Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [14C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12-14-day-old plants was calculated to be 330 mumol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22 degrees C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.     

Adenine nucleotide pool size,adenine nucleotide translocase activity,and respiratory activity in newborn rabbit liver mitochondria

The adenine nucleotide content (ATP+ADP+AMP) of newborn rabbit liver mitochondria was 6.0±0.5 nmol/mg mitochondrial protein at birth, increased rapidly to 14.5±1.7 nmol/mg protein by 2 h postnatal, peaked at 6 h, then decreased gradually to 7.8±0.6 nmol/mg protein by 4 days postnatal. There was a strong positive correlation (r=0.82) between the total adenine nucleotide pool size and adenine nucleotide translocase activity in these mitochondria. In contrast, glutamate + malate-supported State 3 respiratory rates remained constant from birth through the first week of life. State 4 rates also remained constant, as did the respiratory control index and uncoupled respiratory rates. The following conclusions are suggested: (1) The maximum rate of translocase activity is limited by the intramitochondrial adenine nucleotide pool size. (2) In newborn rabbit liver mitochondria, the State 3 respiratory rate is not limited by either the adenine pool size or the maximum capacity for translocase-mediated adenine exchange. (3) In contrast to rat, rabbit liver mitochondria are fully functional at birth with regard to respiratory rates and oxidative phosphorylation. (4) The rapid postnatal accumulation of adenine nucleotides by liver mitochondria, now documented in two species, may be a general characteristic of normal metabolic adjustment in neonatal mammals.     

Phosphoenolpyruvate efflux from kidney cortex mitochondria of rabbit

(1) The relationship between phosphoenolpyruvate formation and its accumulation in kidney cortex mitochondria of rabbit was studied in the presence of glutamate as substrate. (2) In mitochondria incubated in either State 4 or under uncoupled conditions, both 1,2,3-benzenetricarboxylate and atractyloside resulted in a marked elevation of the intramitochondrial phosphoenolpyruvate accompanied by a 2–4-fold decline in production of this compound. The same effect was induced by n-butylmalonate in uncoupled mitochondria, while both phosphoenolpyruvate efflux and its production were inhibited to a smaller extent in mitochondria incubated with 1,2,3-benzenetricarboxylate in State 3. (3) Citrate, malate or 2-phosphoglycerate caused a fast displacement of phosphoenolpyruvate from atractyloside-inhibited mitochondria to the reaction medium. In contrast, on the addition of ATP to mitochondria incubated with 1,2,3-benzenetricarboxylate, the rate of phosphoenolpyruvate efflux was lower than that induced by either malate or citrate. (4) Despite the presence of both 1,2,3-benzenetricarboxylate and atractyloside, arsenite and rotenone plus antimycin resulted in a leakage of phosphoenolpyruvate from the mitochondria, probably via a carrier-independent mechanism. (5) Based on the present results it seems that depending on the metabolic condition, the tricarboxylate carrier and the adenine nucleotide translocase are functioning to different extents in the efflux of phosphoenolpyruvate from rabbit renal mitochondria to the surrounding medium.     

Cyclic electron transport in chloroplasts. The Q-cycle and the site of action of antimycin

Cyclic electron transport systems have been set up in broken chloroplasts, with photochemically reduced ferredoxin or 9,10-anthraquinone-2-sulphonate as cofactor. In good agreement with the literature, only the ferredoxin-catalyzed pathway was found to be inhibited by antimycin; but both pathways were found to have a slow electrogenic reaction, both were inhibited by the cytochrome b-563 oxidation inhibitor 2-heptyl-4-hydroxyquinoline N-oxide (the inhibition being strongest at limiting light intensity), and the two pathways had the same proton/electron stoichiometry at limiting light intensity. It is concluded that a Q-cycle can occur in cyclic electron transport with either cofactor; and therefore that the site of action of antimycin in chloroplasts is not within the Q-cycle, as it is believed to be in mitochondria and bacteria. Instead, a ferredoxin-quinone reductase is proposed as the site of action of antimycin in the ferredoxin-catalyzed cyclic pathway. It is also concluded that the data presented here are consistent with the suggestion that the Q-cycle in photosynthetic electron transport is a facultative one, its degree of engagement depending on competition between the Rieske centre and cytochrome b-563 for reducing equivalents from plastosemiquinone.     

Adenine nucleotide transport in hepatoma mitochondria. Characterization of factors influencing the kinetics of ADP and ATP uptake

Initial velocity measurements of [³H]ADP and [³H]ATP uptake have been made with mitochondria isolated from Morris hepatomas of differing growth rates, and factors known to influence the rates of nucleotide exchange have been examined in an effort to determine whether the elevated rates of aerobic glycolysis in these tumors can be attributed to altered carrier activity. These studies included the determination of the apparent kinetic constants for nucleotide uptake as a function of the mitochondrial energy state and the dependence of transport rates on temperature. Also included in these studies were measurements of the mitochondrial levels of endogenous inhibitors, divalent cations and internal adenine nucleotides. Results obtained showed that with mitochondria isolated from the various tumor lines, the apparent kinetic constants for nucleotide uptake are different from those of control rat or regenerating liver mitochondria; the apparent V_max values for both ADP and ATP uptake are significantly lower. Furthermore, under conditions of a high-energy state, the K_m and V_max values for ATP uptake are greater than the K_m and V_max value for ADP uptake but that under uncoupled conditions, the opposite is observed. Comparison of the levels of mitochondrial Ca²⁺, Mg²⁺, long-chain acyl-CoA ester and adenine nucleotide from the various mitochondria showed that important differences exist between liver and hepatoma mitochondria in the levels of Ca²⁺, long-chain acyl-CoA ester and AMP. Mitochondrial Ca²⁺ levels are elevated 3–5-fold in all tumor lines, and for Morris 7777 hepatoma (a rapidly growing tumor) by a remarkable 70-fold; whereas the levels of acyl-CoA ester and AMP are significantly lower in the more rapidly growing tumors. Arrhenius plots for nucleotide uptake in mitochondria from liver and hepatoma are characterized as being biphasic, having similar activation energies above and below the break point temperature (28–38 and 6–16 kcal/mol, respectively). However, the transition temperature for mitochondria from the various hepatomas is uniformly 4–5°C lower than mitochondria from control liver. The latter difference may reflect a variation in membrane composition, most probably lipid components. It is concluded that the presence of elevated levels of Ca²⁺ and lower levels of AMP in hepatoma mitochondria and difference of membrane compositions may play an important role in limiting adenine nucleotide transport activity in vivo and that the impaired carrier activity may contribute to higher rates of aerobic glycolysis observed in these tumors.     

Apoptotic and anti-proliferative effects of all-trans retinoic acid. Adenine nucleotide translocase sensitizes HeLa cells to all-trans retinoic acid

We examined the apoptotic and anti-proliferative effects of all-trans retinoic acid (atRA) in HeLa cells. Our results demonstrated that HeLa cells were more sensitive to the anti-proliferative effects of atRA than to its apoptotic effects. Furthermore, we demonstrated that caspase inhibition attenuates cell death but does not alter the atRA-dependent reduction in cell proliferation, which suggests that atRA-induced apoptosis is independent of the arrest in cell proliferation. To check whether ANT proteins mediated these atRA effects, we transiently transfected cells with expression vectors encoding for individual ANT (adenine nucleotide translocase 1-3). Our results revealed that ANT1 and ANT3 over-expressing HeLa cells increased their atRA sensitivity. Thus, our results not only demonstrate the different functional activities of ANT isoforms, but also contribute to a better understanding of the properties of atRA as an anti-tumoral agent used in cancer therapy.     

Relation between red cell anion exchange and urea transport

The new distilbene compound, DCMBT (4,4′-dichloromercuric-2,2,2′,2′-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385–389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.     

Resolution of the light-dependent modulation system of pea chloroplasts

The mechanism of the light-dependent inactivation of glucose-6-phosphate dehydrogenase and the light-dependent activation of NADP⁺-malate dehydrogenase has been studied in partially purified extracts of pea (Pisum sativum) chloroplasts. Neither partially purified enzyme could be light modulated by washed thylakoids alone. However, a factor (mol. wt. 50 000) was present in the stroma which could, when added to purified enzyme and thylakoid membranes, reconstitute a light-dependent modulation of either glucose-6-phosphate dehydrogenase or NADP⁺-malate dehydrogenase. This factor, which we term protein-modulating factor, is distinct from ferredoxin-thioredoxin reductase and from thioredoxin, the factors involved in another scheme for light modulation. The scheme proposed here for light modulation involves electron transfer from Photosystem I to a membrane-bound light-effect mediator and then to the soluble protein modulating factor which modulates chloroplast enzyme activity, probably by reduction of a regulatory disulfide bond.     

Isolation and characterization of light- and dithiothreitol-modulatable glucose-6-phosphate dehydrogenase from pea chloroplasts

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) has been purified to electrophoretic homogeneity from pea chloroplasts. The enzyme, which has a Stokes radius of 52 Å, is a tetramer made up of four 56000 Da monomers. The pH optimum is around 8.2. The enzyme is absolutely specific for NADP. The apparent K_m(NADP) is 2.4 ± 0.1 μM. NADPH inhibition of the enzyme is competitive with respect to NADP (mean K_i, 18 ± 5 μM) and is mixed (K_p >K_m, V_max >V_p) with respect to glucose 6-phosphate (mean crossover point, 0.5 ± 0.1 mM). The apparent K_m(glucose 6-phosphate) is 0.37 ± 0.01 mM. The purified enzyme is inactivated in the light in the presence of dilute stroma and washed thylakoids, and by dithiothreitol. Enzyme which has been partially inactivated by treatment with dithiothreitol can be further inactivated in the light in the presence of dilute stroma and washed thylakoids and reactivated in the dark, but only to the extent of the reverse of light inactivation. Dithiothreitol-inactivated enzyme is not reactivated further by addition of crude stroma or oxidized thioredoxin. Dithiothreitol-dependent inactivation of the enzyme follows pseudo-first-order kinetics and shows rate saturation. The enzyme which has been partially inactivated by treatment with dithiothreitol does not differ from the untreated control with respect to thermal and tryptic inactivation. However, enzyme which has been partially light inactivated shows different thermal and tryptic inactivation patterns as compared to the dark control. These observations suggest that the changes in the enzyme brought about by light modulation are not necessarily identical with those brought about by dithiothreitol inactivation.     

Modification of liver mitochondrial lipids and of adenine nucleotide translocase and oxidative phosphorylation by cold adaptation

The decrease in respiration rate following thyroidectomy is preceded by changes in the lipid composition of the mitochondrial membrane (Hoch, F.L., Subramanian, C., Dhopeshwarkar, G.A. and Mead, J.F. (1981) Lipids 16, 328–334) and in concert, changes in the kinetic parameters of the adenine nucleotide translocase (Mak, I.T., Shrago, E. and Elson, C.E. (1981) Fed. Proc. 40, 398). To demonstrate that physiological adaptation also involves this sequence of events, rats were housed at 8°C for 3–4 weeks. Cold adaptation resulted in a modest (5%) increase in the unsaturation index for the mitochondrial fatty acids comprised of a significant increase in arachidonic acid and a reciprocal decrease in linoleic acid. Phospholipid analysis indicated that cold adaptation increased the mitochondrial phosphatidylethanolamine and reciprocally decreased the phosphatidylcholine content. Concomitantly, cold adaptation resulted in 25–30% increases in rat liver mitochondrial respiratory activities without changing the respiratory control or ADP/O ratios. The kinetic parameters of the adenine nucleotide translocase were determined by the back-exchange method (Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66–79). At 0–4 and 10°C, the V_max and K_m of the cold-adapted rat liver adenine nucleotide translocase were not distinguishable from the control values. The K_i values determined by Dixon plot studies for atractylate and palmitoyl-CoA were also comparable between the two groups. However, at 25 and 37°C, cold-adapted rat liver adenine nucleotide translocase exhibited a 20% increase in V_max and a 20% decrease in K_m for external ADP. The results suggest that one adaption to a cold environment involves hormone-mediated changes in the lipid composition in the mitochondrial membranes which in turn modulate the adenine nucleotide translocase and subsequent respiratory activities.     

The influence of anions on oxygen evolution by isolated spinach chloroplasts

A study has been made of the onset of chloride deprivation on the oxygen-evolving characteristics of isolated spinach chloroplasts. Using a modulated oxygen electrode it is found that the type of inhibition depends on the anion replacing chloride in the bathing medium. With nitrate a large increase in phase lag accompanies a relatively small inhibition which can be shown to be consistent with a decrease in the rate constant of the reaction which limits the rate of electron transport between water and Photosystem II. With sulphate there is a very small phase change but a larger inhibition which suggests that replacing chloride with sulphate in an electron-transport chain shuts off that chain. With acetate there is a moderate increase in phase lag and the largest inhibitory effect. The phase-lag increase suggests that acetate is affecting the same chloride-sensitive site as nitrate. However, the inhibition cannot be explained by this effect alone and points to the existence of a second chloride-sensitive site. Of the four forward reactions associated with the Kok model of oxygen evolution (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) only S1₃ → S₀ is slowed down when chloride is replaced by nitrate. This reaction is not slowed down by replacing chloride with sulphate.     

Adenine nucleotide efflux in mitochondria induced by inorganic pyrophosphate

The efflux of mitochondrial adenine nucleotide which is induced by addition of PP_i to suspensions of rat liver mitochondria has been investigated. This efflux of adenine nucleotide is greatly stimulated by the uncoupler FCCP at 1 μM, V_max being 6.7 nmol/min per mg protein as compared to 2.0 nmol/min per mg protein in its absence. The depletion process is inhibited by carboxyatractyloside. The K_m for PP_i of 1.25 mM is essentially unchanged when uncoupler is added. Quantitation of the individual adenine nucleotide species (ATP, ADP and AMP) and their relationship to the rate of efflux suggests that ADP is the predominant species being exchanged for PP_i.     

Accumulation of vanadium by tunicate blood cells occurs via a specific anion transport system

Tunicates, or sea squirts, are known to sequester vanadium to very high concentrations within specialized blood cells. They selectively accumulate the element from seawater against a 10⁶- to 10⁷-fold concentration gradient, and store it mainly as V(III). The mechanism for this selective accumulation involves the facilitated diffusion of vanadate across the blood cell plasma membrane followed by intracellular reduction to a non-transportable cation. Evidence for this mechanism was obtained by studying vanadate and [⁴⁸V]vanadate influx into living blood cells (vanadocytes). Influx of [⁴⁸V]vanadate into the cells is a rapid ( ) process which can be saturated (K_m = 1.4 (±2%) mM). Net vanadate accumulation is equal to isotopic influx, and accumulated vanadate is not released by washing cells with EDTA. Uncouplers of oxidative phosphorylation and glycolytic inhibitors have no effect on the rate of influx. Phosphate competes with vanadate for transport, and is itself taken up by the cell. The similar anions, sulfate and chromate, neither inhibit transport, nor are they taken up by the vanadocyte. Influx is inhibited by those stilbene disulfonate derivatives known to bind specifically to the external transport site of the anion exchange protein in the human erythrocyte membrane. During the influx of vanadate, the electron paramagnetic resonance (EPR) signal of intracellular vanadyl increases, indicating that transported V(V) is reduced upon entering the cell. The EPR signal of the blood cells at room temperature is characteristic of unbound V(IV), in agreement with reports that reduced vanadate is not bound to a protein or other macromolecule in these cells.     

Adenine nucleotide translocase mediates the K(ATP)-channel-openers-induced proton and potassium flux to the mitochondrial matrix

K_ATP channel openers have been shown to protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, although their mechanisms of action have not been fully clarified. In this study we investigated the influence of the adenine nucleotide translocase (ANT) inhibitors–carboxyatractyloside (CAT) and bongkrekic acid (BA)–on the diazoxide- and pinacidil-induced uncoupling of isolated rat heart mitochondria respiring on pyruvate and malate (6 + 6 mM). We found that both CAT (1.3 M) and BA (20 M) markedly reduced the uncoupling of mitochondrial oxidative phosphorylation induced by the K_ATP channel openers. Thus, the uncoupling effect of diazoxide and pinacidil is evident only when ANT is not fixed by inhibitors in neither the C- nor the M-conformation. Moreover, the uncoupling effect of diazoxide and pinacidil was diminished in the presence of ADP or ATP, indicating a competition of K_ATP channel openers with adenine nucleotides. CAT also abolished K⁺-dependent mitochondrial respiratory changes. Thus ANT could also be involved in the regulation of K_ATP-channel-openers-induced K⁺ flux through the inner mitochondrial membrane.     

Adenine nucleotide content and energy charge in dry cells and cysts of Azotobacter vinelandii

Abstract The levels of intracellular adenine nucleotides, energy charge, oxygen consumption and poly-β-hydroxybutyric acid stored, have been investigated in dry vegetative cells and cysts of Azotobacter vinelandii . The data show that under desiccation conditions the cysts retain viability at energy charge values of 0.20 and an ATP/ADP ratio of 0.24, whereas under the same desiccation conditions, vegetative cells die at energy charge values <0.5 and an ATP/ADP ratio of <0.6.     

Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis

Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson–Golabi–Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT.     

Titration of cardiolipin by either 10-<Emphasis Type="Italic">N</Emphasis>-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.     

The flavonoid quercetin induces changes in mitochondrial permeability by inhibiting adenine nucleotide translocase

This study shows the effects of the flavonoid quercetin on diverse mitochondrial functions, among them membrane permeability. Our findings indicate that the addition of 50 μM quercetin did not produce reactive oxygen derived species; however, it inhibited the oxidative stress induced after the addition of Fe²/H₂O₂ by about 38%. At this concentration, quercetin also promoted a fast calcium release, inhibited oxidative phosphorylation, stimulated oxygen consumption, and decreased membrane potential. In addition 50 μM quercetin inhibited the adenine nucleotide translocase (ANT) by 46%. These effects induced the opening of the permeability transition pore and release of cytochrome c, by its interaction with a component of the non-specific pore complex, fixed to the carrier in the conformation c, as carboxyatractyloside does. Quercetin-induced permeability transition pore opening was inhibited by 0.5 μM cyclosporin A, but, interestingly, the release of cytochrome c was not inhibited by the immunosuppressor, as quercetin was found to disrupt the outer membrane.     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [¹⁴C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2′-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5′-nucleotidase (EC 3.1.3.5), reflecting the markedly higher V_max/K_m ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher V_max/K_m ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

Pleiotropic effects of the yeast Sal1 and Aac2 carriers on mitochondrial function via an activity distinct from adenine nucleotide transport

In Saccharomyces cerevisiae, SAL1 encodes a Ca2+ -binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Delta exacerbates the respiratory deficiency and mtDNA instability of ggc1Delta, shy1Delta and mtg1Delta mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+ -binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96 V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria.