Glutamate-mediated inhibition of oxidative phosphorylation in cultured retinal cells

Glutamate is an excitotoxin responsible for causing neuronal damage associated with mitochondria dysfunction. We have analyzed the relationship between the mitochondrial respiratory rate, the membrane potential (delta psi) and the activity of mitochondrial complexes in retinal cells in culture, used as neuronal models. Glutamate (10 microM-10 mM) dose-dependently decreased the O2 consumption and the membrane potential. A linear correlation was found between these parameters, suggesting that the mitochondrial respiratory function was affected. Exposure to glutamate (100 microM) for 10 min, in the absence of Mg2+, inhibited the activity of complex I (26.3%), complexes II/III (22.2%) and complex IV (26.7%). MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), a non-competitive antagonist of the NMDA (N-methyl-D-aspartate) receptors, completely reversed the effect exerted by 100 microM glutamate at the level of complexes I, II/III and IV. These results suggest that NMDA receptor-mediated inhibition of mitochondrial respiratory chain complexes may be responsible for the alteration in the respiratory rate of chick retinal cells submitted to glutamate.     

Proton/electron stoichiometry of mitochondrial complex I estimated from the equilibrium thermodynamic force ratio.

The thermodynamic forces on electrons (delta Eh) and protons (delta p) across mitochondrial complexes I, III and IV were measured in isolated mitochondria respiring on succinate. The force ratio (delta Eh/delta p) across complex I close to equilibrium was found to be about 2. The equilibrium force ratio across complex I was measured during sulphite oxidation and was again close to 2. These results indicate that the proton/electron stoichiometry of complex I is 2, in conditions of high protonmotive force.     

Chemiosmotic systems in bioenergetics: H(+)-cycles and Na(+)-cycles.

The development of membrane bioenergetic studies during the last 25 years has clearly demonstrated the validity of the Mitchellian chemiosmotic H+ cycle concept. The circulation of H+ ions was shown to couple respiration-dependent or light-dependent energy-releasing reactions to ATP formation and performance of other types of membrane-linked work in mitochondria, chloroplasts, some bacteria, tonoplasts, secretory granules and plant and fungal outer cell membranes. A concrete version of the direct chemiosmotic mechanism, in which H+ potential formation is a simple consequence of the chemistry of the energy-releasing reaction, is already proved for the photosynthetic reaction centre complexes. Recent progress in the studies on chemiosmotic systems has made it possible to extend the coupling-ion principle to an ion other than H+. It was found that, in certain bacteria, as well as in the outer membrane of the animal cell, Na+ effectively substitutes for H+ as the coupling ion (the chemiosmotic Na+ cycle). A precedent is set when the Na+ cycle appears to be the only mechanism of energy production in the bacterial cell. In the more typical case, however, the H+ and Na+ cycles coexist in one and the same membrane (bacteria) or in two different membranes of one and the same cell (animals). The sets of delta mu H+ and delta mu Na+ generators as well as delta mu H+ and delta mu Na+ consumers found in different types of biomembranes, are listed and discussed.     

Supercomplexes in the respiratory chains of yeast and mammalian mitochondria

Around 30-40 years after the first isolation of the five complexes of oxidative phosphorylation from mammalian mitochondria, we present data that fundamentally change the paradigm of how the yeast and mammalian system of oxidative phosphorylation is organized. The complexes are not randomly distributed within the inner mitochondrial membrane, but assemble into supramolecular structures. We show that all cytochrome c oxidase (complex IV) of Saccharomyces cerevisiae is bound to cytochrome c reductase (complex III), which exists in three forms: the free dimer, and two supercomplexes comprising an additional one or two complex IV monomers. The distribution between these forms varies with growth conditions. In mammalian mitochondria, almost all complex I is assembled into supercomplexes comprising complexes I and III and up to four copies of complex IV, which guided us to present a model for a network of respiratory chain complexes: a 'respirasome'. A fraction of total bovine ATP synthase (complex V) was isolated in dimeric form, suggesting that a dimeric state is not limited to S.cerevisiae, but also exists in mammalian mitochondria.     

Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol

Two type 2A protein phosphatases, phosphatases I (Mr = 180,000) and III (Mr = 177,000), were purified to near homogeneity from human erythrocyte cytosol. Phosphatase I was composed of alpha (34 kDa), beta (63 kDa), and delta (74 kDa) subunits in a ratio of 1:1:1. Phosphatase III comprised alpha, beta, and gamma (53 kDa) subunits in the same ratio. Heparin-Sepharose column chromatography converted most of phosphatase I and 20% of phosphatase III into alpha 1 beta 1 which were indistinguishable from phosphatase IV (Usui, H., Kinohara, N., Yoshikawa, K., Imazu, M., Imaoka, T., and Takeda, M. (1983) J. Biol. Chem. 258, 10455-10463). The catalytic subunit alpha and the beta subunit of phosphatases I, III, and IV displayed identical V8 and papain peptide maps, respectively, while the peptide maps of the alpha, beta, gamma, and delta subunits were clearly distinct. The molar ratio of phosphatases I, III, and IV in erythrocyte cytosol was estimated to be 6:1:14. Comparison of molecular activities of alpha, alpha 1 beta 1, alpha 1 beta 1 delta 1, and alpha 1 beta 1 gamma 1 revealed that beta suppressed phosphorylase and P-H2B histone phosphatase activities of alpha but stimulated the P-H1 histone phosphatase activity, and delta suppressed all the phosphatase activities of alpha 1 beta 1. The gamma subunit stimulated the P-histone phosphatase activity of alpha 1 beta 1 but inhibited the phosphorylase and P-spectrin phosphatase activities. The beta subunit increased the Mg2+ or Mn2+ requirement for P-H2B histone phosphatase activity of alpha, an effect which was counteracted by delta. The effects of heparin, H1 histone, protamine, and polylysine on the phosphorylase phosphatase activity of phosphatases I, III, IV, and alpha were described and discussed in connection with the functions of the subunits.     

Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1

The respiratory chain in the inner mitochondrial membrane contains three large multi‐enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19‐Å 3D map of the 1.7‐MDa amphipol‐solubilized supercomplex I₁III₂IV₁ from bovine heart obtained by single‐particle electron cryo‐microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X‐ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol‐binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.     

In vivo membrane assembly of the E.coli polytopic protein, melibiose permease, occurs via a Sec-independent process which requires the protonmotive force.

To investigate the mechanism of polytopic membrane protein insertion in Escherichia coli, we have examined the protein and energy requirements for in vivo membrane assembly of the prototypic 12 transmembrane domain sugar co-transporter, melibiose permease (MelB). MelB membrane assembly was analyzed both kinetically, by pulse labeling experiments, and functionally by measuring the activity of the inserted permease. Strikingly, the rate of MelB membrane assembly is decreased approximately 4-fold upon dissipation of the transmembrane electrochemical proton gradient, delta(mu)H+, indicative of a strong requirement for delta(mu)H+. Interestingly, selective dissipation of either the electrical (delta(psi)) or the chemical (delta(pH)) component of delta(mu)H+ demonstrates that either form of energy is required for MelB membrane assembly. In contrast, MelB membrane assembly does not require SecA, SecY or SecE, all three proteins which are strictly required for protein translocation. Neither the rate of MelB membrane assembly nor the amount of functional permease is affected by inactivation or depletion of these Sec proteins. These results strongly suggest that polytopic membrane proteins such as MelB insert into the cytoplasmic membrane by a mechanism fundamentally different from protein translocation.     

The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes

The ratios of the oxidative phosphorylation complexes NADH:ubiquinone reductase (complex I), succinate:ubiquinone reductase (complex II), ubiquinol:cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and F1F0-ATP synthase (complex V) from bovine heart mitochondria were determined by applying three novel and independent approaches that gave consistent results: 1) a spectrophotometric-enzymatic assay making use of differential solubilization of complexes II and III and parallel assays of spectra and catalytic activities in the samples before and after ultracentrifugation were used for the determination of the ratios of complexes II, III, and IV; 2) an electrophoretic-densitometric approach using two-dimensional electrophoresis (blue native-polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis) and Coomassie blue-staining indices of subunits of complexes was used for determining the ratios of complexes I, III, IV, and V; and 3) two electrophoretic-densitometric approaches that are independent of the use of staining indices were used for determining the ratio of complexes I and III. For complexes I, II, III, IV, and V in bovine heart mitochondria, a ratio 1.1 +/- 0.2:1.3 +/- 0.1:3:6.7 +/- 0.8:3.5 +/- 0.2 was determined.     

Intrinsic characteristics of the proton pump in the luminal membrane of a tight urinary epithelium. The relation between transport rate and delta mu H

A number of tight urinary epithelia, as exemplified by the turtle bladder, acidify the luminal solution by active transport of H+ across the luminal cell membrane. The rate of active H+ transport (JH) decreases as the electrochemical potential difference for H+ [delta mu H = mu H(lumen) - mu H(serosa)] across the epithelium is increased. The luminal cell membrane has a low permeability for H+ equivalents and a high electrical resistance compared with the basolateral cell membrane. Changes in JH thus reflect changes in active H+ transport across the luminal membrane. To examine the control of JH by delta mu H in the turtle bladder, transepithelial electrical potential differences (delta psi) were imposed at constant acid-base conditions or the luminal pH was varied at delta psi = 0 and constant serosal PCO2 and pH. When the luminal compartment was acidified from pH 7 to 4 or was made electrically positive, JH decreased as a linear function of delta mu H as previously described. When the luminal compartment was made alkaline from pH 7 to 9 or was made electrically negative, JH reached a maximal value, which was the same whether the delta mu H was imposed as a delta pH or a delta psi. The nonlinear JH vs. delta mu H relation does not result from changes in the number of pumps in the luminal membrane or from changes in the intracellular pH, but is a characteristic of the H+ pumps themselves. We propose a general scheme, which, because of its structural features, can account for the nonlinearity of the JH vs. delta mu H relations and, more specifically, for the kinetic equivalence of the effects of the chemical and electrical components of delta mu H. According to this model, the pump complex consists of two components: a catalytic unit at the cytoplasmic side of the luminal membrane, which mediates the ATP-driven H+ translocation, and a transmembrane channel, which mediates the transfer of H+ from the catalytic unit to the luminal solution. These two components may be linked through a buffer compartment for H+ (an antechamber).     

Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans

Stable supercomplexes of bacterial respiratory chain complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) have been isolated as early as 1985 (Berry, E. A., and Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete "respirasome." Inactivation of the gene for tightly bound cytochrome c552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.     

A high melting structure in DNA distinguishes phases of the cell cycle

Differential scanning microcalorimetry of the nuclei of dividing CHO cells revealed DNA structures that showed structural transitions at 60, 76, 88, and 105 degrees C (transitions I to IV, respectively). In cultures synchronized by isoleucine deprivation the enthalpies of transitions I and II were rather constant throughout the cell cycle. While the sum of the enthalpies of III and IV was nearly constant, the ratio of IV to III varied substantially from one phase of the cycle to another. A high IV:III ratio of 6 characterized G1 while S phase gave a IV:III ratio of about 2. Cells containing metaphase chromosomes also showed a IV:III ratio near 2. The IV:III ratio for CHO cells showed a progressive decrease as the cells were maintained in isoleucine-free medium from 0 to 6 days.     

Mutations in mitochondrial complex III uniquely affect complex I in Caenorhabditis elegans

Mitochondrial supercomplexes containing complexes I, III, and IV of the electron transport chain are now regarded as an established entity. Supercomplex I·III·IV has been theorized to improve respiratory chain function by allowing quinone channeling between complexes I and III. Here, we show that the role of the supercomplexes extends beyond channeling. Mutant analysis in Caenorhabditis elegans reveals that complex III affects supercomplex I·III·IV formation by acting as an assembly or stabilizing factor. Also, a complex III mtDNA mutation, ctb-1, inhibits complex I function by weakening the interaction of complex IV in supercomplex I·III·IV. Other complex III mutations inhibit complex I function either by decreasing the amount of complex I (isp-1), or decreasing the amount of complex I in its most active form, the I·III·IV supercomplex (isp-1;ctb-1). ctb-1 suppresses a nuclear encoded complex III defect, isp-1, without improving complex III function. Allosteric interactions involve all three complexes within the supercomplex and are necessary for maximal enzymatic activities.     

Architecture of active mammalian respiratory chain supercomplexes

In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I1III2 and I1III2IV1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals.     

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.     

Bax releases cytochrome c preferentially from a complex between porin and adenine nucleotide translocator. Hexokinase activity suppresses this effect

The mechanism by which external Bax releases cytochrome c is still controversial and may also depend on the type of mitochondria and the actual localisation of cytochrome c. Outer membrane porin acquires high binding affinity for hexokinase by interacting with the adenine nucleotide translocator (ANT) in the contact sites. (I) The hexokinase protein was thus used as a tool to isolate the contact site forming complex between outer membrane porin and inner membrane ANT from a TritonX100 extract of brain membranes. (II) A significant amount of cytochrome c was co-purified with the isolated hexokinase porin ANT complexes that were reconstituted in phospholipid vesicles. Bax-C released the endogenous cytochrome c from the vesicles without forming unspecific pores. This was shown by loading the vesicles with malate that was not liberated by Bax-C. (III) The Bax-C effect was dependent on a specific association of cytochrome c with the porin ANT complex, as dissociation of the complex by bongkrekate abolished the Bax dependent cytochrome c liberation. (IV) The Bax-C effect was as well suppressed by hexokinase phosphorylating glucose.     

A structural model of the cytochrome C reductase/oxidase supercomplex from yeast mitochondria

Mitochondrial respiratory chain complexes are arranged in supercomplexes within the inner membrane. Interaction of cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) was investigated in Saccharomyces cerevisiae. Projection maps at 15 A resolution of supercomplexes III(2) + IV(1) and III(2) + IV(2) were obtained by electron microscopy. Based on a comparison of our maps with atomic x-ray structures for complexes III and IV we present a pseudo-atomic model of their precise interaction. Two complex IV monomers are specifically attached to dimeric complex III with their convex sides. The opposite sides, which represent the complex IV dimer interface in the x-ray structure, are open for complex IV-complex IV interactions. This could lead to oligomerization of III(2) + IV(2) supercomplexes, but this was not detected. Instead, binding of cytochrome c to the supercomplexes was revealed. It was calculated that cytochrome c has to move less than 40 A at the surface of the supercomplex for electron transport between complex III(2) and complex IV. Hence, the prime function of the supercomplex III(2) + IV(2) is proposed to be a scaffold for effective electron transport between complexes III and IV.     

Subunit arrangement in beef heart complex III

Beef heart mitochondrial complex III was separated into 12 polypeptide bands representing 11 different subunits by using the electrophoresis conditions described by Sch?gger et al. [(1986) Methods Enzymol. 126, 224-237]. Eight of the 12 polypeptide bands were identified from their NH2-terminal sequences as obtained by electroblotting directly from the NaDodSO4-polyacrylamide gel onto a solid support. The topology of the subunits in complex III was explored by three different approaches. (1) Protease digestion experiments of submitochrondrial particles in the presence and absence of detergent showed that subunits II and VI are on the M side of the inner membrane and subunits V and XI on the C side. (2) Labeling experiments with the membrane-intercalated probes [125I]TID and arylazidoPE indicated that cytochrome b is the predominant bilayer embedded subunit of complex III, while the non-heme iron protein appears to be peripherally located. (3) Cross-linking studies with carbodiimides and homobifunctional cleavable reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and VI+VII. The cytochrome c binding site was found to include subunits IV, VIII, and X. The combined data are used to provide an updated model for the topology of beef heart complex III.     

Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of respirasomes in potato mitochondria

Plant mitochondria were previously shown to comprise respiratory supercomplexes containing cytochrome c reductase (complex III) and NADH dehydrogenase (complex I) of I(1)III(2) and I(2)III(4) composition. Here we report the discovery of additional supercomplexes in potato (Solanum tuberosum) mitochondria, which are of lower abundance and include cytochrome c oxidase (complex IV). Highly active mitochondria were isolated from potato tubers and stems, solubilized by digitonin, and subsequently analyzed by Blue-native (BN) polyacrylamide gel electrophoresis (PAGE). Visualization of supercomplexes by in-gel activity stains for complex IV revealed five novel supercomplexes of 850, 1,200, 1,850, 2,200, and 3,000 kD in potato tuber mitochondria. These supercomplexes have III(2)IV(1), III(2)IV(2), I(1)III(2)IV(1), I(1)III(2)IV(2), and I(1)III(2)IV(4) compositions as shown by two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE and BN/BN-PAGE in combination with activity stains for cytochrome c oxidase. Potato stem mitochondria include similar supercomplexes, but complex IV is partially present in a smaller version that lacks the Cox6b protein and possibly other subunits. However, in mitochondria from potato tubers and stems, about 90% of complex IV was present in monomeric form. It was suggested that the I(1)III(2)IV(4) supercomplex represents a basic unit for respiration in mammalian mitochondria termed respirasome. Respirasomes also occur in potato mitochondria but were of low concentrations under all conditions applied. We speculate that respirasomes are more abundant under in vivo conditions.