ATP Export Research Articles

Acute Glutathione Depletion Restricts Mitochondrial ATP Export in Cerebellar Granule Neurons

Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (Deltapsi(m)) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to "state 4" could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na(+) cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with Deltapsi(m) collapse followed by cytoplasmic Ca(2+) deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction.

Cytochrome c, released from cerebellar granule cells undergoing apoptosis or excytotoxic death, can generate protonmotive force and drive ATP synthesis in isolated mitochondria.

In rat cerebellar granule cells, cytochrome c release takes place during glutamate toxicity and apoptosis due to deprivation of depolarising levels of potassium. We show that, as in necrosis, the released cytochrome c present in the cytosolic fraction obtained from cerebellar granule cells undergoing apoptosis can operate as a reactive oxygen species (ROS) scavenger and as a respiratory substrate. The capability of the cytosolic fraction containing cytochrome c, obtained from cerebellar granule cells undergoing either necrosis or apoptosis, to energise coupled mitochondria isolated by the same cells is also investigated. We show that, in both cases, the cytosolic fraction containing cytochrome c, added to mitochondria, can cause proton ejection, and membrane potential generation and can drive ATP synthesis and export in the extramitochondrial phase, as photometrically measured via the ATP detecting system. Cytochrome c, separated immunologically from the cytosolic fraction of apoptotic cells when added to mitochondria, is found to cause proton ejection to generate membrane potential and to drive ATP synthesis and export in a manner not sensitive to the further addition of the cytosolic fraction depleted of cytochrome c, which failed to do this. In the light of these findings we propose that in apoptosis the released cytochrome c can contribute to provide ATP required for the cell programmed death to occur.

Origin and evolution of the mitochondrial proteome.

The endosymbiotic theory for the origin of mitochondria requires substantial modification. The three identifiable ancestral sources to the proteome of mitochondria are proteins descended from the ancestral alpha-proteobacteria symbiont, proteins with no homology to bacterial orthologs, and diverse proteins with bacterial affinities not derived from alpha-proteobacteria. Random mutations in the form of deletions large and small seem to have eliminated nonessential genes from the endosymbiont-mitochondrial genome lineages. This process, together with the transfer of genes from the endosymbiont-mitochondrial genome to nuclei, has led to a marked reduction in the size of mitochondrial genomes. All proteins of bacterial descent that are encoded by nuclear genes were probably transferred by the same mechanism, involving the disintegration of mitochondria or bacteria by the intracellular membranous vacuoles of cells to release nucleic acid fragments that transform the nuclear genome. This ongoing process has intermittently introduced bacterial genes to nuclear genomes. The genomes of the last common ancestor of all organisms, in particular of mitochondria, encoded cytochrome oxidase homologues. There are no phylogenetic indications either in the mitochondrial proteome or in the nuclear genomes that the initial or subsequent function of the ancestor to the mitochondria was anaerobic. In contrast, there are indications that relatively advanced eukaryotes adapted to anaerobiosis by dismantling their mitochondria and refitting them as hydrogenosomes. Accordingly, a continuous history of aerobic respiration seems to have been the fate of most mitochondrial lineages. The initial phases of this history may have involved aerobic respiration by the symbiont functioning as a scavenger of toxic oxygen. The transition to mitochondria capable of active ATP export to the host cell seems to have required recruitment of eukaryotic ATP transport proteins from the nucleus. The identity of the ancestral host of the alpha-proteobacterial endosymbiont is unclear, but there is no indication that it was an autotroph. There are no indications of a specific alpha-proteobacterial origin to genes for glycolysis. In the absence of data to the contrary, it is assumed that the ancestral host cell was a heterotroph.

Identification and Characterization of the Mode of Action of MON 65500: A Novel Inhibitor of ATP Export from Mitochondria of the Wheat “Take-All” Fungus, Gaeumannomyces graminis var. tritici

MON 65500 represents a novel class of fungicide, the hindered silyl amides, that selectively inhibit the causative agent of “take-all” disease in wheat, Gaeumannomyces graminis var. tritici. This paper indicates that the primary mode of action of MON 65500 is the inhibition of transport of the adenine nucleotide, ATP, from the mitochondrial matrix. By preventing the export of ATP to the cytosol, all extramitochondrial energy-dependant processes are disrupted, leading to cell death.

ATP synthesis and export in heart left ventricle mitochondria from spontaneously hypertensive rat.

Use was made of mitochondria isolated from heart left ventricles of either spontaneously hypertensive or age-matched Wistar-Kyoto rats used as a control to find out whether hypertrophy (5-week-old rats) or hypertrophy/hypertension (24-week-old rats) can cause change in the mechanisms by which ATP is synthesised via ATP synthase and subsequently exported via the ADP/ATP translocator outside mitochondria. To do this, photometric measurements were made of the rate of ATP appearance in the extramitochondrial phase, which occurs as a result of ADP addition to mitochondria. In mitochondria from spontaneously hypertensive rats deficit of ATP production was found dependent on changes in the KmADP and Vmax values of both the ADP/ATP translocator and the ATP synthase. The ADP/ATP translocator was found to determine the rate of ATP production outside mitochondria in all the tested samples. In an initial investigation carried out to ascertain how cell ATP deficit can be counterbalanced, an increase in both adenylate kinase and creatine kinase activities was found in both hypertrophy and hypertrophy/hypertension. A possible increase in anaerobic glycolysis was also suggested by the increased lactate dehydrogenase activity.

The controlling influence of ADP, ATP and magnesium on the activities of adenylate kinase, ATP synthase, ADP/ATP translocator and the mitochondrial respiration in plants

In this study we investigated the influence of adenine nucleotides and magnesium on ATP formation by oxidative phosphorylation and adenylate kinase (AK) and on ATP-export by the ADP/ATP translocator in mitochondria of potato ( Solanum tuberosum L.). Our kinetics and inhibitor studies showed that ATP in mitochondria of potato tubers could be provided by two different enzymes, depending on the ADP concentration. ATP was primarily formed by oxidative phosphorylation at low ADP concentrations and by AK at high ADP concentrations. In addition, the extent of ATP formation and the fractions contributed by each of these two systems were controlled by the level of magnesium ions in the medium. At high magnesium concentrations ADP import, ATP-synthesis by ATP synthase and ATP export increased. The effect of increasing ADP (and ATP) and magnesium concentrations on respiratory rates, respiratory control values and P:O ratios were quantified. Respiration rates of mitochondria from potato tubers in state 3 [B. Chance, G.R. Williams, J. Biol Chem 217 (1955) 635–647] increased with rising ADP (and ATP) concentration, when magnesium was present (5 mM). The P:O ratio was a function of the added ADP but it was independent of the presence of magnesium. Applying the test suggested by Liu et al. [X.C. Liu, K. Jones, H.G. Dickinson, Theor. Appl. Genet. 76 (1988) 305–310] to measure ADP/ATP translocator activity we found some inconsistencies.

ATP transport and ABC proteins.

ATP can be exported into the extracellular space, where it has important biological effects. Recent evidence shows that direct ATP export across the plasma membrane is associated with the presence of ABC proteins. Do the ABC proteins pump ATP as well as their other substrates, and if so, why?

Cytoplasmic male sterility in Petunia hybrida: factors affecting mitochondrial ATP export in normal and cytoplasmically male sterile plants

In view of accumulating evidence that cytoplasmic male sterility (CMS) in some species results from an inability to generate the high ATP/ADP ratios required for specific stages of differentiation in the reproductive cycle, a number of aspects of ATP metabolism are being examined in CMS and male fertile plants.In experiments designed to test mitochondrial efficiency in ATP export, organelles from CMS plants performed very poorly when compared with normal lines. It is proposed that although most of the molecules involved in mitochondrial ATP production are nuclear encoded, the lesions in mitochondrial (mt)DNA known to accompany the CMS phenotype may be expressed as small modifications within the architecture of the mitochondrial membrane. To detect whether such changes could affect the ADP-ATP translocator in the membrane, two sets of experiments were carried out to determine a 'Km' for the translocator. The two methods employed were based on different precepts, but nevertheless indicated a 'Km' for the mitochondrial translocator in CMS lines which differed dramatically from that of male fertile plants. The view that CMS in Petunia hybrida thus might result from small differences in mtDNA encoded membrane proteins is considered in the light of the cytological changes seen to accompany CMS in these plants, as well as in the context of current theories advanced to explain CMS in other species.

Rate-controlling steps of oxidative phosphorylation in rat liver mitochondria. A synoptic approach of model and experiment

The contribution of different steps to the control of oxidative phosphorylation in isolated rat liver mitochondria was investigated by a combination of experiments and computer simulations. The parameters of the mathematical model of phosphorylating mitochondria were derived from experimental data. The model correctly describes the competition between ATP utilization inside and outside mitochondria for the ATP generated in mitochondria. On the basis of the good agreement between experiments and simulations, the contribution of different steps to the control of respiration was estimated by computing their control strengths, i.e., the influence of their activities on the rate of respiration. The rate-controlling influences vary depending on the load of oxidative phosphorylation. The predominant steps are: in the fully active state (State 3) — the hydrogen supply to the respiratory chain; in the resting state (State 4) — the proton leak of the mitochondrial inner membrane; in states of non-maximum ATP export — the adenine nucleotide translocator. Titrations of respiration with phenylsuccinate, antimycin, oligomycin and carboxyatractyloside completely support these conclusions.

Distribution of adenine nucleotides in the perfused rat heart.

A computer technique for determination of the distribution of adenine nucleotides among compartmented, protonated, and metal-chelated species has been developed for the perfused rat heart. This procedure requires knowledge of tissue levels of creatine, creatine phosphate, ATP, ADP, and AMP and the glycolytic and respiration rates. The method is applicable to any physiological state of the organ and has been applied to transient behavior in aerobic, anoxic, and ischemic hearts. The results suggest that ADP uptake and ATP export by mitochondria are normally linked and equal in rate during aerobic metabolism or short-term anoxia but become separate and unequal during ischemia, so that mitochondrial adenine nucleotides, primarily AMP, accumulate.