oxaloacetate plus 60 P.M mersalyl plus 5 rnM benzene-1,2,3tricarboxylate; 0, oxaloacetate plus 10 mM phenylsuccinate. Mitochondrial protein was I.6 mg. FIG. 2. Exchange of intramitochondrial malonate with added oxaloacetate, malonate, or P,, and its sensitivity to mersalyl. f”C]Malonate-loaded mitochondria were incubated in the presence of 1 mM N-ethylmaleimide and malonate (a), oxaloacetate (b), or P, (c) for 2 min at 8°C (see Materials and Methods). H, 0, and A, 60 phi mersalyl was also included in the reaction medium. Mitochondrial protein was 1.6 mg.
experiment, it is shown that the oxoglutaratelmalate exchange, i.e., the oxoglutarate carrier, is una.fSected by mersalyl. These data suggest that the oxoglutarate carrier can mediate the exchange between oxaloacetate and malate. A contribution of the tricarboxylate carrier to the oxaloacetate/malate exchange is ruled out by the lack of inhibition by benzene-1,2,3-tricarboxylate, a powerful and specific inhibitor of the tricarboxylate carrier (22, 23). The inhibition of oxaloacetate transport by mersalyl was further analyzed in Fig. 4, where the time course of the PJmalo-
Fm. 4. Effect of mersalyl on the time course of the oxaloacetate/malonat and P,/malonate exchanges. [‘ClMalonate-loaded mitochondria were incubated in the presence of 1 mM N-ethylmaleimide and 5 mM oxaloacetate (0 and W or 5 mM P, (0 and 0) for the time indicated at 8°C (see Materials and Methods). W and 0, 60 pM mersalyl was also included in the reaction medium. Mitochondrial protein was 1.8 mg.
164
PASSARELLA,
PALMIERI,
AND
nate and oxaloacetate/malonate exchanges, in the presence and absence of the inhibitor, is shown. Equilibrium is reached after 1 and 3 min for the Pi/malenate and oxaloacetate/malonate exchanges, respectively. In the presence of mersalyl, the PJmalonate exchange is inhibited, whereas the exchange oxaloacetatelmalonate tends to reach the equilibrium value, although more slowly than its control.
change of oxaloacetate with Pi and that the oxoglutarate carrier catalyzes the exchange of oxaloacetate with oxoglutarate. Thus, phenylsuccinate and butylmalonate are inhibitors of both the dicarboxylate and the oxoglutarate carriers, but phenylsuccinate has a higher affinity than butylmalonate for the oxoglutarate carrier, whereas butylmalonate is more effective than phenylsuccinate against the dicarboxylate carrier (8, 12, 24). The apparent discrepancy between the different ability of phenylsuccinate to inhibit the two exchange reactions, illustrated in Fig. 5, and its inhibition constants [Ki = 0.7 mM for the dicarboxylate carrier and 0.25 mM for the oxoglutarate carrier (see Ref. 811 can be explained on the basis of a higher aftinity of oxaloacetate for the oxoglutarate carrier with respect to the dicarboxylate carrier, as will be shown below.
The Sensitivity of the OxaloacetatelPi and OxaloacetateIOxoglutarate Exchanges to Butylmalonate and Phenylsuccinate
To further elucidate the transport systems for oxaloacetate, the sensitivity of the oxaloacetate/Pi and oxaloacetate/oxoglutarate exchanges to butylmalonate and phenylsuccinate was investigated. Figure 5 shows that the oxaloacetate/Pi exchange is more effectively inhibited by butylmalonate than by phenylsuccinate. Thus, complete inhibition is obtained with butylmalonate at 0.5 mM concentration and with phenylsuccinate at 4 mM. On the other hand, the oxaloacetate/oxoglutarate exchange is more inhibited by phenylsuccinate than by butylmalonate. These results may be interpreted to show that the dicarboxylate carrier catalyzes the ex-
0
1 2 3 LlNHlBlTOR, crnM,
QUAGLIARIELLO
Kinetics
of Oxaloacetate
Exchange
In view of the lack of labeled oxaloacetate, the kinetics of oxaloacetate exchange were measured by following the eMux of [14Cloxoglutarate and 32Pi in the presence of N-ethylmaleimide. Figure 6 shows Lineweaver-Burk plots of oxoglutarate and Pi efflux versus oxaloacetate concentration. In this experiment, mitochondria loaded
4
300I 0
2 4 6 LlNHlBlTOR,,rnM,
FIG. 5. The sensitivity of the oxaloacetate/Pi (a) and the oxaloacetate/oxoglutarate (b) exchanges to the inhibitors phenylsuccinate and butylmalonate. 32Pi-loaded mitochondria (al and [‘4C]oxoglutarate-loaded mitochondria (bl were incubated in the presence of 2 mM arsenite, 1 mM N-ethylmaleimide, and 5 mM oxaloacetate for 2 min at 8°C (see Materials and Methods). W, with butylmalonate; 0, with phenylsuccinate. In the absence of inhibitors, the percentage of exchange was 45 in (a) and 80 in (b). These values are taken as 100%. Mitochondrial protein was 1.7 mg. In the absence of counteranion, the addition of phenylsuccinabs or butylmalonate has no effect.
MITOCHONDRIAL
OXALOACETATE
165
TRANSPORT
varied between 6.5 and 10 mM for the oxaloacetate/Pi exchange and between 0.6 and 1.2 mM for the oxaloacetate/oxoglutarate exchange. Similar values of apparent Km were also obtained when the kinetics of oxaloacetate exchange were measured using mitochondria loaded with either [14C10xoglutarate or 32Pi alone. Inhibition of Exchange oacetate
01
I 0.5
1
~/[~xALoACETATEI(~M-')
FIG. 6. The dependence of the rate of the oxaloacetate/oxoglutarate and oxaloacetate/P, exchanges on the external oxaloacetate concentration. Mitochondria loaded with both [‘%loxoglutarate and 12P, (2.2 mg of protein) were incubated in a medium containing 0.2 M sucrose, 20 mM HEPESTris, pH 7.0, 10 mM KCl, 1 mM MgC12, 1 pg of rotenone, 2 mM arsenite, 3 pg of oligomycin, and 1 mM N-ethylmaleimide. After 1 min of incubation, the assay was started with oxaloacetate, at the concentrations indicated, and was stopped after 6 s by the addition of 20 mM butylmalonate and 20 mM phenylsuccinate. Other conditions were as indicated under Materials and Methods. Aft,er centrifuging the mitochondria, the percentage of exchange obtained with increasing concentrations of oxaloacetate was measured. The amounts of Pi and oxoglutarate present in the mitochondrial matrix prior to the addition of oxaloacetate (20 nmol of PJmg of protein and 15 nmol of oxoglutarate/mg of protein) were measured as described under Materials and Methods. These values were used to calculate the rate of the oxaloacetate/oxoglutarate (0) and oxaloacetate/S (0) exchanges. V is expressed as micromoles per minute per gram of protein.
with both [14Cloxoglutarate and “Pi (see Materials and Methods) were used for comparative purposes. The slopes show that the apparent K, of the oxaloacetate/ Pi exchange (K, = 9 mM> is severalfold higher than that of the oxaloacetate/oxoglutarate exchange (K, = 1.2 mM). In a series of five experiments, the K, values
Reactions
by Oxal-
It was previously shown (8) that oxaloacetate inhibits the rate of oxoglutarate/ malate exchange in a competitive manner (Kj = 1.1 mM). The nature of the inhibition of the oxoglutarate carrier by oxaloacetate has been reinvestigated by studying the effect of oxaloacetate on the rate of oxoglutarate/oxoglutarate exchange. At variance with the previous experiments, arsenite and mersalyl were also present, both in the loading procedure and in the reaction mixture. Experiments that are not shown confirmed that the inhibition of the oxoglutarate carrier by oxaloacetate is competitive with respect to oxoglutarate.
I 0
I 20 IO I, k4elLONATEI (rnW’1
25 ,I I PHOSPHATTEIlrndl
5
FIG. 7. Inhibition of the influx of [Wmalonate (a) and :12P1 (b) by oxaloacetate. The reaction mixture contained 0.2 M sucrose, 20 mM HEPES-Tris, pH 7.0,10 mM KCl, 1 mM MgC12, 1 pg of rotenone, 3 pg of oligomycin, 1 mM N-ethylmaleimide, Pi-loaded mitochondria [1.9 mg of protein in (a) and 1.7 mg in (b)], and (added at time zero) [‘%]malonate or 32Pi at the concentrations indicated. 0 and 0, controls; 0, with 5 mM oxaloacetate and n , with 3 mM oxaloacetate added simultaneously with the labeled substrate. The assay was stopped after 4 s by the addition of 20 mM butylmalonate. Other conditions were as indicated under Materials and Methods. V is expressed as micromoles per minute per gram of protein
166
PASSARELLA,
PALMIERI,
The Kj values varied between 0.8 and 1.2 mM in three experiments. Previous work (12, 25-27) has shown that the dicarboxylate carrier has two separate binding sites, one specific for Pi and the other specific for the dicarboxylates. Figure 7 reports the effect of oxaloacetate on the transport of malonate and phosphate by the dicarboxylate carrier (Nethylmaleimide was added to inhibit Pi/ OH and Pi/Pi exchanges catalyzed by the Pi carrier). The data, presented as doublereciprocal plots, indicate that oxaloacetate competitively increases the K, of the malonate/Pi exchange without changing the V and yields an apparent Ki of 2.5 mM. On the other hand, the oxaloacetate inhibition of the Pi/Pi exchange is noncompetitive. Exchange of Oxaloacetate with chondrial Anions in Heart dria
IntramitoMitochon-
Sluse et al. (28) have shown that, in ratheart mitochondria, the activity of the dicarboxylate carrier is negligible at 4’33, whereas the oxoglutarate carrier is still active at this temperature. To investigate whether or not oxaloacetate is transported by the oxoglutarate carrier, rat-heart mitochondria were loaded with 32Pi or [14Cloxoglutarate and incubated in the presence of 5 mM oxaloacetate at 4°C. It was found that oxaloacetate causes a significant eMux of intramitochondrial oxoglutarate under conditions where no exchange is observed with 32Pi, indicating the occurrence of oxaloacetate transport via the oxoglutarate carrier. DISCUSSION
So far, the uptake of oxaloacetate by isolated mitochondria has been estimated only indirectly by measuring the rate of oxidation of intramitochondrial nicotinamide nucleotides upon addition of oxaloacetate to mitochondria (l-7, but see 8, 9). Using this technique, Gimpel et al. (7) have suggested that oxaloacetate is transported across the mitochondrial membrane by the dicarboxylate but not the oxoglutarate carrier. The use of the oxoglutarate carrier by oxaloacetate is directly shown by the ob-
AND
QUAGLIARIELLO
servations presented in this paper. Addition of oxaloacetate causes oxoglutarate efflux from mitochondria in a phenylsuccinate-sensitive reaction (Fig. 1). Efflux of labeled oxoglutarate was previously reported by us (8,9), but arsenite and mersalyl were not included in the reaction medium. A possible explanation of these results, offered by Gimpel et al. (see Ref. 7), is that, in oxoglutarate-loaded mitochondria, oxaloacetate is not exchanged for oxoglutarate, but for a small amount of Pi or malate (still present in these mitochondria) via the dicarboxylate carrier. Inside the mitochondria, the oxaloacetate would be reduced to malate, which in turn could be exchanged for further oxaloacetate. The extramitochondrial malate would then be taken up in exchange for oxoglutarate. The main indication against this explanation is the lack of inhibition of the oxaloacetate/oxoglutarate exchange by mersalyl, which, at the concentration used, completely inhibits the dicarboxylate carrier. The subsequent experimental data reported give further evidence for the contention that oxaloacetate can be transported via the oxoglutarate carrier. The oxaloacetateloxoglutarate exchange is more effectively inhibited by phenylsuccinate than by butylmalonate, in agreement with their inhibition constants toward the oxoglutarate carrier (8). Mersalyl inhibits the rate, but not the extent, of the exchanges between added oxaloacetate and intramitochondrial malonate or malate. The most straightforward explanation of this result would seem to be that malonate and malate are both substrates of the dicarboxylate and the oxoglutarate carrier, and mersalyl, by inhibiting the dicarboxylate carrier, decreases the rate of the exchanges. In heart mitochondria, under conditions where the dicarboxylate carrier is inactive, the oxaloacetate/oxoglutarate exchange is operating. The present study gives direct evidence that oxaloacetate can also be transported by the dicarboxylate carrier, although less efficiently. The data of Fig. 1 demonstrate that added oxaloacetate exchanges with intramitochondrial Pi, in the presence of N-ethylmaleimide, i.e., under conditions
MITOCHONDRIAL
OXALOACETATE
where Pi transport is catalyzed only by the dicarboxylate carrier. The oxaloacetate/Pi exchange is inhibited by known inhibitors of the dicarboxylate carrier, i.e, mersalyl, butylmalonate, and phenylsuccinate. The proposed existence of different binding sites for Pi and dicarboxylates on the dicarboxylate carrier (12, 25-27) raises the question of the site of oxaloacetate binding. Our data suggest that oxaloacetate binds at the dicarboxylate-binding site and away from the Pi-binding site, since the inhibition of the dicarboxylate carrier by oxaloacetate is competitive with respect to malonate, but noncompetitive with respect to Pi (Fig. 7). The kinetic data of the oxaloacetate exchange (Fig. 6) reveal that, at low substrate concentration, the contribution of the dicarboxylate carrier to the oxaloacetate translocation is relatively minor with respect to that given by the oxoglutarate carrier. It is likely that, due to the presence of a ketonic group, the binding affinity of oxaloacetate to the dicarboxylate carrier and/or the mobility of the carriersubstrate complex are severely limited. The apparent higher capacity of mitochondria to transport oxaloacetate via the oxoglutarate carrier than via the dicarboxylate carrier can account for some of the findings reported in the literature, e.g., the lack of inhibition by Pi of the oxaloacetate-induced oxidation of intramitochondrial NAD(P)H and a higher effectiveness of succinate than malonate in inhibiting oxaloacetate uptake by mitochondria (2). Thus, Pi is not transported on the oxoglutarate carrier, and succinate is a better substrate than malonate for the oxoglutarate carrier (8). It is interesting to note that the efflux of large amounts of oxaloacetate from isolated mitochondria has been observed in the presence of oxoglutarate (29, 30). In these investigations on the energylinked H transfer between malate and oxoglutarate plus NH, in the mitochondria, glutamate is formed and malate is oxidized to oxaloacetate. In view of the present results, it becomes obvious that in these experiments oxaloacetate has been extruded in exchange with oxoglutarate by the oxoglutarate carrier.
TRANSPORT
167
In the intact cell, the concentration of free oxaloacetate is very low in the mitochondria (31-33) and much higher in the cytosol (34). An interesting implication to the intact tissue of the data presented in this paper is that an oxaloacetate influx and malate efflux via the oxoglutarate carrier (either by a direct exchange or by a combined oxaloacetate/oxoglutarate and oxoglutarate/malate exchange) may contribute to the transfer of reducing equivalents from mitochondria to cytosol. In heart, Illingworth et al. (35) have recently postulated an oxaloacetate-malate exchange across the mitochondrial membrane as the only possibility of accounting for the efflux of reducing equivalents. REFERENCES
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9.
10.
*-.
11
12.
13.
14.
HASLAM, J. M., AND KREBS, H. A. (1968) Biothem. J. 107, 659-667. HASLAM, J. M., AND GRIFFITHS, D. E. (1968) Biochem. J. 109, 921-928. WOJTCZAK, A. B. (1969) Biochim. Biophys. Actu 172, 52-65. KUNZ, W., B~HME, G., BOHNENSACK, R., AND LUTZE, G. (19691 in Biochemistry of Intracellular Structures (Wojtczak, L., Drabikowski, W., and Strelecka-Golaszewska, H., eds.), pp. 35-54, Polish Scientific, Warszawa. BOHNENSACK, R., AND KUNZ, W. (1971)Biochim. Biophys. Actu 226, 33-41. ROBINSON, B. H., AND CHAPPELL, J. B. (1967) Biochem. J. 105, 18P. GIMPEL, G. A., DE HAAN, E. J., AND TAGER, J. M. (1973) Biochim. Biophys. Acta 292, 582591. PALMIERI, F., QUAGLIARIELLO, E., AND KLINGENBERG, M. (1972) Eur. J. Biochem. 29, 408416. PALMIERI, F. (1973) in Mechanisms in Bioenergetics (Azzone, G. F., Ernster, L., Papa, S., Quagliariello, E., and Siliprandi, N., eds.), pp. 375-385, Academic Press, New York. KLINGENBERG, M., AND SLENCZKA, W. (1959) Biochem. Z. 331, 486-517. TYLER, D. D., AND G~NZE, J. (1967) in. Methods in Enzymology (Estabrook, R. W., and Pulman, M. E., eds.1, Vol. 10, pp. 75-77, Academic Pres, New York. PALMIERI, F., PREZIOSO, G., QUAGLIARIELLO, E., AND KLINGENBERG, M. (1971) Eur. J. Biothem. 22, 66-74. PALMIERI, F., QUAGLIARIELLO, E., AND KLINGENBERG, M. (1970) Eur. J. Biochem. 17, 230238. KRBGER, A., AND KLINGENBERG, M. (1966) Bio-
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PASSARELLA,
PALMIERI,
them. 2. 344, 317-336. 15. BERGMEYER, H. U., AND BERNT, E. (1963) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 324-327, Academic Press, New York. 16. BEREMBLUM, I., AND CHAIN, E. (1938) Biochem. J. 32, 295-298. 17. MEIJER, A. J., GROOT, G. S. P., AND TAGER, J. M. (1970) FEBS Lett. 8, 41-44. 18. ROBINSON, B. H. (1970) FEBS Lett. 11, 200-204. 19. QIJAGLIARIELLO, E., AND PALMIERI, F. (1971) Biochim. Appl. 18, 191-219. 20. CHAPPELL, J. B. (1968) Brit. Med. Bull. 24, 150157. 21. KLINGENBERG, M. (1970) in Essays in Biochemistry (Campbell, P. N., and Dickens, F., eds.), Vol. 6, pp. 119-159, Academic Press, London. 22. PALMIERI, F., STIPANI, I., QUAGLIARIELLO, E., AND KLINGENBERG, M. (1972) Eur. J. Biothem. 26, 587-594. 23. ROBINSON, B. R., WILLIAMS, G. R., HALPERIN, M. L. AND LEZONOFF, C. C. (1971) Eur. J. Biochem. 20, 65-71. 24. ROBINSON, B. H., AND CHAPPELL, J. B. (1967) Biochim. Biophys. Res. Commun. 28,249-255. 25. PALMIERI, F., PASSARELLA, S., STIPANI, I., AND QUAGLIARIELLO, E. (1974) Biochim. Biophys. A& 333, 195-208. 26. QUAGLIARIELLO, E., PASSARELLA, S., AND PALMIERI, F. (1974) in Dynamics of Energy-Trans-
AND
27.
28. 29.
30. 31. 32.
33.
34. 35.
QUAGLIARIELLO ducing Membranes (Ernster, L., Estabrook, R. W., and Slater, E. C., eds.), p. 483-495, Elsevier, Amsterdam. CROMPTON, M., PALMIERI, F., CAPANO, M., AND QUAGLIARIELLO, E. (1975) B&hem. J. 146, 667-673. SLUSE, F. E., MEIJER, A. J., AND TAGER, J. M. (1971) FEBS Lett. 18, 149-153. KLINGENBERG, M. (1962) in Redoxfunktionen Cytoplasmaticher Strukturen, pp. 163-187, Springer-Verlag, Berlin. KLINGENBERG, M., VON HAFEN, H., AND WENSKE, G. (1965) Biochem. 2. 343, 452-478. WILLIAMSON, J. R., SCHOLZ, R., AND BROWNING, E. T. (1969) J. Biol. Chem. 244, 4617-4627. WILLIAMSON, J. R. (1976) in Gluconeogenesis (Hanson, R. W., and Mehlman, M. A., eds.), pp. 165-220, Wiley, New York. ACKRELL, B. A. C. (1974) in Horizons in Biochemistry and Biophysics (Quagliariello, E., Palmieri, F., and Singer T. P., eds.), Vol. 1, pp. 175-219, Addison-Wesley, Reading, Mass. SAFER, B., AND WILLIAMSON, J. R. (1973)5. Biol. Chem. 248, 2570-2579. ILLINGWORTH, J. A., FORD, W. C. L., KOBAYASHI, K., AND WILLIAMSON, J. R. (1975) in Recent Advances in Studies on Cardiac Structure and Metabolism (Roy, P. E., and Harris, P., eds.), Vol. 8, pp. 271-290, University Park Press, Baltimore.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
The Transport
180,
160-168
of Oxaloacetate
S. PASSARELLA, Department
F. PALMIERI, of Biochemistry, Received
(1977)
in Isolated AND University
July
Mitochondria
E. QUAGLIARIELLO of Bari,
Italy
6, 1976
The mechanism of mitochondrial oxaloacetate transport has been investigated by measuring the rate and the extent of exchange reactions between intramitochondrial anions and added oxaloacetate. The exchange between oxaloacetate and intramitochondrial oxoglutarate is insensitive to mersalyl at a concentration which completely inhibits the dicarboxylate carrier. Oxaloacetate causes efflux of intramitochondrial Pi, malonate, and malate. Mersalyl inhibits completely the oxaloacetate/Pi exchange, but only partially the oxaloacetate/malonat and the oxaloacetate/malate exchanges. The inhibition of the last two reactions decreases on increasing the time of incubation. Butylmalonate inhibits more than phenylsuccinate the exchange oxaloacetate&“P,,. , whereas phenylsuccinate is a more effective inhibitor than butylmalonate of the oxaloacetat~,,/[‘%!]oxoglutarat+~ exchange. The apparent K, values ranged from 0.6 to 1.2 mM for the oxaloacetate/oxoglutarate exchange and from 6.5 to 10 mM for the oxaloacetat.e/Pi exchange. The inhibition of oxoglutarate uptake by oxaloacetate is competitive. Oxaloacetate inhibits the malonate/Pi exchange competitively and it is a noncompetitive inhibitor of the Pi/P, exchange. It is concluded that oxaloacetate may be transported across the mitochondrial membrane by the oxoglutarate carrier and, much less effectively, by the dicarboxylate carrier. The implications of these findings are discussed. acid (sodium salt), [U-‘4Clsucrose, and 3H,0 were obtained from the Radiochemical Center (Amersham, England); rotenone was from F. P. Penick and Co. (New York); N-ethylmaleimide, oligomycin, and mersalyl were from Sigma; 2-phenylsuccinic acid was from K & K Laboratories, Inc. (Plainview, New York); oxaloacetate and glutamate dehydrogenase were from Boehringer; benzene-1,2,3-tricarboxylic acid and 2-butylmalonic acid were from Aldrich (Milwaukee, Wisconsin). Loading of mitochondria with labeled metabolites. Rat-liver or rat-heart mitochondria were isolated as previously described (10, 111, using a medium consisting of 0.25 M sucrose, 1 mM EGTA,’ and 20 mM Tris-HCl, pH 7.2. The mitochondria (40-50 mg of protein) were incubated at 20°C in 10 ml of medium consisting of 100 rnM KCl, 1 rnM EGTA, 20 mM Tris-HCl, pH 6.8, and one of the following metabolites (2 mM): Pi, malonate, malate, or 2-oxoglutarate. In this incubation medium, 1 pg/ml of rotenone was also present to block NADH dehydrogenase and therefore inhibit the oxidation of NAD+dependent substrates. When mitochondria were loaded with P,, the pH was 6.4 and 5 *g/ml of oligomycin was added to inhibit the incorporation of P,
Although several investigations in the last decade have indicated that oxaloacetate can cross the inner mitochondrial membrane (l-91, the mechanism by which the 0x0 acid is transported is still a matter of debate. Gimpel et al. (7) concluded that, in rat-liver mitochondria, oxaloacetate is transported only by the dicarboxylate carrier. However, in a study on the kinetics and the specificity of the oxoglutarate carrier, it was suggested that oxaloacetate is a substrate of the oxoglutarate carrier (8, 9). In this paper, the problem of the permeability of mitochondria to oxaloacetate is reinvestigated by measuring the exchange between intra- and extramitochondrial anions and by testing their sensitivity to inhibitors. The kinetics of oxaloacetate exchange and the competition with other anions are analyzed for possible carrier mechanisms. MATERIALS [32PlPhosphoric C4]malonic acid
acid, (sodium
AND
METHODS
~-lU-~~C]malic acid, salt), 2-ox0-[5-‘~Clglutaric
’ Abbreviations used: EGTA, ethylenenitrilolltetraacetic acid: droxyethyl)-1-piperazineethanesulfonic
ll-
lethylene-bis(oxyHEPES, 4-(2-hyacid.
160 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
MITOCHONDRIAL
OXALOACETATE
into ATP. When 2-oxoglutarate was used, 2 mM arsenite was added to inhibit oxoglutarate dehydrogenase. After 2 min, the mitochondria were washed in the medium without the metabolite and then suspended (40-50 nig of mitochondrial protein/ml). The intramitochondrial metabolite was labeled by adding to the mitochondrial suspension carrier-free labeled metabolite (approximately 1 &i/ml of mitochondrial suspension, i.e., about 0.02 &i/nmol and about 1 nmol/mg of mitochondrial protein, except for ,,Pi, whose activity was about lo” times higher). The mitochondria, loaded in this way and suspended in 1 ml of reaction mixture in the absence of counteranion for 2 min at 8”C, contained labeled metabolites at a concentration ranging from 12 to 24 mM, whereas the extramitochondrial concentration was less than 20 WM. In some experiments, the mitochondria were labeled with both [‘YJloxoglutarate and 32Pi. This was achieved by loading the mitochondria with 2-oxoglutarate, as described above, and by introducing to the mitochondrial suspension carrier-free [“Cloxoglutarate and carrier-free 3zPi, in the amount of 1 &i/ ml each. This is justified, since oxoglutarate does not exchange with Pi and the intramitochondrial content of Pi is still high in oxoglutarate-loaded mitochondria. Equilibration of the radioisotope between the extramitochondrial and intramitochondrial pools of oxoglutarate and Pi is obtained after 10 min at 0°C. Arsenite and oligomycin were present all through the entire procedure. Measurement of the exchange between intra- and extramitochondrial metabolites. Mitochondria loaded with labeled metabolite (about 2 mg of protein) were incubated at 8°C in 1.0 ml of medium consisting of 100 mM KCl, 1 mM EGTA, 20 mM 4-(2hydroxyethyll-l-piperazineethanesulfonic acid-Tris (HEPES-Tris), pH 7.0, in the presence of 2 pg of rotenone and 5 c(g of oligomycin. In some reactions, the medium included the inhibitors arsenite, Nethylmaleimide, mersalyl, phenylsuccinate, butylmalonate, or benzene-1,2,3-tricarboxylate, as indicated in the figure legends. After 1 min, the exchange was initiated by addition of the unlabeled metabolite and terminated 2 min later (unless otherwise specified) by centrifugation in an Eppendorf microcentrifuge. At 2 min, the exchange is at or near the equilibrium. The percentage exchange was calculated according to the equation : percentage exchange = 100 (Ca - Cp)lCa, where Cp and Ca represent the radioactivity measured in the presence and absence of external anions, respectively. Ca was approximately 2 x 10” cpm/mitochondrial pellet. In the absence of external anion, the mitechondria lose less than 10% of their labeled intramitochondrial metabolite in 2 min at 8°C. The results were corrected for this small time-dependent spontaneous leakage of radioactivity. Measurement of the kinetics of metabolite er-
TRANSPORT
161
change. The kinetics of the exchange were studied by using the inhibitor stop method, essentially according to the procedure described previously (12). Mitochondria loaded with the indicated metabolite were incubated at 8°C in 1.0 ml of medium consisting of 0.2 M sucrose, 10 mM KCl, 20 mM HEPESTris, pH 7.0, 1 IIIM MgCIP, 2 mM arsenite, 1 pg of rotenone, and 3 pg of oligomycin. When phosphateloaded mitochondria were used, the suspension also contained 1 mM N-ethylmaleimide. After 1 min, the assay was started by addition of unlabeled (for efflux) or labeled (for uptake) substrate and terminated 4-6 s later by rapid addition of an inhibitor. During this period, the rate of exchange was constant within the limits of experimental error. Butylmalonate (20 mM) and 20 mM phenylsuccinate were used to inhibit the dicarboxylate and the oxoglutarate carrier, respectively. Other methods. After rapidly centrifuging the mitochondria in an Eppendorf microcentrifuge for 1 min at O”C, the radioactivity in the pellets and supernatants was measured as described previously (13). The mitochondrial protein was determined by a modified biuret method (14). In some experiments, oxoglutarate was assayed enzymatically with glutamate dehydrogenase (15). Inorganic phosphate was estimated by the method of Beremblum and Chain (16). For measuring the amount of metabolite in the matrix, the space available to 3H,0 and [‘Vlsucrose was determined, in parallel experiments, as previously described (13). RESULTS
Exchange of Oxaloacetate with Intramitochondrial Oxoglutarate or Pi In Fig. la, the ability of oxaloacetate to exchange with [14C]oxoglutarate in oxoglutarate-loaded mitochondria is shown. Arsenite and rotenone were present to inhibit the oxidation of oxoglutarate. In agreement with previous results (8, 91, externally added oxaloacetate causes a significant efflux of intramitochondrial [14C]oxoglutarate, half maximal exchange being reached at 0.5 mM oxaloacetate. Analyses of supernatants and pellets for oxoglutarate, assayed enzymatically with glutamate dehydrogenase, were in good agreement with the radioactive data. Figure lb shows that oxaloacetate is also able to induce the efflux of Pi from Pi-loaded mitochondria [N-ethylmaleimide was present to inhibit the Pi carrier (17, 18) specifically]. However, the amount of oxaloacetate required for half maximal ex-
162
PASSARELLA,
PALMIERI,
AND QUAGLIARIELLO
FIG. 1. (a) Exchange of intramitochondrial oxoglutarate with added oxaloacetate. [‘4C10xoglutarate-loaded mitochondria were incubated in the presence of oxaloacetate and 2 mM arsenite for 2 min at 8°C (see Materials and Methods). Mitochondrial protein was 1.7 mg. 0, without mersalyl; H, with 60 PM mersalyl; x , with 20 mM phenylsuccinate. In the absence of counteranion, the addition of 20 mM phenylsuccinate has no effect. (b) Exchange of intramitochondrial Pi with added oxaloacetate. 32P,-loaded mitochondria were incubated in the presence of oxaloacetate and 1 mM N-ethylmaleimide for 2 min at 8°C (see Materials and Methods). Mitochondrial protein was 1.9 mg. 0, without mersalyl; 0, with 60 PM mersalyl.
change with Pi is much higher. In the control experiment, the extent of the Pi/Pi exchange was larger and saturated early (not shown). To examine the contribution of the dicarboxylate carrier to the oxaloacetate/Pi and oxaloacetate/oxoglutarate exchanges, these reactions were tested for sensitivity to mersalyl (Fig. 1). While the oxaloacetate/Pi exchange is completely abolished by mersalyl, the oxaloacetateloxoglutarate exchange is unaffected. Since mersalyl at the concentrations used (20-40 nmol/mg of protein) completely inhibits the dicarboxylate carrier without affecting the oxoglutarate carrier (17-191, these results indicate that the dicarboxylate carrier is involved in the oxaloacetate/Pi exchange, but not in the oxaloacetate/oxoglutarate exchange. Figure 1 also shows that the exchange is oxaloacetateloxoglutarate strongly inhibited by phenylsuccinate, a rather powerful inhibitor of the oxoglutarate carrier (8). Exchange of Oxaloacetate with Intramitochondrial Dicarboxylates
In Figs. 2 and 3, the ability of oxaloacetate to promote efflux of dicarboxylates was tested using malonateor malateloaded mitochondria. Malonate and mal-
ate are transported by both the dicarboxylate and the oxoglutarate carriers, whereas malate, but not malonate, is also a substrate for the tricarboxylate carrier (see 20, 21). Figure 2 reports the exchange of intramitochondrial malonate with added Pi, oxaloacetate, or malonate and the sensitivity to mersalyl. All three externally added substrates cause malonate efflux, although Pi and malonate are more active than oxaloacetate: at 1 mM concentration, about 40% exchange is found with oxaloacetate compared to 80% with Pi or malonate. Furthermore, these exchange reactions show different sensitivities to mersalyl. While the PJmalonate exchange is completely abolished by mersalyl, only partial inhibition is seen on the oxaloacetate/malonate and malonate/malonate exchanges. Since the dicarboxylate carrier is completely inhibited by mersalyl, the mersalyl-insensitive part of the oxaloacetate/ malonate and malonatelmalonate exchanges points to the existence of a transport mechanism different from the dicarboxylate carrier. Figure 3 shows that the oxaloacetatel malate exchange is also partially inhibited by mersalyl at a concentration which inhibits completely the PJmalate exchange, i.e., the dicarboxylate carrier. In the same
MITOCHONDRIAL
OXALOACETATE
>---y 0
163
TRANSPORT
'---t
-e
. . . . . . . . . . w-------t ~-
25
5 ISUt3SIRAlEl
-
-----_. 75
A 10
imM)
FIG. 3. Exchange of intramitochondrial malate with added oxaloacetate, oxoglutarate. or P,, and its sensitivity to the inhibitors mersalyl, benzene-1,2,3tricarboxylate, and phenylsuccinate. \‘4C]Malateloaded mitochondria were incubated in the presence of 1 mM N-ethylmaleimide and oxaloacetate (O), oxoglutarate CO), or Pi (A) for 2 min at 8°C (see Materials and Methods). l , oxoglutarate plus 60 FM mersalyl; A, P, plus 60 FM mersalyl; n , oxaloacetate plus 60 PM mersalyl; x, oxaloacetate plus 5 mM benzene-1,2,3tricarboxylate; 0, oxaloacetate plus 60 P.M mersalyl plus 5 rnM benzene-1,2,3tricarboxylate; 0, oxaloacetate plus 10 mM phenylsuccinate. Mitochondrial protein was I.6 mg. FIG. 2. Exchange of intramitochondrial malonate with added oxaloacetate, malonate, or P,, and its sensitivity to mersalyl. f”C]Malonate-loaded mitochondria were incubated in the presence of 1 mM N-ethylmaleimide and malonate (a), oxaloacetate (b), or P, (c) for 2 min at 8°C (see Materials and Methods). H, 0, and A, 60 phi mersalyl was also included in the reaction medium. Mitochondrial protein was 1.6 mg.
experiment, it is shown that the oxoglutaratelmalate exchange, i.e., the oxoglutarate carrier, is una.fSected by mersalyl. These data suggest that the oxoglutarate carrier can mediate the exchange between oxaloacetate and malate. A contribution of the tricarboxylate carrier to the oxaloacetate/malate exchange is ruled out by the lack of inhibition by benzene-1,2,3-tricarboxylate, a powerful and specific inhibitor of the tricarboxylate carrier (22, 23). The inhibition of oxaloacetate transport by mersalyl was further analyzed in Fig. 4, where the time course of the PJmalo-
Fm. 4. Effect of mersalyl on the time course of the oxaloacetate/malonat and P,/malonate exchanges. [‘ClMalonate-loaded mitochondria were incubated in the presence of 1 mM N-ethylmaleimide and 5 mM oxaloacetate (0 and W or 5 mM P, (0 and 0) for the time indicated at 8°C (see Materials and Methods). W and 0, 60 pM mersalyl was also included in the reaction medium. Mitochondrial protein was 1.8 mg.
164
PASSARELLA,
PALMIERI,
AND
nate and oxaloacetate/malonate exchanges, in the presence and absence of the inhibitor, is shown. Equilibrium is reached after 1 and 3 min for the Pi/malenate and oxaloacetate/malonate exchanges, respectively. In the presence of mersalyl, the PJmalonate exchange is inhibited, whereas the exchange oxaloacetatelmalonate tends to reach the equilibrium value, although more slowly than its control.
change of oxaloacetate with Pi and that the oxoglutarate carrier catalyzes the exchange of oxaloacetate with oxoglutarate. Thus, phenylsuccinate and butylmalonate are inhibitors of both the dicarboxylate and the oxoglutarate carriers, but phenylsuccinate has a higher affinity than butylmalonate for the oxoglutarate carrier, whereas butylmalonate is more effective than phenylsuccinate against the dicarboxylate carrier (8, 12, 24). The apparent discrepancy between the different ability of phenylsuccinate to inhibit the two exchange reactions, illustrated in Fig. 5, and its inhibition constants [Ki = 0.7 mM for the dicarboxylate carrier and 0.25 mM for the oxoglutarate carrier (see Ref. 811 can be explained on the basis of a higher aftinity of oxaloacetate for the oxoglutarate carrier with respect to the dicarboxylate carrier, as will be shown below.
The Sensitivity of the OxaloacetatelPi and OxaloacetateIOxoglutarate Exchanges to Butylmalonate and Phenylsuccinate
To further elucidate the transport systems for oxaloacetate, the sensitivity of the oxaloacetate/Pi and oxaloacetate/oxoglutarate exchanges to butylmalonate and phenylsuccinate was investigated. Figure 5 shows that the oxaloacetate/Pi exchange is more effectively inhibited by butylmalonate than by phenylsuccinate. Thus, complete inhibition is obtained with butylmalonate at 0.5 mM concentration and with phenylsuccinate at 4 mM. On the other hand, the oxaloacetate/oxoglutarate exchange is more inhibited by phenylsuccinate than by butylmalonate. These results may be interpreted to show that the dicarboxylate carrier catalyzes the ex-
0
1 2 3 LlNHlBlTOR, crnM,
QUAGLIARIELLO
Kinetics
of Oxaloacetate
Exchange
In view of the lack of labeled oxaloacetate, the kinetics of oxaloacetate exchange were measured by following the eMux of [14Cloxoglutarate and 32Pi in the presence of N-ethylmaleimide. Figure 6 shows Lineweaver-Burk plots of oxoglutarate and Pi efflux versus oxaloacetate concentration. In this experiment, mitochondria loaded
4
300I 0
2 4 6 LlNHlBlTOR,,rnM,
FIG. 5. The sensitivity of the oxaloacetate/Pi (a) and the oxaloacetate/oxoglutarate (b) exchanges to the inhibitors phenylsuccinate and butylmalonate. 32Pi-loaded mitochondria (al and [‘4C]oxoglutarate-loaded mitochondria (bl were incubated in the presence of 2 mM arsenite, 1 mM N-ethylmaleimide, and 5 mM oxaloacetate for 2 min at 8°C (see Materials and Methods). W, with butylmalonate; 0, with phenylsuccinate. In the absence of inhibitors, the percentage of exchange was 45 in (a) and 80 in (b). These values are taken as 100%. Mitochondrial protein was 1.7 mg. In the absence of counteranion, the addition of phenylsuccinabs or butylmalonate has no effect.
MITOCHONDRIAL
OXALOACETATE
165
TRANSPORT
varied between 6.5 and 10 mM for the oxaloacetate/Pi exchange and between 0.6 and 1.2 mM for the oxaloacetate/oxoglutarate exchange. Similar values of apparent Km were also obtained when the kinetics of oxaloacetate exchange were measured using mitochondria loaded with either [14C10xoglutarate or 32Pi alone. Inhibition of Exchange oacetate
01
I 0.5
1
~/[~xALoACETATEI(~M-')
FIG. 6. The dependence of the rate of the oxaloacetate/oxoglutarate and oxaloacetate/P, exchanges on the external oxaloacetate concentration. Mitochondria loaded with both [‘%loxoglutarate and 12P, (2.2 mg of protein) were incubated in a medium containing 0.2 M sucrose, 20 mM HEPESTris, pH 7.0, 10 mM KCl, 1 mM MgC12, 1 pg of rotenone, 2 mM arsenite, 3 pg of oligomycin, and 1 mM N-ethylmaleimide. After 1 min of incubation, the assay was started with oxaloacetate, at the concentrations indicated, and was stopped after 6 s by the addition of 20 mM butylmalonate and 20 mM phenylsuccinate. Other conditions were as indicated under Materials and Methods. Aft,er centrifuging the mitochondria, the percentage of exchange obtained with increasing concentrations of oxaloacetate was measured. The amounts of Pi and oxoglutarate present in the mitochondrial matrix prior to the addition of oxaloacetate (20 nmol of PJmg of protein and 15 nmol of oxoglutarate/mg of protein) were measured as described under Materials and Methods. These values were used to calculate the rate of the oxaloacetate/oxoglutarate (0) and oxaloacetate/S (0) exchanges. V is expressed as micromoles per minute per gram of protein.
with both [14Cloxoglutarate and “Pi (see Materials and Methods) were used for comparative purposes. The slopes show that the apparent K, of the oxaloacetate/ Pi exchange (K, = 9 mM> is severalfold higher than that of the oxaloacetate/oxoglutarate exchange (K, = 1.2 mM). In a series of five experiments, the K, values
Reactions
by Oxal-
It was previously shown (8) that oxaloacetate inhibits the rate of oxoglutarate/ malate exchange in a competitive manner (Kj = 1.1 mM). The nature of the inhibition of the oxoglutarate carrier by oxaloacetate has been reinvestigated by studying the effect of oxaloacetate on the rate of oxoglutarate/oxoglutarate exchange. At variance with the previous experiments, arsenite and mersalyl were also present, both in the loading procedure and in the reaction mixture. Experiments that are not shown confirmed that the inhibition of the oxoglutarate carrier by oxaloacetate is competitive with respect to oxoglutarate.
I 0
I 20 IO I, k4elLONATEI (rnW’1
25 ,I I PHOSPHATTEIlrndl
5
FIG. 7. Inhibition of the influx of [Wmalonate (a) and :12P1 (b) by oxaloacetate. The reaction mixture contained 0.2 M sucrose, 20 mM HEPES-Tris, pH 7.0,10 mM KCl, 1 mM MgC12, 1 pg of rotenone, 3 pg of oligomycin, 1 mM N-ethylmaleimide, Pi-loaded mitochondria [1.9 mg of protein in (a) and 1.7 mg in (b)], and (added at time zero) [‘%]malonate or 32Pi at the concentrations indicated. 0 and 0, controls; 0, with 5 mM oxaloacetate and n , with 3 mM oxaloacetate added simultaneously with the labeled substrate. The assay was stopped after 4 s by the addition of 20 mM butylmalonate. Other conditions were as indicated under Materials and Methods. V is expressed as micromoles per minute per gram of protein
166
PASSARELLA,
PALMIERI,
The Kj values varied between 0.8 and 1.2 mM in three experiments. Previous work (12, 25-27) has shown that the dicarboxylate carrier has two separate binding sites, one specific for Pi and the other specific for the dicarboxylates. Figure 7 reports the effect of oxaloacetate on the transport of malonate and phosphate by the dicarboxylate carrier (Nethylmaleimide was added to inhibit Pi/ OH and Pi/Pi exchanges catalyzed by the Pi carrier). The data, presented as doublereciprocal plots, indicate that oxaloacetate competitively increases the K, of the malonate/Pi exchange without changing the V and yields an apparent Ki of 2.5 mM. On the other hand, the oxaloacetate inhibition of the Pi/Pi exchange is noncompetitive. Exchange of Oxaloacetate with chondrial Anions in Heart dria
IntramitoMitochon-
Sluse et al. (28) have shown that, in ratheart mitochondria, the activity of the dicarboxylate carrier is negligible at 4’33, whereas the oxoglutarate carrier is still active at this temperature. To investigate whether or not oxaloacetate is transported by the oxoglutarate carrier, rat-heart mitochondria were loaded with 32Pi or [14Cloxoglutarate and incubated in the presence of 5 mM oxaloacetate at 4°C. It was found that oxaloacetate causes a significant eMux of intramitochondrial oxoglutarate under conditions where no exchange is observed with 32Pi, indicating the occurrence of oxaloacetate transport via the oxoglutarate carrier. DISCUSSION
So far, the uptake of oxaloacetate by isolated mitochondria has been estimated only indirectly by measuring the rate of oxidation of intramitochondrial nicotinamide nucleotides upon addition of oxaloacetate to mitochondria (l-7, but see 8, 9). Using this technique, Gimpel et al. (7) have suggested that oxaloacetate is transported across the mitochondrial membrane by the dicarboxylate but not the oxoglutarate carrier. The use of the oxoglutarate carrier by oxaloacetate is directly shown by the ob-
AND
QUAGLIARIELLO
servations presented in this paper. Addition of oxaloacetate causes oxoglutarate efflux from mitochondria in a phenylsuccinate-sensitive reaction (Fig. 1). Efflux of labeled oxoglutarate was previously reported by us (8,9), but arsenite and mersalyl were not included in the reaction medium. A possible explanation of these results, offered by Gimpel et al. (see Ref. 7), is that, in oxoglutarate-loaded mitochondria, oxaloacetate is not exchanged for oxoglutarate, but for a small amount of Pi or malate (still present in these mitochondria) via the dicarboxylate carrier. Inside the mitochondria, the oxaloacetate would be reduced to malate, which in turn could be exchanged for further oxaloacetate. The extramitochondrial malate would then be taken up in exchange for oxoglutarate. The main indication against this explanation is the lack of inhibition of the oxaloacetate/oxoglutarate exchange by mersalyl, which, at the concentration used, completely inhibits the dicarboxylate carrier. The subsequent experimental data reported give further evidence for the contention that oxaloacetate can be transported via the oxoglutarate carrier. The oxaloacetateloxoglutarate exchange is more effectively inhibited by phenylsuccinate than by butylmalonate, in agreement with their inhibition constants toward the oxoglutarate carrier (8). Mersalyl inhibits the rate, but not the extent, of the exchanges between added oxaloacetate and intramitochondrial malonate or malate. The most straightforward explanation of this result would seem to be that malonate and malate are both substrates of the dicarboxylate and the oxoglutarate carrier, and mersalyl, by inhibiting the dicarboxylate carrier, decreases the rate of the exchanges. In heart mitochondria, under conditions where the dicarboxylate carrier is inactive, the oxaloacetate/oxoglutarate exchange is operating. The present study gives direct evidence that oxaloacetate can also be transported by the dicarboxylate carrier, although less efficiently. The data of Fig. 1 demonstrate that added oxaloacetate exchanges with intramitochondrial Pi, in the presence of N-ethylmaleimide, i.e., under conditions
MITOCHONDRIAL
OXALOACETATE
where Pi transport is catalyzed only by the dicarboxylate carrier. The oxaloacetate/Pi exchange is inhibited by known inhibitors of the dicarboxylate carrier, i.e, mersalyl, butylmalonate, and phenylsuccinate. The proposed existence of different binding sites for Pi and dicarboxylates on the dicarboxylate carrier (12, 25-27) raises the question of the site of oxaloacetate binding. Our data suggest that oxaloacetate binds at the dicarboxylate-binding site and away from the Pi-binding site, since the inhibition of the dicarboxylate carrier by oxaloacetate is competitive with respect to malonate, but noncompetitive with respect to Pi (Fig. 7). The kinetic data of the oxaloacetate exchange (Fig. 6) reveal that, at low substrate concentration, the contribution of the dicarboxylate carrier to the oxaloacetate translocation is relatively minor with respect to that given by the oxoglutarate carrier. It is likely that, due to the presence of a ketonic group, the binding affinity of oxaloacetate to the dicarboxylate carrier and/or the mobility of the carriersubstrate complex are severely limited. The apparent higher capacity of mitochondria to transport oxaloacetate via the oxoglutarate carrier than via the dicarboxylate carrier can account for some of the findings reported in the literature, e.g., the lack of inhibition by Pi of the oxaloacetate-induced oxidation of intramitochondrial NAD(P)H and a higher effectiveness of succinate than malonate in inhibiting oxaloacetate uptake by mitochondria (2). Thus, Pi is not transported on the oxoglutarate carrier, and succinate is a better substrate than malonate for the oxoglutarate carrier (8). It is interesting to note that the efflux of large amounts of oxaloacetate from isolated mitochondria has been observed in the presence of oxoglutarate (29, 30). In these investigations on the energylinked H transfer between malate and oxoglutarate plus NH, in the mitochondria, glutamate is formed and malate is oxidized to oxaloacetate. In view of the present results, it becomes obvious that in these experiments oxaloacetate has been extruded in exchange with oxoglutarate by the oxoglutarate carrier.
TRANSPORT
167
In the intact cell, the concentration of free oxaloacetate is very low in the mitochondria (31-33) and much higher in the cytosol (34). An interesting implication to the intact tissue of the data presented in this paper is that an oxaloacetate influx and malate efflux via the oxoglutarate carrier (either by a direct exchange or by a combined oxaloacetate/oxoglutarate and oxoglutarate/malate exchange) may contribute to the transfer of reducing equivalents from mitochondria to cytosol. In heart, Illingworth et al. (35) have recently postulated an oxaloacetate-malate exchange across the mitochondrial membrane as the only possibility of accounting for the efflux of reducing equivalents. REFERENCES
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