Biogenesis of mitochondrial membranes in Neurospora crassa. Mitochondrial protein synthesis during conidial germination.

The conidia of Neurospora crassa entered logarithmic growth after a 1-h lag period at 30 degrees C. Although [14C]leucine is incorporated quickly early in growth, cellular protein data indicated that no net protein synthesis occurred until after 2 h of growth. Neurospora is known to produce ethanol during germination even though respiratory enzymes are present. Also, Neurospora mitochondria isolated from cells less than 3-h old are uncoupled. Since oxygen uptake increased during germination, was largely cyanide-sensitive, and reached a maximum at 3 h, it is hypothesized that during early germination the uncoupled electron transport chain merely functions to dispose of reducing equivalents generated by substrate level ATP production. The rate of protein synthesis in vitro by mitochondria isolated from 0-8-h-old cells increased as did cell age. Mitochondrial protein synthesis in vivo, assayed in the presence of 100 mug cycloheximide/ml, increased from low levels in the cinidia to peak levels at 3-4 h of age and then slowly decreased. The rate of mitochondrial protein synthesis in vivo was linear for at least 90 min in 0-4-h-old cells, but declined after 15 min of incorporation in 6 and 8-h-old cells. The products of mitochondrial protein synthesis in vivo were analyzed with dodecylsulfate gel electrophoresis and autoradiography. Early in germination 80% of the synthesis was of two small proteins (molecular weights 7200 and 9000). At 8 h 85% of the radioactivity was in 10 larger proteins (12 200 to 80 000). Within the high-molecular-weight class, proteins of between 12 000 and 21 500 molecular weight were preferentially lavelled early in germination, whereas after 8 h of growth proteins of 27 500 to 80 000 molecular weight were preferentially labelled. It is hypothesized that the 7200 and 9000-molecular-weight products of mitochondrial protein synthesis combine with other proteins to form the larger proteins found later in growth. The availability of these other proteins in cells of different ages could affect the rate of mitochondrial protein synthesis in vivo.     

Composition and synthesis of cellular lipids in Neurospora crassa during cellular differentiation.

The synthesis of cellular lipids of Neurospora crassa was measured during growth on low (2% sucrose)- and high (15% glucose)-carbohydrate supplementation. The amount of lipid per dry weight of cells does not change during the germination and early logarithmic growth periods, but the percentage of phospholipid in the lipid does increase, reaching a maximal value of 90% at 4 to 5 h after inoculation, at which time the phospholipid content of the cells is approximately 60 mumol/g (dry weight). The content of the anionic phospholipids, as a percentage of the lipid fraction, is relatively constant during the growth period, but the contents of the zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine change in a reciprocal fashion. During the first 8 h of growth, phosphatidylcholine falls from 53% of the phospholipid to 43%, whereas phosphatidylethanolamine rises from 29 to 38%. The total of these two phospholipids is approximately 83% during the growth period studied. The synthesis of cellular phospholipids, measured either by [32P]H3PO4 or [14C]glucose incorporation, reached maximal levels between 3 and 5 h of growth. The effect of the high-carbohydrate supplement on cellular lipids was minimal. Inclusion of 15% glucose decreased the labeling of phospholipid by [32P]H3PO4, but did not affect lipid composition. This observation is in contrast to the effects of high glucose on mitochondrial phospholipid synthesis.     

Essential roles of mitochondrial and heme function in lung cancer bioenergetics and tumorigenesis

Contrary to Warburg’s hypothesis, mitochondrial oxidative phosphorylation (OXPHOS) contributes significantly to fueling cancer cells. Several recent studies have demonstrated that radiotherapy-resistant and chemotherapy-resistant cancer cells depend on OXPHOS for survival and progression. Several cancers exhibit an increased risk in association with heme intake. Mitochondria are widely known to carry out oxidative phosphorylation. In addition, mitochondria are also involved in heme synthesis. Heme serves as a prosthetic group for several proteins that constitute the complexes of mitochondrial electron transport chain. Therefore, heme plays a pivotal role in OXPHOS and oxygen consumption. Further, lung cancer cells exhibit heme accumulation and require heme for proliferation and invasion in vitro. Abnormalities in mitochondrial biogenesis and mutations are implicated in cancer. This review delves into mitochondrial OXPHOS and lesser explored area of heme metabolism in lung cancer.     

Changes in thermal phase transition of various membranes during temperature acclimation in Tetrahymena

Changes in the thermal phase transition temperature of membrane lipids were studied by X-ray wide-angle diffraction during adaptation of Tetrahymena pyriformis to a lower growth temperature. After a shift in growth temperature from 39 to 15 degrees C, the phase transition temperature was lowered gradually in microsomal and pellicular phospholipids, whereas that in mitochondrial phospholipids was unchanged for 10 h after the temperature shift. Only a small decrease in the transition temperature of mitochondrial phospholipids was observed, even after 24 h following the shift. Transition temperatures of microsomal, pellicular and mitochondrial phospholipids reached the growth temperature (15 degrees C) about 6, 10 and 24 h after the temperature shift. The temperature dependence of the solid phase in membrane phospholipids was estimated from the 4.2 A peak of the X-ray diffraction pattern. In the case of the phospholipids extracted from cells grown at 39 degrees C, the solid phase was increased upon lowering temperature in a similar manner in all three membrane fractions: mitochondria, pellicles and microsomes. However, in the case of the phospholipids from cells exposed to a lower growth temperature (15 degrees C) for 10 h, the increase in the solid phase was significantly smaller in mitochondrial phospholipids than in two other membrane fractions. The difference in the thermal behaviour of mitochondrial lipid from pellicular and microsomal lipids is discussed in terms of phase transition and phase separation.     

Inhibition of mitochondrial respiration by phosphoenolpyruvate

The cytosolic factors that influence mitochondrial oxidative phosphorylation rates are relatively unknown. In this report, we examine the effects of phosphoenolpyruvate (PEP), a glycolytic intermediate, on mitochondrial function. It is reported here that in rat heart mitochondria, PEP delays the onset of state 3 respiration in mitochondria supplied with either NADH-linked substrates or succinate. However, the maximal rate of state 3 respiration is only inhibited when oxidative phosphorylation is supported by NADH-linked substrates. The capacity of PEP to delay and/or inhibit state 3 respiration is dependent upon the presence or absence of ATP. Inhibition of state 3 is exacerbated in uncoupled mitochondria, with a 40% decrease in respiration seen with 0.1mM PEP. In contrast, ATP added exogenously or produced by oxidative phosphorylation completely prevents PEP-mediated inhibition. Mechanistically, the results support the conclusion that the main effects of PEP are to impede ADP uptake and inhibit NADH oxidation. By altering the NADH/NAD(+) status of mitochondria, it is demonstrated that PEP enhances succinate dehydrogenase activity and increase free radical production. The results of this study indicate PEP may be an important modulator of mitochondrial function under conditions of decreased ATP.     

Biochemical and cytological studies of mitochondrial development in chloramphenicol-induced rice coleoptiles.

The ultrastructure and respiratory activity of mitochondria in rice coleoptile grown in the presence and in the absence of an inibitor of mitochondrial protein synthesis (chloramphenicol) have been studied. It is shown that during the first 48 h of germination a rapid development of mitochondrial cristae takes place without notable influence of chloramphenicol on biogenesis of mitochondria. But the presence of the inhibitor has a significant effect in the subsequent period (48-144 h): a gradual and almost complete reduction of mitochondrial cristae is observed. These unusual "noncristate" mitochondria, although greatly lacking cytochrome oxidase, have a high respiratory activity. The respiration of "noncristate" mitochondria is resistant to KCN. It is supposed that chloramphenicol-induced rice coleoptile can be used as a new convenient object for studies of the nature of alternative oxidase as well as the biogenesis of mitochondria with cyanide-insensitive respiration.     

Genetic determination of the mitochondrial adenine nucleotide translocation system and ITS role in the eukaryotic cell

On integrating experimental data published previously, the following picture of the mitochondrial adenine nucleotide (AdN) translocation system is being presented: 1. The AdN translocation system serves not only to transport ATP synthesized within mitochondria into the cytosol but also to transport cytosolic ATP into the mitochondria when oxidative phosphorylation is not functioning. 2. The AdN translocator is coded for by nuclear genes and the mitochondrial protein synthesis is not involved in its formation. 3. The AdN translocation system must be preserved and functioning even in cells which could dispense with oxidative phosphorylation. It assures appropriate concentrations of intramitochondrial ATP. 4. The intramitochondrial ATP is required for normal replication of mitochondrial DNA. Tis supports the view that the mitochondrion is a self-replicating semi-autonomous organelle. 5. The appropriate concentration of ATP must be present in mitochondria to make possible cell growth or multiplication. This points to a direct or indirect role of mitochondria in the control of cell proliferation.     

Biochemical and cytological studies of mitochondrial development in chloramphenicol-induced rice coleoptiles

The ultrastructure and respiratory activity of mitochondria in rice coleoptile grown in the presence and in the absence of an inhibitor of mitochondrial protein synthesis (chloramphenicol) have been studied. It is shown that during the first 48 h of germination a rapid development of mitochondrial cristae takes place without notable influence of chloramphenicol on biogenesis of mitochondria. But the presence of the inhibitor has a significant effect in the subsequent period (48–144 h): a gradual and almost complete reduction of mitochondrial cristae is observed. These unusual “noncristate” mitochondria, although greatly lacking cytochrome oxidase, have a high respiratory activity. The respiration of “noncristate” mitochondria is resistant to KCN. It is supposed that chloramphenicol-induced rice coleoptile can be used as a new convenient object for studies of the nature of alternative oxidase as well as the biogenesis of mitochondria with cyanide-insensitive respiration.     

Changes in mitochondrial membrane composition and oxidative status during rapid growth,maturation and aging in zebrafish, Danio rerio

Considering membranes and membrane components as possible pacemakers of the main processes taking place inside mitochondria, changes in phospholipids or fatty acids could play a central role linking different mechanisms involved in cumulative damage to cell molecules and dysfunction during periods of high stress, such as rapid growth and aging. Changes affecting either lipid class or fatty acid compositions could affect phospholipid and membrane properties and alter mitochondrial function and cell viability. In the present study, mitochondrial oxidative status and mitochondrial membrane phospholipid compositions were analyzed throughout the life-cycle of zebrafish. TBARS content significantly increased in 18-month-old fish while aconitase activity decreased in 24-month-old fish, which have been related with oxidative damage to molecules. Mitochondria-specific superoxide dismutase decreased in 24-month-old animals although this change was not statistically significant. Age affected both mitochondrial phospholipid content and the peroxidation index of most phospholipid classes suggesting that oxidative damage to mitochondrial lipids was occurring.     

Regulation of energy metabolism in Saccharomyces cerevisiae. Relationships between catabolite repression, trehalose synthesis, and mitochondrial development.

Changes in trehalose accumulation and in cytochromes during diauxic growth in glucose medium were examined in a normal Saccharomyces cerevisiae strain. While no appreciable disaccharide accumulation occurred during most of the logarithmic phase, a rapid synthesis took place during the final stages. The intrinsic capacity of cells to accumulate trehalose was also determined under nonproliferating conditions, in glucose medium lacking a nitrogen source. Cells harvested at an early growth stage had a much lower trehalose accumulation capacity than cells taken after glucose was exhausted from the culture medium. A high trehalose accumulation capacity could also be obtained at any growth stage by using maltose or galactose as carbon source. Since cells grown under various conditions exhibit a correlated change in cytochrome development and in trehalose accumulation capacity, it was concluded that the level of glucose repression determines the concentration and/or state of activation of the trehalose synthetase-trehalase complex. Independent control of trehalose accumulation capacity and mitochondrial biogenesis by the level of glucose repression was shown in two ways: by demonstrating derepression of trehalose accumulation without development of cytochromes a and c in microaerobic cells, and by showing repression-dependent changes in a cytoplasmic respiration-deficient (ρ^?) mutant, which lacked functional mitochondria. Therefore, the capacity of a cell to accumulate trehalose is not regulated solely by the supply of ATP generated by oxidative phosphorylation.     

Dependence of mitochondrial oxidative phosphorylation on activity of the adenine nucleotide translocase

The coupled reactions of electron transport and ATP synthesis for the first two sites of mitochondrial oxidative phosphorylation have been previously reported to be near equilibrium in isolated respiring pigeon heart (Erecińska, M., Veech, R. L., and Wilson, D. F. (1974) Arch. Biochem. Biophys. 160, 412-421) and rat liver mitochondria (Forman, N. G., and Wilson, D. F. (1982) J. Biol. Chem. 257, 12908-12915). Measurements are presented in this paper which demonstrate that the same relationship exists for both forward and reverse electron transport in rat heart mitochondria. This conclusion implies that adenine nucleotide translocation, a partial reaction of the system, is also near equilibrium, contrasting with proposals that the translocase is rate-limiting for oxidative phosphorylation. To resolve this controversy, the respiratory rates of suspensions of isolated rat liver and rat heart mitochondria were controlled by varying either the added [ATP]/[ADP][Pi] ratios ratios or [ADP] (by varying hexokinase in a regenerating system). Titrations with carboxyatractyloside, a high affinity inhibitor of the translocase which is noncompetitive with ADP, were carried out to assess the dependence of the respiratory rate on translocase activity. Plots of respiratory rate versus [carboxyatractyloside] were all strongly sigmoidal. In liver mitochondria, 40%-70% and in heart mitochondria 66% of the sites could be blocked with carboxyatractyloside before a 10% decrease in the respiratory rate was observed. Further analysis showed that liver and heart mitochondria have translocase/cytochrome a ratios of 1.52 and 3.20, respectively, and that at 23 degrees C the maximal turnover numbers for the translocases were 65 s-1 and 23 s-1. In all states of controlled respiration (no added inhibitor), a substantial excess of translocase activity was present, suggesting that the translocase was not normally rate-limiting in oxidative phosphorylation.     

Protection of cellular and mitochondrial functions against anoxic damage by fructose in perfused liver.

In anoxic perfused liver, conversion of fructose to lactate was greatly increased to about 3 mumol/min per g liver. This increase in lactate implied that the same amount of ATP was also produced. The rate of metabolism of glucose was less than 10% of that of fructose, as judged by rate of production of lactate. In anoxic liver perfused with fructose, the ATP levels of both the tissue and mitochondria remained high, despite lack of oxygen, thus preventing enzyme leakage and preserving processes requiring ATP, such as bile excretion and urea formation. The mitochondrial oxidative phosphorylation capacity of anoxic liver perfused with fructose was also unimpaired. Spectral analysis of light transmitted through the liver revealed that the mitochondrial electron transfer system was in the completely reduced state during anoxia, indicating that the mitochondria were incapable of synthesizing ATP. These results suggest that fructose metabolism during anoxia resulted in sufficient production of ATP for maintaining the physiological functions of the cells and the oxidative phosphorylation capacity of their mitochondria.     

Role of Unc104/KIF1-related motor proteins in mitochondrial transport in Neurospora crassa

Eukaryotic cells use diverse cytoskeleton-dependent machineries to control inheritance and intracellular positioning of mitochondria. In particular, microtubules play a major role in mitochondrial motility in the filamentous fungus Neurospora crassa and in mammalian cells. We examined the role of two novel Unc104/KIF1-related members of the kinesin family, Nkin2 and Nkin3, in mitochondrial motility in Neurospora. The Nkin2 protein is required for mitochondrial interactions with microtubules in vitro. Mutant hyphae lacking Nkin2 show mitochondrial motility defects in vivo early after germination of conidiospores. Nkin3, a member of a unique fungal-specific subgroup of small Unc104/KIF1-related proteins, is not associated with mitochondria in wild-type cells. However, it is highly expressed and recruited to mitochondria in Deltankin-2 mutants. Mitochondria lacking Nkin2 require Nkin3 for binding to microtubules in vitro, and mitochondrial motility defects in Deltankin-2 mutants disappear with up-regulation of Nkin3 in vivo. We propose that mitochondrial transport is mediated by Nkin2 in Neurospora, and organelle motility defects in Deltankin-2 mutants are rescued by Nkin3. Apparently, a highly versatile complement of organelle motors allows the cell to efficiently respond to exogenous challenges, a process that might also account for the great variety of different mitochondrial transport systems that have evolved in eukaryotic cells.     

Inhibition of mitochondrial protein synthesis influences the glucocorticoid sensitivity of lymphoid cells

Inhibition of mitochondrial protein synthesis impairs the formation of the 13 polypeptides encoded on the mitochondrial genome. These polypeptides are part of enzyme complexes involved in oxidative phosphorylation. Prolonged inhibition of mitochondrial protein synthesis thus reduces the oxidative phosphorylation capacity which ultimately results in impairment of energy-requiring processes. Via a different mechanism glucocorticoid hormones also decrease the oxidative phosphorylation capacity of, e.g., lymphoid cells. The present study shows that inhibition of mitochondrial protein synthesis influences glucocorticoid-induced responses of lymphoid cells in two opposing manners. (a) It is enhanced after induction in cells with a reduced oxidative phosphorylation capacity resulting from preceding inhibition of mitochondrial protein synthesis. This can be explained by the synergistic effects of glucocorticoids and prolonged inhibition of mitochondrial protein synthesis on energy-producing processes. (b) It is counteracted when mitochondrial protein synthesis is impaired during induction of the response. The latter observation suggests that mitochondrial protein synthesis is involved in the generation of glucocorticoid-induced effects on lymphoid cells.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.     

The effects of altered sterol composition on the mitochondrial adenine nucleotide transporter of Saccharomyces cerevisiae.

1. The membrane sterol composition of mitochondria of the ole-3 mutant of Saccharomyces cerevisiae was manipulated by growing the organism in the presence of Tween 80 (1%, W/V) plus defined supplements o- delta-aminolaevulinate. 2. Changes in mitochondrial sterol content induced considerable changes in the adenine nucleotide transporter. 3. As the sterol content was decreased, the affinity of the transporter for ATP did not alter significantly, but the rate of ATP uptake was greatly decreased, the total number of atractylate-sensitive binding sites diminished, and the proportion of high-affinity binding sites was decreased. 4. Since sterol depletion also uncouples oxidative phosphorylation [Astin & Haslam (1977) Biochem. J., 166, 287-298] and prevents the intramitochondrial generation of ATP, the decrease in the rate of ATP uptake by sterol-depleted mitochondria will cause a decrease in intramitochondrial ATP concentrations in vivo. This probably explains the inhibition of mitochondrial macromolecular synthesis that has previously been reported in lipid-depleted yeast mitochondria.     

Hexose metabolism in pancreatic islets: preferential utilization of mitochondrial ATP for glucose phosphorylation

The respective contribution of exogenous and intramitochondrially formed ATP to D-glucose phosphorylation by mitochondria-bound hexokinase was examined in both rat liver and pancreatic islet mitochondria by comparing the generation of D-glucose 6-[32P]phosphate from exogenous [gamma-32P]ATP to the total rate of D-[U-14C]glucose phosphorylation. In liver mitochondria, the fractional contribution of exogenous ATP to D-glucose phosphorylation ranged from 4 to 74%, depending on the availability of endogenous ATP formed by either oxidative phosphorylation or in the reaction catalyzed by adenylate kinase. Likewise, in islet mitochondria exposed to exogenous ATP but deprived of exogenous nutrient, about 60% of D-glucose phosphorylation was supported by mitochondrial ATP. Such a fractional contribution was further increased in the presence of ADP and succinate, and suppressed by mitochondrial poisons. It is concluded that, in islet like in liver mitochondria, mitochondrial ATP is used preferentially to exogenous ATP as a substrate for D-glucose phosphorylation by mitochondria-bound hexokinase. This may favour the maintenance of a high cytosolic ATP concentration in glucose-stimulated islet cells.     

Generation of adenosine triphosphate in cytochrome-deficient mutants of Neurospora.

The fungus Neurospora crassa is known to possess a branched respiratory system consisting of the standard cytochrome chain and a cyanide-insensitive alternate oxidase. In the present experiments, the physiological function of the alternate oxidase has been analyzed by taking advantage of a number of cytochrome-deficient mutants, particularly poky f. Respiration, cellular ATP levels, and growth have been examined under the influence of three classes of inhibitors: inhibitors of the cytochrome chain (antimycin, cyanide), an inhibitor of the laternate oxidase (salicyl hydroxamic acid), and an uncoupling agent (carbonyl cyanide m-chlorophenylhydrazone). The results indicate that the over-all efficiency of the alternate oxidase in producing ATP and supporting growth is much less than that of the cytochrome chain. Depending upon the amount of oxidative phosphorylation at Sites II and III in the cytochrome chain, which varies from strain to strain, the efficiency of the alternate oxidase relative to that of the cytochrome chain ranges from 13% in wild type Neurospora to 18 to 21% in poky f, 35% in mi-3, and 57% in cyt-2. A comparison of the short term effects of cyanide and carbonyl cyanide m-chlorophenylhydrazone on cellular ATP in poky f suggests that, during respiration through the alternate oxidase, ATP can be produced both by substrate-level phosphorylation (accompanying glycolysis and the oxidation of alpha-ketoglutarate) and by oxidative phosphorylation at Site I. When cells are grown on sucrose, as much as 22% of ATP synthesis in the presence of cyanide occurs at Site I. When cells are grown on acetate to diminish the rate of glycolysis, the contribution of Site I becomes proportionately larger. Both the growth experiments and the short term inhibitor experiments reveal that ATP levels in Neurospora are kept high be a feedback process which depresses ATP breakdown (and growth) very quckly after ATP synthesis is inhibited. Thus, poky f grows more slowly that wild type Neurospora and is inhibited still further when either the cytochrome chain or the alternate oxidase is blocked. Under all of these conditions, however, cellular ATP in poky f is maintained at a high level (about 3 mmol per kg of cell water, slightly above the values measured in the wild type strain). Continue.     

Heat-induced changes in respiratory pathways and mitochondrial structure during microcycle conidiation of Neurospora crassa

Changes in both respiratory pathways and mitochondrial structure of Neurospora crassa occurred under conditions of microcycle conidiation. Upon heat-treatment at 46°C, conidia developed a highly cyanide-insensitive, hydroxamate-sensitive respiration associated with morphological alterations in mitochondrial membranes; such changes were time-dependent. When heat-treated conidia were shifted down to 25°C, the alternate, hydroxamate-sensitive respiration decreased significantly, paralleling the recovery of well-cristated mitochondria with an electron-dense matrix in the germ tubes. The decrease in hydroxamate-sensitivity was associated with two periods of increase in cyanide sensitivity corresponding to the events of germination and precocious proconidial budding.