Mitochondrial adenine nucleotide transport and its role in the economy of the cell

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90 Mitchell, J. R., Potter, W. Z., Hinson, J. A., Snodgrass, W. R., Timbrell, J. A. and Gillette, J. R. (1975) in Handbook of Experimental Pharmacology, Vol. 28, part 3, pp. 383-419, Springer-Verlag, Heidelberg Wood, A. W., Levin, W., Lu, A. Y. H., Yagi, H., Hernandez, O., Jerina, D. M. and Conney, A. H. (1976) J. Biol. Chem. 251, 4882-4890 Owens, I. S. (1978) in ref. [l] DeBaun, J. R., Rowley, J. Y., Miller, E. C. and Miller, J. A. (1968) Proc. Sot. Exp. Biol. Med. 129, 268-273 Irving, C. C. (1971) Xenobioiica 1, 387-398

9 Gillette, J. R. (1974) Biochem. Pharmacol. 23, 2785-2794,2927-2938 10 Mulder, G. J., Hinson, J. A. and Gillette, J. R. (1978) Biochem. Pharmacol. 27, 16411650

11 Singer, B. (1977) Trends Biochem. Sci. 2, 180-183 12 Ames, B. N., McCann, J. and Yamasahi, E. (1975) Mutation Res. 31, 347-364 13 King, C. M. and Allaben, W. T. (1978) in ref. [l] 14 Mulder, G. J., Hinson, J. A., Nelson, W. and Thorgeirsson, S. S. (1977) Biochem. Pharmacol. 26, 1356-1358 15 Miller, E. C. (1978) Cancer Res. 38, 1479-1496

Mitochondrial adenine n~cleotide transport and its role in the economy of the cell Pierre V. Vignais and Guy J. M. Lauquin Current understanding of the process of mitochondrial adenine nucleotide transport is expanding along two principal lines of research, namely, the molecular propertiesofthe carrier protein and its physiological significance. During normal cell function, adenine nucleotide transport is probably not a rate-limiting process. It provides the oxidative phosphorylation system with the required spectjicity for cytosolic ADP and drives the asymmetric exchange of cytosolic ADP for mitochondrial ATP. The adenine nucleotide carrier, located in the inner mitochondrial membrane, provides a mechanism for the export of the ATP generated by oxidative phosphorylation from the mitochondria to the rest of the cell where the ATP provides the source of energy required to drive energyconsuming reactions (synthesis of biomolecules, active transport, mechanical work in muscle tissue, etc.). The ADP which results from these reactions enters the mitochondria in exchange for ATP. As the two processes, namely ATP synthesis in mitochondria and ATP consumption in the cytosol, are linked by the adenine nucleotide carrier, the turnover of this carrier can be assessed from the oxygen consumption of cells. If we consider a human adult with a caloric expenditure of 2200 kcal/day, and assuming that six ATP molecules are formed for each 0, molecule used, the turnover of ATP amounts to 120 mol or roughly 60 kg/day [l]. Two trends of research on ADP/ATP transport have now become evident. First, the molecular properties of the adenine nucleotide carrier are currently under investigation (see reviews [2-51). Much of The authors are at the Laboratory of Biochemistry, Department of Fundamental Research, Nuclear Research Center, 38041 Grenoble, France.

the present knowledge concerning structural and functional aspects of the adenine nucleotide carrier has been obtained through the use of the inhibitors, atractyloside, carboxyatractyloside and bongkrekic acid, which bind specifically to the carrier. The carrier protein is small. Estimates obtained by SDS polyacrylamide gel electrophoresis reveal molecular weights ranging from 30,000 for the liver and the heart carrier, to 37,000 for the carrier isolated from mitochondria of cerevisiae. Saccharomyces Evidence obtained from spin labelling studies [6] indicates that the carrier is in direct contact with the lipid core of the mitochondrial membrane. Consequently, the mobility of this protein would depend on the degree of fluidity of the surrounding lipids. Recent molecular advances concern the immunological and chemical characterization of the carrier protein. Immunological studies have revealed an antigenic determinant in the carrier protein, probably related to the conformation assumed by the carrier, once it is bound to carboxyatractyloside [5,7]. The carrier protein has been labeled with photoactivable derivatives of radiolabeled ADP and atractyloside [8,9]. Such covalent photolabeling may prove invaluable for mapping the adenine nucleotide carrier.

16 Mulder,

G. J., Hinson, J. A. and Gillette, J. R. (1977) Biochem. Pharmacol. 26, 189-

196 17 Kadlubar,

F. F., Miller, J. E. C. (1976) Cancer. Res. 36, 18 Radomski, J. L., Hearn, W. T., Moreno, H. and Scott,

A. and Miller, 2350-2359

L., Radomski, W. E. (1977)

Cancer Res. 37, 1757-1762 19 Mitchell, J. R. (1976) Ann.

Int. Med. 84, 181-192 20 Rannug, U., Sundvall, A. and Ramel, C. (1978) Chem.-Biof. Interactions 20, l-16 21 Hill, D. L., Shih, T. W., Johnston, T. P. and Struck, R. F. (1978) Cancer Res. 38, 2438-2442

In addition, with the purified carrier protein, it has been possible to reconstitute the ADP/ATP transport by incorporating the protein into liposomes [lO,ll]. The second trend of research concerns the study of the adenine nucleotide carrier, considered from the standpoint of its role in the cell economy. This article aims to discuss some physiological aspects related to this latter point. Is the adenine nucleotide carrier a ratelimiting factor for ATP-requiring reactions in the cytosol? Attempts to mimic with isolated liver mitochondria the in vivo conditions where the cytosol contains both ADP and ATP have been achieved by adding purified F,-ATPase [ 121 or glucose plus hexokinase [13], as ATP-utilizing systems, to a medium supplemented with ATP, and oxidizable substrate. With a more or less stable concentration of Pi, the rate of respiration and of the coupled phosphorylation appears to be dictated by the external rather than by the internal ATP/ ADP ratio. For example [13], when the external ATP/ADP ratio is lower than 5, the rate of ATP synthesis is maximal, and the adenine nucleotide car&r is probably not rate-limiting. For ratios ranging from 5 to 10, there is only a 10% decrease in the rate of ATP synthesis. When the ATP/ ADP ratio is increased from 10 to 100, the phosphorylation rate drops to zero, possibly because of competition between ATP and ADP for the carrier, and thus to the carrier becoming a rate-limiting factor. Reported ATP/ADP ratios in the cytosol of isolated rat liver cells range from 5 to 9 [14-161, which would suggest, on the basis of the above in vitro studies, that the adenine nucleotide carrier is not a rate-limiting factor in those cells. Considering these relatively high ATP/ ADP ratios in the cytosol, one may ask why AT? cioes not compete with ADP for access to the carrier. In fact, in rat liver cells the cytosolic concentrations of the 0

Elscvier/North-Holland

Biomedical Press 1979

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uncomplexed forms of ADP and ATP, which are the true substrates for the carrier, are 115 and 170 PM respectively [ 151. The concentration of free ADP in the cytosol is therefore at least 10 times higher than the Km for ADP; this is high enough to prevent a significant inhibition of ADP transport by ATP, the Ki for ATP being close to 200 /LM [ 171. The following data obtained with rat liver cells also agree with the concept-that the adenine nucleotide carrier does not limit the rate of oxidative phosphorylation of cytosolic ADP. First, the rate at which ATP is delivered to the cytosol of liver cells incubated under conditions of maximum metabolic activity is lower than the capacity of ADP/ATP transport assayed with isolated liver mitochondria. In a typical experiment [ 181, isolated rat liver cells rapidly synthetizing both urea and glucose from appropriate precursors at 37”C, respired at a rate as high as 9.65 pmol O,/min/g (wet wt). The rate of glucose synthesis was 1.72 pmol/min/g and that of urea synthesis 4.93 pmol/min/g. The ATP transported to the cytosol via the adenine nucleotide carrier was equal to the ATP formed by oxidative phosphorylation (57.90 pmol/min/g) minus the ATP required for the mitochondrial reactions for the synthesis of glucose (3.44 I*.mol/min/g) and urea (9.86 pmol/ min/g), i.e. 44.60 pmol/min/g. Based on an extrapolated value of 1 pmol of ADP/ mitochondrial exchange/min/g ATP protein at 37°C (calculated from data obtained with isolated rat liver mitochondria [2]), and assuming 60 mg mitochondrial protein/g of cells [19], the rate of ADP/ATP exchange is approximately 60 pmol/min/g of liver cells at 37°C. This simple calculation shows that even when the energy demand for cytosolic reactions is very large, the capacity of the carrier is still sufficient to cope with the demand. Under conditions of lower metabolic activity, the capacity of the carrier is obviously in excess. Other data on liver cells concern the relationship between the activity of the respiratory chain and the phosphorylation state, i.e. the ratio (ATP)/ (ADP)(Pi). They show that changes in the rate of respiration parallel changes in the phosphorylation state [20], and that a state of near equilibrium exists between the difference in the redox state across the first two sites of oxidative phosphorylation on the one hand and the cytosolic phosphorylation state on the other [21]. If indeed oxidative phosphorylation of cytosolic ADP is near equilibrium, then all intermediary reactions and notably

91

ADP/ATP transport must also be at ADP/ATP transport by long chain acylequilibrium. From these criteria the CoAs based on in vitro studies. In these adenine nucleotide carrier is unlikely to in vivo experiments, oleyl-CoA was allowed be a rate-limiting factor in in vivo con- to accumulate in large amounts in the ditions. isolated cells upon incubation with oleate; Referring to inhibition of glucose syn- the newly formed oleyl-CoA exhibited thesis by atractyloside in rat liver cells only a slight inhibitory effect on the and to the dose-effect curve which is adenine nucleotide carrier [22]. The possilinear even at low concentrations of the bility remains that long chain acyl-CoA inhibitor, Akerboom et al. [22] concluded esters may play a regulatory function in other tissues, for example, the heart that ADP/ATP transport is a rate-limiting muscle for which fatty acids are a primary factor in the delivery of ATP to cytosol. This conclusion must be qualified. Indeed, fuel. the oxidative phosphorylation of cytosolic ADP can be considered as a cyclic multi- The basis of the difference in phosphate enzyme system in which the bulk of ADP potentials in the cytosol and mitochondria: arising in the cytosol as a result of ATP- a result of the electrogenicity of ADP/ATP consuming processes is transported in transport or a reflection of mitochondrial exchange for mitochondrial ATP to feed , compartmentalization? the oxidative phosphorylation system. In It is a remarkable feature of cell a cyclic system, any inhibition of a compartmentation that the ATP/ADP component enzyme is expected to depress ratio and consequently the phosphorylathe cycle rate to some extent [23]. tion potential, which is a function of the In summary, it is not the capacity of phosphorylation state (ATP)/(ADP)(P,), the carrier (at least in liver cells), but is significantly higher in the cytosol than rather the rate at which ADP (and Pi) are in mitochondria. Calculatipns of the phosfed into the oxidative phosphorylation phate potential in the cytosol and mitosystem that determines the overall rate of chondria of isolated liver cells reveal a oxidative phosphorylation, and this, in difference of about 2 kcal/mol [15]. Similar turn, depends on the rate of the ATP- differences inside and outside of the consuming reactions taking place in the mitochondria have been obtained in in cytosol. If not rate-limiting, the physiowith isolated mitovitro experiments logical significance of the adenine-nucleochondria incubated with oxidizable tide carrier rests initially on its specificity substrate, ADP and Pt [26]. Since such for ADP and ATP, the selection of ADP differences are abolished by uncouplers or among cytosolic nucleoside-diphosphates respiratory inhibitors, it was inferred that conferring indirectly the specificity of respiratory energy is used not only for the synthesis of ATP in mitochondria, but ADP for the oxidative phosphorylation system. A second important function of also for the preferential exchange of the the carrier is to drive the asymmetrical mitochondrial ATP against cytosolic ADP. exchange of cytosolic ADP against mito- The way in which energy controls the chondrial ATP. This will be discussed asymmetric exchange of ADPex against later. ATPin may be rationalized as follows [4]. The exchange of cytosolic ADP3- against Are long chain -acyl-CoAs physiological mitochondrial ATP4- is electrogenic and inhibitors of ADP/ATP transport ? is favoured by the membrane potential positive outside. Energy is indirectly Assuming that the capacity of ADP/ATP transport is large enough not to limit the required for this asymmetrical exchange, rate of the overall process of oxidative to maintain the membrane potential which otherwise would be dissipated by the net phosphorylation, one may wonder whether there exist physiological inhibitors which outward movement of negative charge are able to decrease the rate of ADP/ATP associated with each turnover of the transport and to render it rate-limiting. carrier. Another way by which energy can Possible candidates are the long chain control the asymmetry of adenine nucleotide transport is by maintenance of a acyl-CoA esters. In fact at low concentrations, they competitively and efficiently conformation of the carrier which shows inhibit ADP/ATP transport in isolated more affinity for ADP,, than for ATPin mitochondria (KI = 1 PM) (for review see [17]. In fact, upon uncoupling, mito[24]). Furthermore, they’ act with equal chondria exhibit similar affinities for ADP efficiency from both sides of the mito- and ATP, as if in the absence of energy, chondrial membrane [25]. However, in the carrier assumes another conformation. In accordance with the conformational vivo experiments in rat liver cells have not confirmed the expected inhibition of hypothesis there is the observation of

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discrete chemical changes in/ the mitochondrial membrane resulting from the functioning of the adenine nucleotide carrier. For example, a few SH groups are unmasked when ADP/ATP transport is initiated in respiring mitochondria by addition of ADP; there is no SH unmasking when the mitochondria are pretreated with an uncoupler, cf. [3]. Most likely, the unmasked SH groups belong to the carrier protein. The above explanations concerning the difference in phosphate potentials between the cytosol and mitochondria require that the activities of ATP and ADP within the mitochondrial matrix and the cytosol reflect their true concentrations. However, the possibility must be considered that the phosphate potential of the mitochondrial matrix, calculated from the mitochondrial nucleotide pool, does not correctly represent that part of the pool which operates during the overall reaction of oxidative phosphorylation. This is reminiscent of recent reports [27,28] which point to the microcompartmentalization of the ATPase complex with the adenine nucleotide carrier in liver mitochondria. In these reports, referring particularly to respiring, coupled mitochondria, the ATPase complex and the adenine nucleotide carrier appeared to behave kinetically as if they belonged to the same unit, the functioning of which would depend more on the extramitochondrial than on the intramitochondrial phosphorylation state. Presumably, the carrier and the ATPase complex have access to a common microaqueous space of the matrix, which does not equilibrate rapidly with the rest of the matrix gel, possibly because of the high viscosity of this gel. Uncoupling or lack of metabolic energy would disorganize the matrix gel and result in the abolition of compartmentalization. Compartmentalization could also explain some puzzling effects of combined inhibitions by atractyloside and cyanide on respiration and on synthesis of glucose and urea by rat liver cells [18]. In this experiment, the rate of respiration was decreased to different levels by increasing concentrations of cyanide and the effect of atractyloside was assayed on the remaining respiration. Unexpectedly, a of atractyloside given concentration inhibited a constant fraction of the remaining respiration. Similar results were obtained for glucose and urea synthesis. Such a behaviour is typical of enzyme entities interacting in a multi-enzyme system [29]; it can be further explained by a limited diffusion in the matrix gel of

ADP and ATP, acting as substrates of the adenine nucleotide carrier and the ATPase complex. Coupling of mitochondrial phosphotransferases with the adenine nucleotide carrier for the delivery of energy to the cytosol

ATP is the main form in which mitochondrial energy is delivered to the cytosol. In fact ATP is used as such in a large number of energy-consuming reactions in the cytosol. There are, however, cases where, in mitochondria, the terminal phosphate bond of ATP is transferred to an acceptor and the phosphorylated acceptor is released to the cytosol. This transfei takes place in the intermembrane space and is catalysed by specific phosphotransferases, nucleoside diphosphokinase and creatine phosphokinase. Both enzymes have a dual localization, mitochondrial and cytosolic. In heart muscle, the mitochondrial creatine phosphokinase isoenzyme is located on the outer face of the inner mitochondrial membrane [30]. In liver the mitochondrial nucleoside diphosphokinase is mainly located in the intermembrane space [31]. Consequently, a significant fraction of the high energy phosphate groups generated during respiration is directed into formation of either phosphocreatine or GTP, UTP and CTP before leaving the mitochondria [32,33]. The close proximity of the adenine nucleotide carrier and the responsible kinases presumably facilitates this process. The cytosolic phosphotransferase, hexokinase, which binds to the outer mitochondrial membrane, may also provide a link between the ATP delivered by the mitochondria and the cytosolic glycolytic pathway [34]. References

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