Interaction between the oxa1 and rmp1 genes modulates respiratory complex assembly and life span in Podospora anserina

A causal link between deficiency of the cytochrome respiratory pathway and life span was previously shown in the filamentous fungus Podospora anserina. To gain more insight into the relationship between mitochondrial function and life span, we have constructed a strain carrying a thermosensitive mutation of the gene oxa1. OXA1 is a membrane protein conserved from bacteria to human. The mitochondrial OXA1 protein is involved in the assembly/insertion of several respiratory complexes. We show here that oxa1 is an essential gene in P. anserina. The oxa1(ts) mutant exhibits severe defects in the respiratory complexes I and IV, which are correlated with an increased life span, a strong induction of the alternative oxidase, and a reduction in ROS production. However, there is no causal link between alternative oxidase level and life span. We also show that in the oxa1(ts) mutant, the extent of the defects in complexes I and IV and the life-span increase depends on the essential gene rmp1. The RMP1 protein, whose function is still unknown, can be localized in the mitochondria and/or the cytosolic compartment, depending on the developmental stage. We propose that the RMP1 protein could be involved in the process of OXA1-dependent protein insertion.     

A mutation in the gene encoding cytochrome c1 leads to a decreased ROS content and to a long-lived phenotype in the filamentous fungus Podospora anserina

We present here the properties of a complex III loss-of-function mutant of the filamentous fungus Podospora anserina. The mutation corresponds to a single substitution in the second intron of the gene cyc1 encoding cytochrome c(1), leading to a splicing defect. The cyc1-1 mutant is long-lived, exhibits a defect in ascospore pigmentation, has a reduced growth rate and a reduced ROS production associated with a stabilisation of its mitochondrial DNA. We also show that increased longevity is linked with morphologically modified mitochondria and an increased number of mitochondrial genomes. Overexpression of the alternative oxidase rescues all these phenotypes and restores aging. Interestingly, the absence of complex III in this mutant is not paralleled with a deficiency in complex I activity as reported in mammals although the respiratory chain of P. anserina has recently been demonstrated to be organized according to the "respirasome" model.     

Inhibition of Oxidative Phosphorylation Induces a Rapid Death of GA-Pretreated Aleurone Cells,But Not of ABA-Pretreated Aleurone Cells

Reactive oxygen species (ROS) mediate programmed cell death in aleurone cells, which is promoted by gibberellic acid (GA) and prevented by abscisic acid (ABA). Plant mitochondria contain two distinct respiratory pathways: respiration through cytochrome c oxidase increases ROS production, whereas respiration through the alternative oxidase pathway lowers it. While studying the effects of GA and ABA on partitioning of respiration between those two pathways during the germinating process, we discovered that oxidative phosphorylation inhibitors like sodium azide and 2, 4-dinitrophenol induce rapid death of GA-pretreated aleurone cells but not of ABA-pretreated cells. Functional aerobic respiration was required for GA signaling, and 6 to 12 hours of GA signaling altered the cellular state of aleurone cells to be extremely susceptible to inhibition of oxidative phosphorylation. Anaerobic conditions were also able to mimic the effects of respiratory inhibitors in specifically inducing cell death in GA-treated cells, but cell death was provoked much more slowly. Cotreatment with various antioxidants did not prevent this process at all, suggesting that no ROS are responsible for this respiratory inhibitor-induced cell death. Our observation implicates that GA may partition all the electrons produced during mitochondrial respiration only to the cytochrome oxidase pathway, which would at least partly contribute to cellular accumulation of ROS.     

Melatonin-related mitochondrial respiration responses are associated with growth promotion and cold tolerance in plants

Melatonin has the ability to improve plant growth and strengthened plant tolerance to environmental stresses; however, the effects of melatonin on mitochondrial respiration in plants and the underlying biochemical and molecular mechanisms are still unclear. The objective of the study is to determine possible effects of melatonin on mitochondrial respiration and energy efficiency in maize leaves grown under optimum temperature and cold stress and to reveal the relationship between melatonin-induced possible alterations in mitochondrial respiration and cold tolerance. Melatonin and cold stress, alone and in combination, caused significant increases in activities and gene expressions of pyruvate dehydrogenase, citrate synthase, and malate dehydrogenase, indicating an acceleration in the rate of tricarboxylic acid cycle. Total mitochondrial respiration rate, cytochrome pathway rate, and alternative respiration rate were increased by the application of melatonin and/or cold stress. Similarly, gene expression and protein levels of cytochrome oxidase and alternative oxidase were also enhanced by melatonin and/or cold stress. The highest values for all these parameters were obtained from the seedlings treated with the combined application of melatonin and cold stress. The activity and gene expression of ATP synthase and ATP concentration were augmented by melatonin under control and cold stress. On the other hand, cold stress reduced markedly plant growth parameters, including root length, plant height, leaf surface area, and chlorophyll content and increased the content of reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide and oxidative damage, including malondialdehyde content and electrolyte leakage level; however, melatonin significantly promoted the plant growth parameters and reduced ROS content and oxidative damage under control and cold stress. These data revealed that melatonin-induced growth promotion and cold tolerance in maize is associated with its modulating effect on mitochondrial respiration.     

Alternative oxidase dependent respiration leads to an increased mitochondrial content in two long-lived mutants of the aging model Podospora anserina

The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms.     

The concomitant expression and availability of conventional and alternative, cyanide-insensitive, respiratory pathways in Candida albicans

The opportunistic oral pathogen Candida albicans expresses a cyanide-insensitive alternative oxidase (AOX) upon exposure to respiratory inhibitors that act downstream from coenzyme Q, and upon ageing of cells. To investigate whether the conventional pathway is retained when the alternative pathway is induced, cells were grown in the presence of sodium cyanide, a reversible inhibitor of cytochrome oxidase. AOX expression was monitored by Western blotting and the presence of cytochromes associated with complexes III and IV of the conventional pathway was monitored by recording spectra between 500 and 650 nm at 77K. The activities of complexes III and IV were determined in polarographic and enzyme-kinetic experiments using specific respiratory substrates and inhibitors. Results indicated that complexes III and IV are constitutively expressed and are functional in cells expressing AOX. Furthermore, the enzymatic activities of complexes III and IV were similar in mitochondrial preparations from cells grown with or without cyanide. We next investigated whether both pathways are simultaneously available for electron transfer from the Q pool to molecular oxygen. Respiration was virtually completely inhibited by the combination of cyanide and salicyl hydroxamic acid (SHAM) or antimycin A and SHAM, but only partly inhibited by either of these inhibitors alone. This indicates that electrons can in principle flow either through the conventional or the alternative respiratory pathway. The availability of two electron pathways in C. albicans and the potential use of either pathway endows this pleomorphic fungus with another level at which it can rapidly adjust to altered environmental conditions.     

Lack of respiratory chain complex I impairs alternative oxidase engagement and modulates redox signaling during elicitor-induced cell death in tobacco

Alternative oxidase (AOX) functions in stress resistance by preventing accumulation of reactive oxygen species (ROS), but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. We investigated the relationships between AOX expression and in vivo activity in Nicotiana sylvestris and the complex I-deficient CMSII mutant in response to a cell death elicitor. While a specific AOX1 isoform in the active reduced state was constitutively overexpressed in CMSII, partitioning through the alternative pathway was similar to the wild type. Lack of correlation between AOX content and activity indicates severe metabolic constraints in nonstressed mutant leaves. The bacterial elicitor harpin N(Ea) induced similar timing and extent of cell death and a twofold respiratory burst in both genotypes with little change in AOX amounts. However, partitioning to AOX was increased twofold in the wild type but remained unchanged in CMSII. Oxidative phosphorylation modeling indicated a twofold ATP increase in both genotypes. By contrast, mitochondrial superoxide dismutase activity and reduced forms of ascorbate and glutathione were higher in CMSII than in the wild type. These results demonstrate genetically programmed flexibility of plant respiratory routes and antioxidants in response to elicitors and suggest that sustained ATP production, rather than AOX activity by itself or mitochondrial ROS, might be important for in planta cell death.     

Mitochondrial metabolism and aging in the filamentous fungus Podospora anserina

The filamentous fungus Podospora anserina has a limited lifespan. In this organism, aging is systematically associated to mitochondrial DNA instability. We recently provided evidence that the respiratory function is a key determinant of its lifespan. Loss of function of the cytochrome pathway leads to the compensatory induction of an alternative oxidase, to a decreased production of reactive oxygen species and to a striking increase in lifespan. These changes are associated to the stabilization of the mitochondrial DNA. Here we review and discuss the links between these different parameters and their implication in the control of lifespan. Since we demonstrated the central role of mitochondrial metabolism in aging, the same relationship has been evidenced in several model systems from yeast to mice, confirming the usefulness of simple organisms as P. anserina for studying lifespan regulation.     

Altered Mitochondrial Respiration in a Chromosomal Mutant of Neurospora crassa

A mutant of Neurospora crassa (cni-1) has been isolated that has two pathways of mitochondrial respiration. One pathway is sensitive to cyanide and antimycin A, the other is sensitive only to salicyl hydroxamic acid. Respiration can proceed through either pathway and both pathways together in this mutant account for greater than 90% of all mitochondrial respiration. The cni-1 mutation segregates as a nuclear gene in crosses to other strains of Neurospora. Absorption spectra of isolated mitochondria from cni-1 show typical b- and c-type cytochromes but the absorption peaks corresponding to cytochrome aa(3) are not detectable. Extraction of soluble cytochrome c-546 from these mitochondria followed by reduction with ascorbate reveals a new absorption peak at 426 nm that is not present in wild-type mitochondria. This peak may be due to an altered cytochrome oxidase with abnormal spectral properties. Mitochondria from cni-1 have elevated levels of succinate-cytochrome c reductase but reduced levels of nicotinamide adenine dinucleotide reduced form cytochrome c reductase and of cyanide- and azide-sensitive cytochrome c oxidase. These studies suggest that the cni-1 mutation results in the abnormal assembly of cytochrome c oxidase so that the typical cytochrome aa(3) spectrum is lost and the enzyme activity is reduced. As a consequence of this alteration, a cyanide-insensitive respiratory pathway is elaborated by these mitochondria which may serve to stimulate adenosine 5'-triphosphate production via substrate level phosphorylation by glycolysis and the Krebs cycle.     

During the stationary growth phase, Yarrowia lipolytica prevents the overproduction of reactive oxygen species by activating an uncoupled mitochondrial respiratory pathway

In the branched mitochondrial respiratory chain from Yarrowia lipolytica there are two alternative oxido-reductases that do not pump protons, namely an external type II NADH dehydrogenase (NDH2e) and the alternative oxidase (AOX). Direct electron transfer between these proteins is not coupled to ATP synthesis and should be avoided in most physiological conditions. However, under low energy-requiring conditions an uncoupled high rate of oxygen consumption would be beneficial, as it would prevent overproduction of reactive oxygen species (ROS). In mitochondria from high energy-requiring, logarithmic-growth phase cells, most NDH2e was associated to cytochrome c oxidase and electrons from NADH were channeled to the cytochromic pathway. In contrast, in the low energy requiring, late stationary-growth phase, complex IV concentration decreased, the cells overexpressed NDH2e and thus a large fraction of this enzyme was found in a non-associated form. Also, the NDH2e-AOX uncoupled pathway was activated and the state IV external NADH-dependent production of ROS decreased. Association/dissociation of NDH2e to/from complex IV is proposed to be the switch that channels electrons from external NADH to the coupled cytochrome pathway or allows them to reach an uncoupled, alternative, ΔΨ-independent pathway.     

Alternative Oxidase Dependent Respiration Leads to an Increased Mitochondrial Content in Two Long-Lived Mutants of the Ageing Model Podospora anserina

The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms.     

Metabolic regulation of pathways of carbohydrate oxidation in potato (Solanum tuberosum) tubers

In the present article we evaluate the consequence of tuber-specific expression of yeast invertase, on the pathways of carbohydrate oxidation, in potato ( Solanum tuberosum L. cv. Desiree). We analysed the relative rates of glycolysis and the oxidative pentose phosphate pathway that these lines exhibited as well as the relative contributions of the cytochrome and alternative pathways of mitochondrial respiration. Enzymatic and protein abundance analysis revealed concerted upregulation of the glycolytic pathway and of specific enzymes of the tricarboxylic acid cycle and the alternative oxidase but invariant levels of enzymes of the oxidative pentose phosphate pathway and proteins of the cytochrome pathway. When taken together these experiments suggest that the overexpression of a cytosolic invertase (EC 3.2.1.26) results in a general upregulation of carbohydrate oxidation with increased flux through both the glycolytic and oxidative pentose phosphate pathways as well as the cytochrome and alternative pathways of oxidative phosphorylation. Moreover these data suggest that the upregulation of respiration is a consequence of enhanced efficient mitochondrial metabolism.     

Impact of a disruption of a pathway delivering copper to mitochondria on Podospora anserina metabolism and life span

A global depletion of cellular copper as the result of a deficiency in high-affinity copper uptake was previously shown to affect the phenotype and life span of the filamentous fungus Podospora anserina. We report here the construction of a strain in which the delivery of copper to complex IV of the mitochondrial respiratory chain is affected. This strain, PaCox17::ble, is a PaCox17-null mutant that does not synthesize the molecular chaperone targeting copper to cytochrome c oxidase subunit II. PaCox17::ble is characterized by a decreased growth rate, a reduction in aerial hyphae formation, reduced female fertility, and a dramatic increase in life span. The mutant respires via a cyanide-resistant alternative pathway, displays superoxide dismutase (SOD) activity profiles significantly differing from those of the wild-type strain and is characterized by a stabilization of the mitochondrial DNA. Collectively, the presented data define individual components of a molecular network effective in life span modulation and copper as an element with a dual effect. As a cofactor of complex IV of the respiratory chain, it is indirectly involved in the generation of reactive oxygen species (ROS) and thereby plays a life span-limiting role. In contrast, Cu/Zn SOD as a ROS-scavenging enzyme lowers molecular damage and thus positively affects life span. Such considerations explain the reported differences in life span of independent mutants and spread more light on the delicate tuning of the molecular network influencing biological ageing.     

Direct effect of Taxol on free radical formation and mitochondrial permeability transition

To elucidate the potential role of mitochondria in Taxol-induced cytotoxicity, we studied its direct mitochondrial effects. In Percoll-gradient purified liver mitochondria, Taxol induced large amplitude swelling in a concentration-dependent manner in the microM range. Opening of the permeability pore was also confirmed by the access of mitochondrial matrix enzymes for membrane impermeable substrates in Taxol-treated mitochondria. Taxol induced the dissipation of mitochondrial membrane potential (DeltaPsi) determined by Rhodamine123 release and induced the release of cytochrome c from the intermembrane space. All these effects were inhibited by 2.5 microM cyclosporine A. Taxol significantly increased the formation of reactive oxygen species (ROS) in both the aqueous and the lipid phase as determined by dihydrorhodamine123 and resorufin derivative. Cytochrome oxidase inhibitor CN(-), azide, and NO abrogated the Taxol-induced mitochondrial ROS formation while inhibitors of the other respiratory complexes and cyclosporine A had no effect. We confirmed that the Taxol-induced collapse of DeltaPsi and the induction of ROS production occurs in BRL-3A cells. In conclusion, Taxol-induced adenine nucleotide translocase-cyclophilin complex mediated permeability transition, and cytochrome oxidase mediated ROS production. Because both cytochrome c release and mitochondrial ROS production can induce suicide pathways, the direct mitochondrial effects of Taxol may contribute to its cytotoxicity.     

Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin

Reactive oxygen species (ROS) are considered a key factor in mitochondrial dysfunction associated with brain aging process. Mitochondrial respiration is an important source of ROS and hence a potential contributor to brain functional changes with aging. In this study, we examined the effect of aging on cytochrome c oxidase activity and other bioenergetic processes such as oxygen consumption, membrane potential and ROS production in rat brain mitochondria. We found a significant age-dependent decline in the cytochrome c oxidase activity which was associated with parallel changes in state 3 respiration, membrane potential and with an increase in H₂O₂ generation. The cytochrome aa₃ content was practically unchanged in mitochondria from young and aged animals. The age-dependent decline of cytochrome c oxidase activity could be restored, in situ, to the level of young animals, by exogenously added cardiolipin. In addition, exposure of brain mitochondria to peroxidized cardiolipin resulted in an inactivation of this enzyme complex. It is suggested that oxidation/depletion of cardiolipin could be responsible, at least in part, for the decline of cytochrome c oxidase and mitochondrial dysfunction in brain aging. Melatonin treatment of old animals largely prevented the age-associated alterations of mitochondrial bioenergetic parameters. These results may prove useful in elucidating the molecular mechanisms underlying mitochondrial dysfunction associated with brain aging process, and may have implications in etiopathology of age-associated neurodegenerative disorders and in the development of potential treatment strategies.