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BBA - Bioenergetics (v.1797, #6-7)

Editorial Board (pp. i).

Thirty years of EBEC by Lech Wojtczak Guest Editor; Krzysztof Zabłocki Guest Editor (pp. 577-578).

Wanderings in bioenergetics and biomembranes by Martin Klingenberg (pp. 579-594).

Having worked for 55years in the center and at the fringe of bioenergetics, my major research stations are reviewed in the following wanderings: from microsomes to mitochondria, from NAD to CoQ, from reversed electron transport to reversed oxidative phosphorylation, from mitochondrial hydrogen transfer to phosphate transfer pathways, from endogenous nucleotides to mitochondrial compartmentation, from transport to mechanism, from carrier to structure, from coupling by AAC to uncoupling by UCP, and from specific to general transport laws. These wanderings are recalled with varying emphasis paid to the covered science stations.

Keywords: Abbreviations; AAC; ADP-ATP carrier; UCP; uncoupling protein; FA; fatty acid; CL; cardiolipin; ITF; induced transition fit; NDP; nucleoside diphosphate; NTP; nucleoside triphosphate; DAN–; dimethylamino-naphthoyl-; DANS–; dimethylamino-naphthalene-sulfonyl; DNP; dinitrophenol; DPN; NAD; TPN; NADP; G1P; glycerol-1-phosphate; cyt; cytochrome; BAT; brown adipose tissue; UQ; ubiquinone; MQ; menaquinone; AA; antimycin A; FeCN; ferricyanide; OxPhos; oxidative phosphorylationMitochondria; Bioenergetics; Biomembranes; Oxidative phosphorylation; Transport; Uncoupling protein

Wanderings in bioenergetics and biomembranes by Martin Klingenberg (pp. 579-594).

The fateful encounter of mitochondria with calcium: How did it happen? by Ernesto Carafoli (pp. 595-606).

A number of findings in the 1950s had offered indirect indications that mitochondria could accumulate Ca²⁺. In 1961, the phenomenon was directly demonstrated using isolated mitochondria: the uptake process was driven by respiratory chain activity or by the hydrolysis of added ATP. It could be accompanied by the simultaneous uptake of inorganic phosphate, in which case precipitates of hydroxyapatite were formed in the matrix, buffering its free Ca²⁺ concentration. The properties of the uptake process were established in the 1960s and 1970s: the uptake of Ca²⁺ occurred electrophoretically on a carrier that has not yet been molecularly identified, and was released from mitochondria via a Na⁺/Ca²⁺ antiporter. A H⁺/Ca²⁺ release exchanger was also found to operate in some mitochondrial types. The permeability transition pore was later also found to mediate the efflux of Ca²⁺ from mitochondria. In the mitochondrial matrix two TCA cycle dehydrogenases and pyruvate dehydrogenase phosphate phosphatase were found to be regulated in the matrix by the cycling of Ca²⁺ across the inner membrane. In conditions of cytoplasmic Ca²⁺ overload mitochondria could store for a time large amounts of precipitated Ca²⁺-phosphate, thus permitting cells to survive situations of Ca²⁺ emergency. The uptake process was found to have very low affinity for Ca²⁺: since the bulk concentration of Ca²⁺ in the cytoplasm is in the low to mid-nM range, it became increasingly difficult to postulate a role of mitochondria in the regulation of cytoplsmic Ca²⁺. A number of findings had nevertheless shown that energy linked Ca²⁺ transport occurred efficiently in mitochondria of various tissues in situ. The paradox was only solved in the 1990s, when it was found that the concentration of Ca²⁺ in the cytoplasm is not uniform: perimitochondrial micropools are created by the agonist-promoted discharge of Ca²⁺ from vicinal stores in which the concentration of Ca²⁺ is high enough to activate the low affinity mitochondrial uniporter. Mitochondria thus regained center stage as important regulators of cytoplasmic Ca²⁺ (not only of their own internal Ca²⁺). Their Ca²⁺ uptake systems was found to react very rapidly to cytoplasmic Ca²⁺ demands, even in the 150-200 msec time scale of processes like the contraction and relaxation of heart. An important recent development in the area of mitochondrial Ca²⁺ transport is its involvement in the disease process. Ca²⁺ signaling defects are now gaining increasing importance in the pathogenesis of diseases, e.g., neurodegenerative diseases. Since mitochondria have now regained a central role in the regulation of cytoplasmic Ca²⁺, dysfunctions of their Ca²⁺ controlling systems have expectedly been found to be involved in the pathogenesis of numerous disease processes.

Keywords: Mitochondrial calcium; Uniporter; Sodium-calcium exchanger; Calcium micropools

The fateful encounter of mitochondria with calcium: How did it happen? by Ernesto Carafoli (pp. 595-606).

Keywords: Mitochondrial calcium; Uniporter; Sodium-calcium exchanger; Calcium micropools

The fateful encounter of mitochondria with calcium: How did it happen? by Ernesto Carafoli (pp. 595-606).

Keywords: Mitochondrial calcium; Uniporter; Sodium-calcium exchanger; Calcium micropools

Mitochondria: The calcium connection by Laura Contreras; Ilaria Drago; Enrico Zampese; Tullio Pozzan (pp. 607-618).

Calcium handling by mitochondria is a key feature in cell life. It is involved in energy production for cell activity, in buffering and shaping cytosolic calcium rises and also in determining cell fate by triggering or preventing apoptosis. Both mitochondria and the mechanisms involved in the control of calcium homeostasis have been extensively studied, but they still provide researchers with long-standing or even new challenges. Technical improvements in the tools employed for the investigation of calcium dynamics have been–and are still–opening new perspectives in this field, and more prominently for mitochondria. In this review we present a state-of-the-art toolkit for calcium measurements, with major emphasis on the advantages of genetically encoded indicators. These indicators can be efficiently and selectively targeted to specific cellular sub-compartments, allowing previously unavailable high-definition calcium dynamic studies. We also summarize the main features of cellular and, in more detail, mitochondrial calcium handling, especially focusing on the latest breakthroughs in the field, such as the recent direct characterization of the calcium microdomains that occur on the mitochondrial surface upon cellular stimulation. Additionally, we provide a major example of the key role played by calcium in patho-physiology by briefly describing the extensively reported–albeit highly controversial–alterations of calcium homeostasis in Alzheimer's disease, casting lights on the possible alterations in mitochondrial calcium handling in this pathology.

Keywords: Abbreviations; Aβ; amyloid beta; Δ; Ψ; _m; inner mitochondrial membrane potential; APP; amyloid precursor protein; BRET; bioluminescence resonance energy transfer; [Ca; ²⁺; ]; _c; cytosolic Ca; ²⁺; concentration; [Ca; ²⁺; ]; _ER; endoplasmic reticulum Ca; ²⁺; concentration; [Ca; ²⁺; ]; _m; mitochondrial Ca; ²⁺; concentration; CaM; calmodulin; CCE; capacitative calcium entry; CFP; cyan fluorescent protein; Cyp D; cyclophilin D; mCU; mitochondrial calcium uniporter; ER; endoplasmic reticulum; FAD; familiar Alzheimer disease; FRET; fluorescence resonance energy transfer; GECI; genetically encoded Ca; ²⁺; indicator; GFP; green fluorescent protein; IMM; inner mitochondrial membrane; IMS; intermembrane space; KO; knock out; IP; ₃; inositol 1,4,5-trisphosphate; IP; ₃; R; IP; ₃; receptor; OMM; outer mitochondrial membrane; PM; plasma membrane; PMCA; plasma membrane Ca; ²⁺; ATPase; PS1; Presenilin-1, PS2, presenilin-2; PTP; permeability transition pore; RyR; ryanodine receptor; RR; Ruthenium Red; SERCA; sarco/endoplasmic reticulum Ca; ²⁺; ATPase; SPCA; secretory pathway Ca; ²⁺; ATPase; SR; sarcoplasmic reticulum; VOCC; voltage-operated Ca; ²⁺; channel; YFP; yellow fluorescent proteinCa; ²⁺; mitochondria; microdomain; neurodegeneration; apoptosis

Mitochondria: The calcium connection by Laura Contreras; Ilaria Drago; Enrico Zampese; Tullio Pozzan (pp. 607-618).

Blocking the K-pathway still allows rapid one-electron reduction of the binuclear center during the anaerobic reduction of the aa₃-type cytochrome c oxidase from Rhodobacter sphaeroides by Krithika Ganesan; Robert B. Gennis (pp. 619-624).

The K-pathway is one of the two proton-input channels required for function of cytochrome c oxidase. In the Rhodobacter sphaeroides cytochrome c oxidase, the K-channel starts at Glu101 in subunit II, which is at the surface of the protein exposed to the cytoplasm, and runs to Tyr288 at the heme a₃/Cu_B active site. Mutations of conserved, polar residues within the K-channel block or inhibit steady state oxidase activity. A large body of research has demonstrated that the K-channel is required to fully reduce the heme/Cu binuclear center, prior to the reaction with O₂, presumably by providing protons to stabilize the reduced metals (ferrous heme a₃ and cuprous Cu_B). However, there are conflicting reports which raise questions about whether blocking the K-channel blocks both electrons or only one electron from reaching the heme/Cu center. In the current work, the rate and extent of the anaerobic reduction of the heme/Cu center were monitored by optical and EPR spectroscopies, comparing the wild type and mutants that block the K-channel. The new data show that when the K-channel is blocked, one electron will still readily enter the binuclear center. The one-electron reduction of the resting oxidized (“O”) heme/Cu center of the K362M mutant, results in a partially reduced binuclear center in which the electron is distributed about evenly between heme a₃ and Cu_B in the R. sphaeroides oxidase. Complete reduction of the heme/Cu center requires the uptake of two protons which must be delivered through the K-channel.

Keywords: Cytochrome oxidase; R. sphaeroides; Respiration; Protons; K-pathway

The role of a conserved tyrosine in the 49-kDa subunit of complex I for ubiquinone binding and reduction by Maja A. Tocilescu; Uta Fendel; Klaus Zwicker; Drose Stefan Dröse; Stefan Kerscher; Ulrich Brandt (pp. 625-632).

Iron–sulfur cluster N2 of complex I (proton pumping NADH:quinone oxidoreductase) is the immediate electron donor to ubiquinone. At a distance of only ∼7Å in the 49-kDa subunit, a highly conserved tyrosine is found at the bottom of the previously characterized quinone binding pocket. To get insight into the function of this residue, we have exchanged it for six different amino acids in complex I from Yarrowia lipolytica. Mitochondrial membranes from all six mutants contained fully assembled complex I that exhibited very low dNADH:ubiquinone oxidoreductase activities with n-decylubiquinone. With the most conservative exchange Y144F, no alteration in the electron paramagnetic resonance spectra of complex I was detectable. Remarkably, high dNADH:ubiquinone oxidoreductase activities were observed with ubiquinones Q₁ and Q₂ that were coupled to proton pumping. Apparent K_m values for Q₁ and Q₂ were markedly increased and we found pronounced resistance to the complex I inhibitors decyl-quinazoline-amine (DQA) and rotenone. We conclude that Y144 directly binds the head group of ubiquinone, most likely via a hydrogen bond between the aromatic hydroxyl and the ubiquinone carbonyl. This places the substrate in an ideal distance to its electron donor iron–sulfur cluster N2 for efficient electron transfer during the catalytic cycle of complex I.

Keywords: Abbreviations; ACMA; 9-amino-6-chloro-2-methoxyacridine; BN-PAGE; blue-native polyacrylamide gel electrophoresis; C; ₁₂; E; ₈; n-alkyl-polyoxyethylene-ether; DBQ; n; -decylubiquinone; dNADH; deamino-nicotinamide-adenine-dinucleotide (reduced form); DQA; 2-decyl-4-quinazolinyl amine; EPR; electron paramagnetic resonance; FCCP; carbonyl-cyanide-; p; -trifluoromethoxyphenylhydrazone; HAR; hexa-ammine-ruthenium(III)-chloride; Hepes; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; log; P; decadic logarithm of the partition coefficient; Mops; 3-(; N; -morpholino) propanesulphonic acid; PMSF; phenylmethylsulfonyl fluoride; Q; ₁; ubiquinone-1 (2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone); Q; ₂; ubiquinone-2 (2,3-dimethoxy-5-methyl-6-geranyl-1,4-benzoquinone); Q; ₉; ubiquinone-9; Tris; Tris(hydroxymethyl)aminomethaneMitochondria; Complex I; Ubiquinone; Inhibitor resistance; Mutagenesis; Yarrowia lipolytica

Mitochondrial respiratory chain super-complex I–III in physiology and pathology by Giorgio Lenaz; Alessandra Baracca; Giovanna Barbero; Christian Bergamini; Maria Elena Dalmonte; Marianna Del Sole; Marco Faccioli; Anna Falasca; Romana Fato; Maria Luisa Genova; Gianluca Sgarbi; Giancarlo Solaini (pp. 633-640).

Recent investigations by native gel electrophoresis showed the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis demonstrated that Complex I and Complex III in mammalian mitochondria kinetically behave as a single unit with control coefficients approaching unity for each component, suggesting the existence of substrate channeling within the super-complex. The formation of this supramolecular unit largely depends on the lipid content and composition of the inner mitochondrial membrane. The function of the super-complexes appears not to be restricted to kinetic advantages in electron transfer: we discuss evidence on their role in the stability and assembly of the individual complexes, particularly Complex I, and in preventing excess oxygen radical formation. There is increasing evidence that disruption of the super-complex organization leads to functional derangements responsible for pathological changes, as we have found in K-ras-transformed fibroblasts.

Keywords: Abbreviations; BHM; Beef Heart Mitochondria; BN-PAGE; Blue Native Polyacrylamide Gel Electrophoresis; CL; Cardiolipin; C; _n; Metabolic Flux Coefficient; CoQ; Coenzyme Q, Ubiquinone; mtDNA; mitochondrial DNA; OXPHOS; Oxidative Phosphorylation System; POM; Potato Tuber Mitochondria; RLM; Rat Liver Mitochondria; ROS; Reactive Oxygen Species; SDS-PAGE; Sodium Dodecyl-Sulfate Polyacrylamide Gel ElectrophoresisMitochondria; Super-complex; Flux control; Channeling; Complex I

The flitting of electrons in complex I: A stochastic approach by Stéphane Ransac; Clément Arnarez; Jean-Pierre Mazat (pp. 641-648).

A stochastic approach based on the Gillespie algorithm is particularly well adapted to describe the time course of the redox reactions that occur inside the respiratory chain complexes because they involve the motion of single electrons between the individual unique redox centres of a given complex. We use this approach to describe the molecular functioning of the peripheral arm of complex I based on its known crystallographic structure and the rate constants of electron tunnelling derived from the Moser and Dutton phenomenological equations. There are several possible electrons pathways but we show that most of them take the route defined by the successive sites and redox centres: NADH⁺ site – FMN – N3 – N1b – N4 – N5 – N6a – N6b – N2 – Q site. However, the electrons do not go directly from NADH towards the ubiquinone molecule. They frequently jump back and forth between neighbouring redox centres with the result that the net flux of electrons through complex I (i.e. net number of electrons reducing a ubiquinone) is far smaller than the number of redox reactions which actually occur. While most of the redox centres are reduced in our simulations the degree of reduction can vary according to the individual midpoint potentials. The high turnover number observed in our simulation seems to indicate that, in the whole complex I, one or several slower step(s) follow(s) the redox reactions involved in the peripheral arm. It also appears that the residence time of FMNH• and SQ• (possible producers of ROS) is low (around 4% and between 1.6% and 5% respectively according to the values of the midpoint potentials). We did not find any evidence for a role of N7 which remains mainly reduced in our simulations. The role of N1a is complex and depends upon its midpoint potential. In all cases its presence slightly decreases the life time of the flavosemiquinone species. These simulations demonstrate the interest of this type of model which links the molecular physico-chemistry of the individual redox reactions to the more global level of the reaction, as is observed experimentally.

Keywords: Gillespie algorithm; Complex I; Stochastic modelling

The flitting of electrons in complex I: A stochastic approach by Stéphane Ransac; Clément Arnarez; Jean-Pierre Mazat (pp. 641-648).

Keywords: Gillespie algorithm; Complex I; Stochastic modelling

The flitting of electrons in complex I: A stochastic approach by Stéphane Ransac; Clément Arnarez; Jean-Pierre Mazat (pp. 641-648).

Keywords: Gillespie algorithm; Complex I; Stochastic modelling

cAMP-dependent protein kinase regulates post-translational processing and expression of complex I subunits in mammalian cells by Sergio Papa; Salvatore Scacco; Domenico De Rasmo; Anna Signorile; Francesco Papa; Damiano Panelli; Annarita Nicastro; Raffaella Scaringi; Arcangela Santeramo; Emilio Roca; Raffaella Trentadue; Maria Larizza (pp. 649-658).

Work is presented on the role of cAMP-dependent protein phosphorylation in post-translational processing and biosynthesis of complex I subunits in mammalian cell cultures. PKA-mediated phosphorylation of the NDUFS4 subunit of complex I promotes in cell cultures in vivo import/maturation in mitochondria of the precursor of this protein. The import promotion appears to be associated with the observed cAMP-dependent stimulation of the catalytic activity of complex I. These effects of PKA are counteracted by activation of protein phosphatase(s). PKA and the transcription factor CREB play a critical role in the biosynthesis of complex I subunits. CREB phosphorylation, by PKA and/or CaMKs, activates at nuclear and mitochondrial level a transcriptional regulatory cascade which promotes the concerted expression of nuclear and mitochondrial encoded subunits of complex I and other respiratory chain proteins.

Keywords: Abbreviations; ATRA; all; trans; retinoic acid; CREB; cyclic-AMP response element binding protein; DMEM; high glucose Dulbecco's modified Eagle's medium; NDHFn; normal human dermal fibroblasts-neonatal; KB; human mouth epidermoid cells carcinoma; NHEK; normal human epidermal keratinocyte; PKA; cAMP-dependent protein kinase; PP2A; Protein phosphatase 2AComplex I; cAMP cascade; PKA; Mitochondrial import; All; trans; retinoic acid; Cell growth

Redox-induced conformational changes within the Escherichia coli NADH ubiquinone oxidoreductase (complex I): An analysis by mutagenesis and FT-IR spectroscopy by Thorsten Friedrich; Petra Hellwig (pp. 659-663).

The proton-pumping NADH:ubiquinone oxidoreductase couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. This process is suggested to be accompanied by conformational changes of the enzyme that may be monitored by redox-induced FT-IR difference spectroscopy. Signals observed in the amide I range are partially attributed to local rearrangements that occur as an electrostatic response to the redox reactions of the FeS clusters. In addition, conformational changes can be reported that depend on pH and at the same time can be perturbed by site-directed mutagenesis of residue E67 on subunit B (the bacterial homologue of the mitochondrial PSST subunit). This residue is located in the vicinity of the cluster N2. Re-evaluating these previous data we here discuss a mechanism, by which the redox reaction of N2 induces conformational changes possibly leading to proton translocation.

Keywords: Complex I; NADH:ubiquinone oxidoreductase; Fe/S cluster; FT-IR spectroscopy; Conformational changes; Escherichia coli

Structure and function of mitochondrial supercomplexes by Natalya V. Dudkina; Roman Kouřil; Katrin Peters; Hans-Peter Braun; Egbert J. Boekema (pp. 664-670).

The five complexes (complexes I–V) of the oxidative phosphorylation (OXPHOS) system of mitochondria can be extracted in the form of active supercomplexes. Single-particle electron microscopy has provided 2D and 3D data describing the interaction between complexes I and III, among I, III and IV and in a dimeric form of complex V, between two ATP synthase monomers. The stable interactions are called supercomplexes which also form higher-ordered oligomers. Cryo-electron tomography provides new insights on how these supercomplexes are arranged within intact mitochondria. The structure and function of OXPHOS supercomplexes are discussed.

Keywords: Oxidative phosphorylation; Mitochondria; Respirasome; ATP synthase; Electron microscopy; Supercomplex

Structure and function of mitochondrial supercomplexes by Natalya V. Dudkina; Roman Kouřil; Katrin Peters; Hans-Peter Braun; Egbert J. Boekema (pp. 664-670).

Keywords: Oxidative phosphorylation; Mitochondria; Respirasome; ATP synthase; Electron microscopy; Supercomplex

Structure and function of mitochondrial supercomplexes by Natalya V. Dudkina; Roman Kouřil; Katrin Peters; Hans-Peter Braun; Egbert J. Boekema (pp. 664-670).

Keywords: Oxidative phosphorylation; Mitochondria; Respirasome; ATP synthase; Electron microscopy; Supercomplex

Electron competition process in respiratory chain: Regulatory mechanisms and physiological functions by Michel Rigoulet; Arnaud Mourier; Anne Galinier; Louis Casteilla; Anne Devin (pp. 671-677).

In mitochondria isolated from the yeast Saccharomyces cerevisiae, under non-phosphorylating conditions, we have previously shown that there is a right of way for electrons coming from the external NADH dehydrogenase, Nde1p. In this work, we show that the electron competition process is identical under more physiological conditions i.e. oxidative phosphorylation. Such a competition generates a priority for cytosolic NADH reoxidation. Furthermore, this electron competition process is associated with an energy wastage (the “active leak”) that allows an increase in redox equivalent oxidation when the redox pressure increases. When this redox pressure is decreased, i.e. under phosphorylating conditions, most of this energy wastage is alleviated. By studying mutant strains affected either in respiratory chain supramolecular organization or in electron competition activity, we show that the respiratory chain supramolecular organization is not responsible for the electron competition processes. Moreover, we show two distinct relationships between the respiratory rate and the quinone redox state that seem to indicate two quinone pools that are involved in the electron right of way. Indeed, the more reduced pool would be associated to the electron right of way for the external dehydrogenases whereas the less reduced pool would be associated to the electron right of way for the internal dehydrogenases.

Keywords: Mitochondria; Yeast; Electron competition; Respiratory chain supramolecular organization; Proton leak; Dehydrogenases

Electron competition process in respiratory chain: Regulatory mechanisms and physiological functions by Michel Rigoulet; Arnaud Mourier; Anne Galinier; Louis Casteilla; Anne Devin (pp. 671-677).

Keywords: Mitochondria; Yeast; Electron competition; Respiratory chain supramolecular organization; Proton leak; Dehydrogenases

Electron competition process in respiratory chain: Regulatory mechanisms and physiological functions by Michel Rigoulet; Arnaud Mourier; Anne Galinier; Louis Casteilla; Anne Devin (pp. 671-677).

Keywords: Mitochondria; Yeast; Electron competition; Respiratory chain supramolecular organization; Proton leak; Dehydrogenases

Structure–function relationships in feedback regulation of energy fluxes in vivo in health and disease: Mitochondrial Interactosome by Valdur Saks; Rita Guzun; Natalja Timohhina; Kersti Tepp; Minna Varikmaa; Claire Monge; Nathalie Beraud; Tuuli Kaambre; Andrey Kuznetsov; Lumme Kadaja; Margus Eimre; Enn Seppet (pp. 678-697).

The aim of this review is to analyze the results of experimental research of mechanisms of regulation of mitochondrial respiration in cardiac and skeletal muscle cells in vivo obtained by using the permeabilized cell technique. Such an analysis in the framework of Molecular Systems Bioenergetics shows that the mechanisms of regulation of energy fluxes depend on the structural organization of the cells and interaction of mitochondria with cytoskeletal elements. Two types of cells of cardiac phenotype with very different structures were analyzed: adult cardiomyocytes and continuously dividing cancerous HL-1 cells. In cardiomyocytes mitochondria are arranged very regularly, and show rapid configuration changes of inner membrane but no fusion or fission, diffusion of ADP and ATP is restricted mostly at the level of mitochondrial outer membrane due to an interaction of heterodimeric tubulin with voltage dependent anion channel, VDAC. VDAC with associated tubulin forms a supercomplex, Mitochondrial Interactosome, with mitochondrial creatine kinase, MtCK, which is structurally and functionally coupled to ATP synthasome. Due to selectively limited permeability of VDAC for adenine nucleotides, mitochondrial respiration rate depends almost linearly upon the changes of cytoplasmic ADP concentration in their physiological range. Functional coupling of MtCK with ATP synthasome amplifies this signal by recycling adenine nucleotides in mitochondria coupled to effective phosphocreatine synthesis. In cancerous HL-1 cells this complex is significantly modified: tubulin is replaced by hexokinase and MtCK is lacking, resulting in direct utilization of mitochondrial ATP for glycolytic lactate production and in this way contributing in the mechanism of the Warburg effect. Systemic analysis of changes in the integrated system of energy metabolism is also helpful for better understanding of pathogenesis of many other diseases.

Keywords: Permeabilized cell; Respiration; Mitochondria; Creatine kinase; Tubulin; Systems biology; Energy metabolism

Multi-site control and regulation of mitochondrial energy production by G. Benard; N. Bellance; C. Jose; S. Melser; K. Nouette-Gaulain; R. Rossignol (pp. 698-709).

With the extraordinary progress of mitochondrial science and cell biology, novel biochemical pathways have emerged as strategic points of bioenergetic regulation and control. They include mitochondrial fusion, fission and organellar motility along microtubules and microfilaments (mitochondrial dynamics), mitochondrial turnover (biogenesis and degradation), and mitochondrial phospholipids synthesis. Yet, much is still unknown about the mutual interaction between mitochondrial energy state, biogenesis, dynamics and degradation. Meanwhile, clinical research into metabolic abnormalities in tumors as diverse as renal carcinoma, glioblastomas, paragangliomas or skin leiomyomata, has designated new genes, oncogenes and oncometabolites involved in the regulation of cellular and mitochondrial energy production. Furthermore, the examination of rare neurological diseases such as Charcot-Marie Tooth type 2a, Autosomal Dominant Optic Atrophy, Lethal Defect of Mitochondrial and Peroxisomal Fission, or Spastic Paraplegia suggested involvement of MFN2, OPA1/3, DRP1 or Paraplegin, in the auxiliary control of mitochondrial energy production. Lastly, advances in the understanding of mitochondrial apoptosis have suggested a supplementary role for Bcl2 or Bax in the regulation of mitochondrial respiration and dynamics, which has fostered the investigation of alternative mechanisms of energy regulation. In this review, we discuss the regulatory mechanisms of cellular and mitochondrial energy production, and we emphasize the importance of the study of rare neurological diseases in addition to more common disorders such as cancer, for the fundamental understanding of cellular and mitochondrial energy production.

Keywords: Mitochondria; Energy metabolism; Oxidative phosphorylation; Regulation; Dynamics; Biogenesis

Multi-site control and regulation of mitochondrial energy production by G. Benard; N. Bellance; C. Jose; S. Melser; K. Nouette-Gaulain; R. Rossignol (pp. 698-709).

Keywords: Mitochondria; Energy metabolism; Oxidative phosphorylation; Regulation; Dynamics; Biogenesis

Multi-site control and regulation of mitochondrial energy production by G. Benard; N. Bellance; C. Jose; S. Melser; K. Nouette-Gaulain; R. Rossignol (pp. 698-709).

Keywords: Mitochondria; Energy metabolism; Oxidative phosphorylation; Regulation; Dynamics; Biogenesis

Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase by Peter Brzezinski; Ann-Louise Johansson (pp. 710-723).

Cytochrome c oxidase is a multisubunit membrane-bound enzyme, which catalyzes oxidation of four molecules of cytochrome c²⁺ and reduction of molecular oxygen to water. The electrons are taken from one side of the membrane while the protons are taken from the other side. This topographical arrangement results in a charge separation that is equivalent to moving one positive charge across the membrane for each electron transferred to O₂. In this reaction part of the free energy available from O₂ reduction is conserved in the form of an electrochemical proton gradient. In addition, part of the free energy is used to pump on average one proton across the membrane per electron transferred to O₂. Our understanding of the molecular design of the machinery that couples O₂ reduction to proton pumping in oxidases has greatly benefited from studies of so called “uncoupled” structural variants of the oxidases. In these uncoupled oxidases the catalytic O₂-reduction reaction may display the same rates as in the wild-type Cyt cO, yet the electron/proton transfer to O₂ is not linked to proton pumping. One striking feature of all uncoupled variants studied to date is that the (apparent) p K_a of a Glu residue, located deeply within a proton pathway, is either increased or decreased (from 9.4 in the wild-type oxidase). The altered p K_a presumably reflects changes in the local structural environment of the residue and because the Glu residue is found near the catalytic site as well as near a putative exit pathway for pumped protons these changes are presumably important for controlling the rates and trajectories of the proton transfer. In this paper we summarize data obtained from studies of uncoupled structural oxidase variants and present a hypothesis that in quantitative terms offers a link between structural changes, modulation of the apparent p K_a and uncoupling of proton pumping from O₂ reduction.

Keywords: Abbreviations; Cyt; c; O; cytochrome; c; oxidase; R; ²; Cyt; c; O with a two-electron reduced catalytic site; P; ³; (also; P; _R; ); the “peroxy” intermediate formed at the catalytic site upon reaction of the 4-electron reduced Cyt; c; O with O; ₂; F; ³; “oxo-ferryl” intermediate; O; ⁰; (also; O; ⁴; ); fully oxidized Cyt; c; O; n; -side; negative side of the membrane; p; -side; positive side of the membrane. If not otherwise indicated amino-acid residues are numbered according to the; Rhodobacter sphaeroides; CytcO sequence and the residues are found in subunit IRespiration; Electron transfer; Cytochrome; aa; ₃; Mitochondria; Membrane protein; Electrostatics; Energy transduction

Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase by Peter Brzezinski; Ann-Louise Johansson (pp. 710-723).

Substrate binding and the catalytic reactions in cbb₃-type oxidases: The lipid membrane modulates ligand binding by Yafei Huang; Joachim Reimann; Laila M.R. Singh; Adelroth Pia Ädelroth (pp. 724-731).

Heme–copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme–copper oxidases of the cbb₃-type are only found in bacteria, often pathogenic ones since they have a low K_m for O₂, enabling the bacteria to colonize semi-anoxic environments. Cbb₃-type (C) oxidases are highly divergent from the mitochondrial-like aa₃-type (A) oxidases, and within the heme–copper oxidase family, cbb₃ is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N₂O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme–copper oxidases are manifested in the lack in cbb₃ of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O₂ and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O₂ and NO reduction as well as the proton-pumping mechanism in all heme–copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein–lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein–membrane interactions in cbb₃ oxidases, and possible physiological consequences for this behavior are discussed.

Keywords: Abbreviations; NOR; bacterial nitric oxide reductase; HCuO; heme–copper oxidaseLipid; Liposome; Nitric oxide reduction; Oxygen reduction; Carbon monoxide; Proton transfer

Mutagenesis of the Sauromatum guttatum alternative oxidase reveals features important for oxygen binding and catalysis by Paul G. Crichton; Mary S. Albury; Charles Affourtit; Anthony L. Moore (pp. 732-737).

The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in mitochondria of all higher plants studied to date. To investigate the role of highly conserved amino acid residues in catalysis we have expressed site-directed mutants of Cys-172, Thr-179, Trp-206, Tyr-253, and Tyr-299 in AOX in the yeast Schizosaccharomyces pombe. Assessment of AOX activity in isolated yeast mitochondria reveals that mutagenesis of Trp-206 to phenylalanine or tyrosine abolishes activity, in contrast to that observed with either Tyr-253 or 299 both mutants of which retained activity. None of the mutants exhibited sensitivity to Q-like inhibitors that differed significantly from the wild type AOX. Interestingly, however, mutagenesis of Thr-179 or Cys-172 (a residue implicated in AOX regulation by α-keto acids) to alanine not only resulted in a decrease of maximum AOX activity but also caused a significant increase in the enzyme's affinity for oxygen (4- and 2-fold, respectively). These results provide important new insights in the mechanism of AOX catalysis and regulation by pyruvate.

Keywords: Abbreviations; Q(H; ₂; ); (reduced) ubiquinone; OG; octyl gallate; SHAM; salicylic hydroxamic acidAlternative oxidase; Oxygen affinity; α-keto acid regulation; Structure–function relations; Site-directed mutagenesis; Schizosaccharomyces pombe; mitochondria

Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump by Michael I. Verkhovsky; Alexander V. Bogachev (pp. 738-746).

The Na⁺-translocating NADH:ubiquinone oxidoreductase (Na⁺-NQR) is a component of the respiratory chain of various bacteria. This enzyme is an analogous but not homologous counterpart of mitochondrial Complex I. Na⁺-NQR drives the same chemistry and also uses released energy to translocate ions across the membrane, but it pumps Na⁺ instead of H⁺. Most likely the mechanism of sodium pumping is quite different from that of proton pumping (for example, it could not accommodate the Grotthuss mechanism of ion movement); this is why the enzyme structure, subunits and prosthetic groups are completely special. This review summarizes modern knowledge on the structural and catalytic properties of bacterial Na⁺-translocating NADH:quinone oxidoreductases. The sequence of electron transfer through the enzyme cofactors and thermodynamic properties of those cofactors is discussed. The resolution of the intermediates of the catalytic cycle and localization of sodium-dependent steps are combined in a possible molecular mechanism of sodium transfer by the enzyme.

Keywords: Abbreviations; E; _m; midpoint redox potential; Fl; oxidized flavin; FlH; ₂; neutral form of reduced flavin; FlH; ⁻; anionic form of reduced flavin; FlH; ^•; neutral flavosemiquinone; Fl; ^•−; anionic flavosemiquinone; HQNO; 2-heptyl-4-hydroxyquinoline N-oxide; Na; ⁺; -NQR; Na; ⁺; -translocating NADH:quinone oxidoreductase; Na; ⁺; /e ̅; number of sodium ions transferred across the membrane by the respiratory chain enzyme normalized to the number of electrons; Q; ubiquinone; QH; ₂; ubiquinol; Rf; riboflavin; Δμ̅; _H; ⁺; transmembrane difference of H; ⁺; electrochemical potentials; SHE; standard hydrogen electrode; Δμ̅; _Na; ⁺; transmembrane difference of Na; ⁺; electrochemical potentials; Δψ; transmembrane electric potentialNa; ⁺; -translocating NADH:quinone oxidoreductase; Sodium transport; Vibrio; Respiratory chain; Flavin

Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump by Michael I. Verkhovsky; Alexander V. Bogachev (pp. 738-746).

Mutation of the heme axial ligand of Escherichia coli succinate–quinone reductase: Implications for heme ligation in mitochondrial complex II from yeast by Elena Maklashina; Sany Rajagukguk; William S. McIntire; Gary Cecchini (pp. 747-754).

A b-type heme is conserved in membrane-bound complex II enzymes (SQR, succinate–ubiquinone reductase). The axial ligands for the low spin heme b in Escherichia coli complex II are SdhC His84 and SdhD His71. E. coli SdhD His71 is separated by 10 residues from SdhD Asp82 and Tyr83 which are essential for ubiquinone catalysis. The same His-10x-AspTyr motif dominates in homologous SdhD proteins, except for Saccharomyces cerevisiae where a tyrosine is at the axial position (Tyr-Cys-9x-AspTyr). Nevertheless, the yeast enzyme was suggested to contain a stoichiometric amount of heme, however, with the Cys ligand in the aforementioned motif acting as heme ligand. In this report, the role of Cys residues for heme coordination in the complex II family of enzymes is addressed. Cys was substituted to the SdhD-71 position and the yeast Tyr71Cys72 motif was also recreated. The Cys71 variant retained heme, although it was high spin, while the Tyr71Cys72 mutant lacked heme. Previously the presence of heme in S. cerevisiae was detected by a spectral peak in fumarate-oxidized, dithionite-reduced mitochondria. Here it is shown that this method must be used with caution. Comparison of bovine and yeast mitochondrial membranes shows that fumarate induced reoxidation of cytochromes in both SQR and the bc₁ complex (ubiquinol–cytochrome c reductase). Thus, this report raises a concern about the presence of low spin heme b in S. cerevisiae complex II.

Keywords: Succinate dehydrogenase; Complex II; Cytochrome; b; Heme; Saccharomyces cerevisiae

ATP hydrolysis in ATP synthases can be differently coupled to proton transport and modulated by ADP and phosphate: A structure based model of the mechanism by Manuela D'Alessandro; B. Andrea Melandri (pp. 755-762).

In the ATP synthases of Escherichia coli ADP and phosphate exert an apparent regulatory role on the efficiency of proton transport coupled to the hydrolysis of ATP. Both molecules induce clearly biphasic effects on hydrolysis and proton transfer. At intermediate concentrations (∼0.5–1µM and higher) ADP inhibits hydrolysis and proton transfer; a quantitative analysis of the fluxes however proves that the coupling efficiency remains constant in this concentration range. On the other hand at nanomolar concentrations of ADP (a level obtainable only using an enzymatic ATP regenerating system) the efficiency of proton transport drops progressively, while the rate of hydrolysis remains high. Phosphate, at concentrations ≥0.1mM, inhibits hydrolysis only if ADP is present at sufficiently high concentrations, keeping the coupling efficiency constant. At lower ADP levels phosphate is, however, necessary for an efficiently coupled catalytic cycle. We present a model for a catalytic cycle of ATP hydrolysis uncoupled from the transport of protons. The model is based on the available structures of bovine and yeast F₁ and on the known binding affinities for ADP and P_i of the catalytic sites in their different functional states. The binding site related to the inhibitory effects of P_i (in association with ADP) is identified as the α_HCβ_HC site, the pre-release site for the hydrolysis products. We suggest, moreover, that the high affinity site, associated with the operation of an efficient proton transport, could coincide with a conformational state intermediate between the α_TPβ_TP and the α_DPβ_DP (similar to the transition state of the hydrolysis/synthesis reaction) that does not strongly bind the ligands and can exchange them rather freely with the external medium. The emptying of this site can lead to an unproductive hydrolysis cycle that occurs without a net rotation of the central stalk and, consequently, does not translocate protons.

Keywords: Abbreviations; Δ; ψ; bulk-to-bulk electrical potential difference; ΔpH; transmembrane difference of pH (pH; _out; −; pH; _in; ); Δ; µ̃; H; _⁺; difference of electrochemical potential of protons, (µ̃; H; _⁺; , in ·µ̃; H; _⁺; , out); ACMA; 9-amino-6-chloro-2-methoxyacridine; AMP-PNP; 5-adenylyl-imidodiphosphate; EF; _O; F; ₁; ATP synthase of; E. coli; Phenol Red; 4,4′-(3; H; -2,1-benzoxathiol-3-ylidene) bis-phenol, S,S-dioxide; Tricine; N; -[2-Hydroxy-1,1-bis(hydroxymethyl)ethyl] glycineATP synthase; Proton transport; Modulation by ADP and phosphate; Model for uncoupled hydrolysis; Escherichia coli; Rhodobacter capsulatus

Structural and energetic basis for H⁺ versus Na⁺ binding selectivity in ATP synthase F_o rotors by Alexander Krah; Denys Pogoryelov; Julian D. Langer; Peter J. Bond; Thomas Meier; Faraldo-Gomez José D. Faraldo-Gómez (pp. 763-772).

The functional mechanism of the F₁F_o ATP synthase, like many membrane transporters and pumps, entails a conformational cycle that is coupled to the movement of H⁺ or Na⁺ ions across its transmembrane domain, down an electrochemical gradient. This coupling is an efficient means of energy transduction and regulation, provided that ion binding to the membrane domain, known as F_o, is appropriately selective. In this study we set out to establish the structural and energetic basis for the ion-binding selectivity of the membrane-embedded F_o rotors of two representative ATP synthases. First, we use a biochemical approach to demonstrate the inherent binding selectivity of these rotors, that is, independently from the rest of the enzyme. We then use atomically detailed computer simulations of wild-type and mutagenized rotors to calculate and rationalize their selectivity, on the basis of the structure, dynamics and coordination chemistry of the binding sites. We conclude that H⁺ selectivity is most likely a robust property of all F_o rotors, arising from the prominent presence of a conserved carboxylic acid and its intrinsic chemical propensity for protonation, as well as from the structural plasticity of the binding sites. In H⁺-coupled rotors, the incorporation of hydrophobic side chains to the binding sites enhances this inherent H⁺ selectivity. Size restriction may also favor H⁺ over Na⁺, but increasing size alone does not confer Na⁺ selectivity. Rather, the degree to which F_o rotors may exhibit Na⁺ coupling relies on the presence of a sufficient number of suitable coordinating side chains and/or structural water molecules. These ligands accomplish a shift in the relative binding energetics, which under some physiological conditions may be sufficient to provide Na⁺ dependence.

Keywords: F; ₁; F; _o; ATP synthase; c-subunit ring rotor; F; _o; rotor; Membrane protein structure; Membrane bioenergetics; Proton-motive force; Sodium-motive force; Ion selectivity; Molecular dynamics simulation; Free-energy calculation; Mass spectrometry; Dicyclohexylcarbodiimide modification

Uncoupling protein-1 is not leaky by Irina G. Shabalina; Mario Ost; Natasa Petrovic; Marek Vrbacky; Jan Nedergaard; Barbara Cannon (pp. 773-784).

The activity of uncoupling protein-1 (UCP1) is rate-limiting for nonshivering thermogenesis and diet-induced thermogenesis. Characteristically, this activity is inhibited by GDP experimentally and presumably mainly by cytosolic ATP within brown-fat cells. The issue as to whether UCP1 has a residual proton conductance even when fully saturated with GDP/ATP (as has recently been suggested) has not only scientific but also applied interest, since a residual proton conductance would make overexpressed UCP1 weight-reducing even without physiological/pharmacological activation. To examine this question, we have here established optimal conditions for studying the bioenergetics of wild-type and UCP1(−/−) brown-fat mitochondria, analysing UCP1-mediated differences in parallel preparations of brown-fat mitochondria from both genotypes. Comparing different substrates, we find that pyruvate (or palmitoyl-l-carnitine) shows the largest relative coupling by GDP. Comparing albumin concentrations, we find the range 0.1–0.6% optimal; higher concentrations are inhibitory. Comparing basic medium composition, we find 125mM sucrose optimal; an ionic medium (50–100mM KCl) functions for wild-type but is detrimental for UCP1(−/−) mitochondria. Using optimal conditions, we find no evidence for a residual proton conductance (not a higher post-GDP respiration, a lower membrane potential or an altered proton leak at highest common potential) with either pyruvate or glycerol-3-phosphate as substrates, nor by a 3–4-fold alteration of the amount of UCP1. We could demonstrate that certain experimental conditions, due to respiratoty inhibition, could lead to the suggestion that UCP1 possesses a residual proton conductance but find that under optimal conditions our experiments concur with implications from physiological observations that in the presence of inhibitory nucleotides, UCP1 is not leaky.

Keywords: Abbreviations; ANT; adenine nucleotide translocase; BSA; bovine serum albumin; EDTA; ethylenediamine tetraacetic acid; FCCP; carbonyl cyanide; p; -(trifluoromethoxy)phenylhydrazone; GDP; guanosine 5′-diphosphate; UCP1; uncoupling protein 1Uncoupling protein 1; Brown adipose tissue mitochondria; Cold acclimation; Thermogenesis; Basal proton leak; Medium tonicity

Uncoupling protein-1 is not leaky by Irina G. Shabalina; Mario Ost; Natasa Petrovic; Marek Vrbacky; Jan Nedergaard; Barbara Cannon (pp. 773-784).

The regulation and turnover of mitochondrial uncoupling proteins by Vian Azzu; Martin Jastroch; Ajit S. Divakaruni; Martin D. Brand (pp. 785-791).

Uncoupling proteins (UCP1, UCP2 and UCP3) are important in regulating cellular fuel metabolism and as attenuators of reactive oxygen species production through strong or mild uncoupling. The generic function and broad tissue distribution of the uncoupling protein family means that they are increasingly implicated in a range of pathophysiological processes including obesity, insulin resistance and diabetes mellitus, neurodegeneration, cardiovascular disease, immunity and cancer. The significant recent progress describing the turnover of novel uncoupling proteins, as well as current views on the physiological roles and regulation of UCPs, is outlined.

Keywords: Abbreviations; ANT; adenine nucleotide translocase; ATF1; Cyclic AMP-dependent transcription factor; ATP; adenosine triphosphate; BAT; brown adipose tissue; GDP; guanosine diphosphate; ORF; open reading frame; PPAR; peroxisome proliferator-activated receptor; SREBP-1c; sterol regulatory element-binding protein-1c; TRE; thyroid response element; UCP; uncoupling protein; UTR; untranslated regionMitochondria; Uncoupling protein; UCP1; UCP2; UCP3; Turnover; Degradation; Regulation

The regulation and turnover of mitochondrial uncoupling proteins by Vian Azzu; Martin Jastroch; Ajit S. Divakaruni; Martin D. Brand (pp. 785-791).

Mitochondrial uncoupling proteins in unicellular eukaryotes by Wieslawa Jarmuszkiewicz; Andrzej Woyda-Ploszczyca; Nina Antos-Krzeminska; Francis E. Sluse (pp. 792-799).

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH₂) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs ( A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species.

Keywords: Abbreviations; AcUCP; uncoupling protein of; A. castellanii; AcAOX; alternative oxidase of; A. castellanii; AOX; alternative oxidase; FCCP; p; -trifluoromethoxyphenylhydrazone; FFA; free fatty acid; HNE; 4-hydroxy-2-nonenal; PN; purine nucleotide; Q; coenzyme Q, ubiquinone; QH; ₂; reduced Q, ubiquinol; Q reduction level or Q redox state; QH; ₂; /total Q in the inner mitochondrial membrane; ROS; reactive oxygen species; UCP; uncoupling protein; ΔΨ; mitochondrial membrane electrical potential; ΔμH; ⁺; electrochemical proton gradientAmoeboid eukaryotes; Energy dissipation; Fungi; Mitochondria; Uncoupling proteins; Ubiquinone redox state

Mitochondrial uncoupling proteins in unicellular eukaryotes by Wieslawa Jarmuszkiewicz; Andrzej Woyda-Ploszczyca; Nina Antos-Krzeminska; Francis E. Sluse (pp. 792-799).

Lipotoxicity, fatty acid uncoupling and mitochondrial carrier function by Eduardo Rial; Rodriguez-Sanchez Leonor Rodríguez-Sánchez; Eunate Gallardo-Vara; Pilar Zaragoza; Eva Moyano; Gonzalez-Barroso M. Mar González-Barroso (pp. 800-806).

Diseases like obesity, diabetes or generalized lipodystrophy cause a chronic elevation of circulating fatty acids that can become cytotoxic, a condition known as lipotoxicity. Fatty acids cause oxidative stress and alterations in mitochondrial structure and function. The uncoupling of the oxidative phosphorylation is one of the most recognized deleterious fatty acid effects and several metabolite transporters are known to mediate in their action. The fatty acid interaction with the carriers leads to membrane depolarization and/or the conversion of the carrier into a pore. The result is the opening of the permeability transition pore and the initiation of apoptosis. Unlike the other members of the mitochondrial carrier superfamily, the eutherian uncoupling protein UCP1 has evolved to achieve its heat-generating capacity in the physiological context provided by the brown adipocyte and therefore it is activated by the low fatty acid concentrations generated by the noradrenaline-stimulated lipolysis.

Keywords: Abbreviations; FA; fatty acid; IMM; inner mitochondrial membrane; OMM; outer mitochondrial membrane; PTP; permeability transition pore; PIC; phosphate carrier; ANT; adenine nucleotide translocator; ROS; reactive oxygen species; UCP; uncoupling protein; CPT-1; carnitine palmitoyl transferase-1; BAT; brown adipose tissueFatty acid; Lipotoxicity; Uncoupling; UCP; Oxidative stress; Mitochondria; Transport

Absence of mitochondrial uncoupling protein 1 affects apoptosis in thymocytes, thymocyte/T-cell profile and peripheral T-cell number by Alison E. Adams; Orlagh M. Kelly; Richard K. Porter (pp. 807-816).

Our laboratory has previously demonstrated the presence of constitutively expressed mitochondrial uncoupling protein 1 in mouse thymocytes. In our endeavours to understand the role of mitochondrial uncoupling protein 1 in thymocyte function, we compared cell profiles in thymus and spleen of wild-type with those of UCP 1 knock-out mice, which in turn led to comparative investigations of apoptotic potential in thymocytes from these mice. We demonstrate that spleen cell numbers were reduced ∼3-fold in UCP 1 knock-out mice compared to wild-type mice. We record a halving of CD8 single positive cell numbers in thymus with a significant incremental increase in CD4/CD8 double positives cell numbers in the thymus of UCP 1 knock-out mice compared to wild-type mice. These data are mirrored by an approximate halving of CD8 single positive cell numbers and a doubling of CD4/CD8 double positive cell numbers in the spleen of UCP 1 knock-out mice compared to wild-type mice. These differences are most probably explained by our observations of decreased apoptotic potential and higher ATP levels in thymocytes of UCP 1 knock-out mice when compared to wild-type controls. We conclude that constitutively expressed UCP 1 is a factor in determining T-cell population selection in mice.

Keywords: Abbreviations; BAT; brown adipose tissue; BSA; bovine serum albumin; DP; CD4; ⁺; /CD8; ⁺; double positive; DN; CD4; ⁻; /CD8; ⁻; double negative; FBS; fetal bovine serum; PBS; phosphate buffered saline; SP; single positive; PI; propidium iodide; PDH; pyruvate dehydrogenase; DEX; dexamethasone; UCP; uncoupling proteinMitochondria; Uncoupling protein; Thymus; Spleen

Mitochondrial carriers function as monomers by Edmund R.S. Kunji; Paul G. Crichton (pp. 817-831).

Mitochondrial carriers link biochemical pathways in the mitochondrial matrix and cytosol by transporting metabolites, inorganic ions, nucleotides and cofactors across the mitochondrial inner membrane. Uncoupling proteins that dissipate the proton electrochemical gradient also belong to this protein family. For almost 35years the general consensus has been that mitochondrial carriers are dimeric in structure and function. This view was based on data from inhibitor binding studies, small-angle neutron scattering, electron microscopy, differential tagging/affinity chromatography, size-exclusion chromatography, analytical ultracentrifugation, native gel electrophoresis, cross-linking experiments, tandem-fusions, negative dominance studies and mutagenesis. However, the structural folds of the ADP/ATP carriers were found to be monomeric, lacking obvious dimerisation interfaces. Subsequently, the yeast ADP/ATP carrier was demonstrated to function as a monomer. Here, we revisit the data that have been published in support of a dimeric state of mitochondrial carriers. Our analysis shows that when critical factors are taken into account, the monomer is the only plausible functional form of mitochondrial carriers. We propose a transport model based on the monomer, in which access to a single substrate binding site is controlled by two flanking salt bridge networks, explaining uniport and strict exchange of substrates.

Keywords: Abbreviations; LAPAO; dodecylamido-N,N′-dimethylpropyl amine oxide; C; ₁₂; E; ₈; dodecyl octaoxyethylene; BN-PAGE; blue native poly-acrylamide gel electrophoresis; CATR; carboxyatractyloside; BKA; bongkrekic acid; SDS-PAGE; sodium dodecyl-sulphate poly-acrylamide gel electrophoresis; AFM; atomic force microscopyMembrane protein; Oligomeric state; Transport mechanism; Detergent; Lipid; Protein–protein interaction

Mitochondrial carriers function as monomers by Edmund R.S. Kunji; Paul G. Crichton (pp. 817-831).

Mitochondrial ion transport pathways: Role in metabolic diseases by Ariel R. Cardoso; Bruno B. Queliconi; Alicia J. Kowaltowski (pp. 832-838).

Mitochondria are the central coordinators of energy metabolism and alterations in their function and number have long been associated with metabolic disorders such as obesity, diabetes and hyperlipidemias. Since oxidative phosphorylation requires an electrochemical gradient across the inner mitochondrial membrane, ion channels in this membrane certainly must play an important role in the regulation of energy metabolism. However, in many experimental settings, the relationship between the activity of mitochondrial ion transport and metabolic disorders is still poorly understood. This review briefly summarizes some aspects of mitochondrial H⁺ transport (promoted by uncoupling proteins, UCPs), Ca²⁺ and K⁺ uniporters which may be determinant in metabolic disorders.

Keywords: Uncoupling protein; Ca; ²⁺; uniporter; ATP-sensitive K; ⁺; channel; Reactive oxygen species

Mitochondrial ion transport pathways: Role in metabolic diseases by Ariel R. Cardoso; Bruno B. Queliconi; Alicia J. Kowaltowski (pp. 832-838).

Keywords: Uncoupling protein; Ca; ²⁺; uniporter; ATP-sensitive K; ⁺; channel; Reactive oxygen species

Mitochondrial ion transport pathways: Role in metabolic diseases by Ariel R. Cardoso; Bruno B. Queliconi; Alicia J. Kowaltowski (pp. 832-838).

Keywords: Uncoupling protein; Ca; ²⁺; uniporter; ATP-sensitive K; ⁺; channel; Reactive oxygen species

Site-directed mutagenesis of charged amino acids of the human mitochondrial carnitine/acylcarnitine carrier: Insight into the molecular mechanism of transport by Nicola Giangregorio; Annamaria Tonazzi; Lara Console; Cesare Indiveri; Ferdinando Palmieri (pp. 839-845).

The structure/function relationships of charged residues of the human mitochondrial carnitine/acylcarnitine carrier, which are conserved in the carnitine/acylcarnitine carrier subfamily and exposed to the water-filled cavity of carnitine/acylcarnitine carrier in the c-state, have been investigated by site-directed mutagenesis. The mutants were expressed in Escherichia coli, purified and reconstituted in liposomes, and their transport activity was measured as³H-carnitine/carnitine antiport. The mutants K35A, E132A, D179A and R275A were nearly inactive with transport activities between 5 and 10% of the wild-type carnitine/acylcarnitine carrier. R178A, K234A and D231A showed transport function of about 15% of the wild-type carnitine/acylcarnitine carrier. The substitutions of the other residues with alanine had little or no effect on the carnitine/acylcarnitine carrier activity. Marked changes in the kinetic parameters with three-fold higher Km and lower Vmax values with respect to the wild-type carnitine/acylcarnitine carrier were found when replacing Lys-35, Glu-132, Asp-179 and Arg-275 with alanine. Double mutants exhibited transport activities and kinetic parameters reflecting those of the single mutants; however, lack of D179A activity was partially rescued by the additional mutation R178A. The results provide evidence that Arg-275, Asp-179 and Arg-178, which protrude into the carrier's internal cavity at about the midpoint of the membrane, are the critical binding sites for carnitine. Furthermore, Lys-35 and Glu-132, which are very probably involved in the salt-bridge network located at the bottom of the cavity, play a major role in opening and closing the matrix gate.

Keywords: Abbreviations; DTE; dithioerythritol; NEM; N; -ethylmaleimide; Pipes; 1,4-piperazinediethanesulfonic acid; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; CAC; carnitine/acylcarnitine carrier; WT; wild-typeCarnitine; Mitochondria; Transport; Site-directed mutagenesis; Mitochondrial carrier

Structural divergence between the two subgroups of P5 ATPases by Sorensen Danny Mollerup Sørensen; Morten J. Buch-Pedersen; Michael Gjedde Palmgren (pp. 846-855).

Evolution of P5 type ATPases marks the origin of eukaryotes but still they remain the least characterized pumps in the superfamily of P-type ATPases. Phylogenetic analysis of available sequences suggests that P5 ATPases should be divided into at least two subgroups, P5A and P5B. P5A ATPases have been identified in the endoplasmic reticulum and seem to have basic functions in protein maturation and secretion. P5B ATPases localize to vacuolar/lysosomal or apical membranes and in animals play a role in hereditary neuronal diseases. Here we have used a bioinformatical approach to identify differences in the primary sequences between the two subgroups. P5A and P5B ATPases appear have a very different membrane topology from other P-type ATPases with two and one, respectively, additional transmembrane segments inserted in the N-terminal end. Based on conservation of residues in the transmembrane region, the two P5 subgroups most likely have different substrate specificities although these cannot be predicted from their sequences. Furthermore, sequence differences between P5A and P5B ATPases are identified in the catalytic domains that could influence key kinetic properties differentially. Together these findings indicate that P5A and P5B ATPases are structurally and functionally different.

Keywords: P5 ATPase; P-type ATPase; Spf1; Ypk9; Parkinson disease

Structural divergence between the two subgroups of P5 ATPases by Sorensen Danny Mollerup Sørensen; Morten J. Buch-Pedersen; Michael Gjedde Palmgren (pp. 846-855).

Keywords: P5 ATPase; P-type ATPase; Spf1; Ypk9; Parkinson disease

Structural divergence between the two subgroups of P5 ATPases by Sorensen Danny Mollerup Sørensen; Morten J. Buch-Pedersen; Michael Gjedde Palmgren (pp. 846-855).

Keywords: P5 ATPase; P-type ATPase; Spf1; Ypk9; Parkinson disease

The ups and downs of mitochondrial calcium signalling in the heart by Elinor J. Griffiths; Dirki Balaska; Wendy H.Y. Cheng (pp. 856-864).

Regulation of intramitochondrial free calcium ([Ca²⁺]_m) is critical in both physiological and pathological functioning of the heart. The full extent and importance of the role of [Ca²⁺]_m is becoming apparent as evidenced by the increasing interest and work in this area over the last two decades. However, controversies remain, such as the existence of beat-to-beat mitochondrial Ca²⁺ transients; the role of [Ca²⁺]_m in modulating whole-cell Ca²⁺ signalling; whether or not an increase in [Ca²⁺]_m is essential to couple ATP supply and demand; and the role of [Ca²⁺]_m in cell death by both necrosis and apoptosis, especially in formation of the mitochondrial permeability transition pore. The role of [Ca²⁺]_m in heart failure is an area that has also recently been highlighted. [Ca²⁺]_m can now be measured reasonably specifically in intact cells and hearts thanks to developments in fluorescent indicators and targeted proteins and more sensitive imaging technology. This has revealed interactions of the mitochondrial Ca²⁺ transporters with those of the sarcolemma and sarcoplasmic reticulum, and has gone a long way to bringing the mitochondrial Ca²⁺ transporters to the forefront of cardiac research. Mitochondrial Ca²⁺ uptake occurs via the ruthenium red sensitive Ca²⁺ uniporter (mCU), and efflux via an Na⁺/Ca²⁺ exchanger (mNCX). The purification and cloning of the transporters, and development of more specific inhibitors, would produce a step-change in our understanding of the role of these apparently critical but still elusive proteins. In this article we will summarise the key physiological roles of [Ca²⁺]_m in ATP production and cell Ca²⁺ signalling in both adult and neonatal hearts, as well as highlighting some of the controversies in these areas. We will also briefly discuss recent ideas on the interactions of nitric oxide with [Ca²⁺]_m.

Keywords: Mitochondria; Calcium; Cardiomyocyte; Heart; Calcium uniporter; Sodium calcium exchanger; Nitric oxide; Neonatal

The ups and downs of mitochondrial calcium signalling in the heart by Elinor J. Griffiths; Dirki Balaska; Wendy H.Y. Cheng (pp. 856-864).

Keywords: Mitochondria; Calcium; Cardiomyocyte; Heart; Calcium uniporter; Sodium calcium exchanger; Nitric oxide; Neonatal

The ups and downs of mitochondrial calcium signalling in the heart by Elinor J. Griffiths; Dirki Balaska; Wendy H.Y. Cheng (pp. 856-864).

Keywords: Mitochondria; Calcium; Cardiomyocyte; Heart; Calcium uniporter; Sodium calcium exchanger; Nitric oxide; Neonatal

Redox-optimized ROS balance: A unifying hypothesis by M.A. Aon; S. Cortassa; B. O'Rourke (pp. 865-877).

While it is generally accepted that mitochondrial reactive oxygen species (ROS) balance depends on the both rate of single electron reduction of O₂ to superoxide (O^₂⁻) by the electron transport chain and the rate of scavenging by intracellular antioxidant pathways, considerable controversy exists regarding the conditions leading to oxidative stress in intact cells versus isolated mitochondria. Here, we postulate that mitochondria have been evolutionarily optimized to maximize energy output while keeping ROS overflow to a minimum by operating in an intermediate redox state. We show that at the extremes of reduction or oxidation of the redox couples involved in electron transport (NADH/NAD⁺) or ROS scavenging (NADPH/NADP⁺, GSH/GSSG), respectively, ROS balance is lost. This results in a net overflow of ROS that increases as one moves farther away from the optimal redox potential. At more reduced mitochondrial redox potentials, ROS production exceeds scavenging, while under more oxidizing conditions (e.g., at higher workloads) antioxidant defenses can be compromised and eventually overwhelmed. Experimental support for this hypothesis is provided in both cardiomyocytes and in isolated mitochondria from guinea pig hearts. The model reconciles, within a single framework, observations that isolated mitochondria tend to display increased oxidative stress at high reduction potentials (and high mitochondrial membrane potential, ∆ Ψ_m), whereas intact cardiac cells can display oxidative stress either when mitochondria become more uncoupled (i.e., low ∆ Ψ_m) or when mitochondria are maximally reduced (as in ischemia or hypoxia). The continuum described by the model has the potential to account for many disparate experimental observations and also provides a rationale for graded physiological ROS signaling at redox potentials near the minimum.

Keywords: Mitochondrial membrane potential; Mild uncoupling; Oxidative phosphorylation; Reverse electron transport; Forward electron transport; Redox potential; Hypoxia; Oxidative stress

Redox-optimized ROS balance: A unifying hypothesis by M.A. Aon; S. Cortassa; B. O'Rourke (pp. 865-877).

Keywords: Mitochondrial membrane potential; Mild uncoupling; Oxidative phosphorylation; Reverse electron transport; Forward electron transport; Redox potential; Hypoxia; Oxidative stress

Redox-optimized ROS balance: A unifying hypothesis by M.A. Aon; S. Cortassa; B. O'Rourke (pp. 865-877).

Keywords: Mitochondrial membrane potential; Mild uncoupling; Oxidative phosphorylation; Reverse electron transport; Forward electron transport; Redox potential; Hypoxia; Oxidative stress

Prevention of cardiolipin oxidation and fatty acid cycling as two antioxidant mechanisms of cationic derivatives of plastoquinone (SkQs) by Vladimir P. Skulachev; Yury N. Antonenko; Dmitry A. Cherepanov; Boris V. Chernyak; Denis S. Izyumov; Ludmila S. Khailova; Sergey S. Klishin; Galina A. Korshunova; Konstantin G. Lyamzaev; Olga Yu. Pletjushkina; Vitaly A. Roginsky; Tatiana I. Rokitskaya; Fedor F. Severin; Inna I. Severina; Ruben A. Simonyan; Maxim V. Skulachev; Natalia V. Sumbatyan; Evgeniya I. Sukhanova; Vadim N. Tashlitsky; Tatyana A. Trendeleva; Mikhail Yu. Vyssokikh; Renata A. Zvyagilskaya (pp. 878-889).

The present state of the art in studies on the mechanisms of antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SkQs) is reviewed. Our experiments showed that these compounds can operate as antioxidants in two quite different ways, i.e. (i) by preventing peroxidation of cardiolipin [Antonenko et al., Biochemistry (Moscow) 73 (2008) 1273–1287] and (ii) by fatty acid cycling resulting in mild uncoupling that inhibits the formation of reactive oxygen species (ROS) in mitochondrial State 4 [Severin et al. Proc. Natl. Acad. Sci. USA 107 (2009), 663–668]. The quinol and cationic moieties of SkQ are involved in cases (i) and (ii), respectively. In case (i) SkQH₂ interrupts propagation of chain reactions involved in peroxidation of unsaturated fatty acid residues in cardiolipin, the formed SkQ⁻ being reduced back to SkQH₂ by heme b_H of complex III in an antimycin-sensitive way. Molecular dynamics simulation showed that there are two stable conformations of SkQ1 with the quinol residue localized near peroxyl radicals at C₉ or C₁₃ of the linoleate residue in cardiolipin. In mechanism (ii), fatty acid cycling mediated by the cationic SkQ moiety is involved. It consists of (a) transmembrane movement of the fatty acid anion/SkQ cation pair and (b) back flows of free SkQ cation and protonated fatty acid. The cycling results in a protonophorous effect that was demonstrated in planar phospholipid membranes and liposomes. In mitochondria, the cycling gives rise to mild uncoupling, thereby decreasing membrane potential and ROS generation coupled to reverse electron transport in the respiratory chain. In yeast cells, dodecyltriphenylphosphonium (С₁₂TPP), the cationic part of SkQ1, induces uncoupling that is mitochondria-targeted since С₁₂TPP is specifically accumulated in mitochondria and increases the H⁺ conductance of their inner membrane. The conductance of the outer cell membrane is not affected by С₁₂TPP.

Keywords: Abbreviations; ∆; ψ; transmembrane electric potential; AAPH; 2,2′-azobis(2-amidinopropane)dihydrochloride; BLM; planar bilayer phospholipid membrane; Cart; carboxyatractyloside; CCCP; carbonyl cyanide; m; -chlorophenylhydrazone; CTMA; cetyltrimethylammonium; C; ₁₀; TPP; decyltriphenylphosphonium; C; ₁₂; R1; decylrhodamine 19; C; ₁₂; R4; decylrhodamine B; C; ₁₂; TPP; dodecyltriphenylphosphonium; DCF; 2′,7′-dichlorodihydrofluorescein diacetate; DMQ; 3-demethoxy ubiquinonyl decyltriphenylphosphonium; DPQ; decylplastoquinone; FCCP; carbonylcyanide p-trifluoromethoxyphenylhydrazone; MDA; malondialdehyde; MitoQ; ubiquinonyl decyltriphenylphosphonium; ML; methyl linoleate; NAC; N-acetyl cysteine; ROS; reactive oxygen species; SkQ; a compound composed of plastoquinone or methylplastoquinone and decyl (or amyl) triphenylphosphonium, Rhodamine 19, or Rhodamine B; SkQ1; plastoquinonyl decyltriphenylphosphonium; SkQ3; 5-methylplastoquinonyl decyltriphenylphosphonium; SkQ5; plastoquinonyl amyltriphenylphosphonium; SkQR1; plastoquinonyl decylrhodamine 19; SkQR4; plastoquinonyl decylrhodamine B; TMRM; tetramethylrhodamine methyl ester; TPP; tetraphenylphosphoniumMitochondria; Reactive oxygen species; SkQ; Antioxidant; Cardiolipin; Fatty acid cycling; Mild uncoupling

Antioxidant defence systems and generation of reactive oxygen species in osteosarcoma cells with defective mitochondria: Effect of selenium by Marta Wojewoda; Jerzy Duszyński; Joanna Szczepanowska (pp. 890-896).

Mitochondrial diseases originate from mutations in mitochondrial or nuclear genes encoding for mitochondrial proteome. Neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) syndrome is associated with the T8993G transversion in ATP6 gene which results in substitution at the very conservative site in the subunit 6 of mitochondrial ATP synthase. Defects in the mitochondrial respiratory chain and the ATPase are considered to be accompanied by changes in the generation of reactive oxygen species (ROS). This study aimed to elucidate effects of selenium on ROS and antioxidant system of NARP cybrid cells with 98% of T8993G mutation load. We found that selenium decreased ROS generation and increased the level and activity of antioxidant enzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Therefore, we propose selenium to be a promising therapeutic agent not only in the case of NARP syndrome but also other diseases associated with mitochondrial dysfunctions and oxidative stress.

Keywords: Mitochondria; NARP; Selenium; ROS; Antioxidant enzyme

Redox signaling (cross-talk) from and to mitochondria involves mitochondrial pores and reactive oxygen species by Andreas Daiber (pp. 897-906).

This review highlights the important role of redox signaling between mitochondria and NADPH oxidases. Besides the definition and general importance of redox signaling, the cross-talk between mitochondrial and Nox-derived reactive oxygen species (ROS) is discussed on the basis of 4 different examples. In the first model, angiotensin-II is discussed as a trigger for NADPH oxidase activation with subsequent ROS-dependent opening of mitochondrial ATP-sensitive potassium channels leading to depolarization of mitochondrial membrane potential followed by mitochondrial ROS formation and respiratory dysfunction. This concept was supported by observations that ethidium bromide-induced mitochondrial damage suppressed angiotensin-II-dependent increase in Nox1 and oxidative stress. In another example hypoxia was used as a stimulator of mitochondrial ROS formation and by using pharmacological and genetic inhibitors, a role of mitochondrial ROS for the induction of NADPH oxidase via PKCɛ was demonstrated. The third model was based on cell death by serum withdrawal that promotes the production of ROS in human 293T cells by stimulating both the mitochondria and Nox1. By superior molecular biological methods the authors showed that mitochondria were responsible for the fast onset of ROS formation followed by a slower but long-lasting oxidative stress condition based on the activation of an NADPH oxidase (Nox1) in response to the fast mitochondrial ROS formation. Finally, a cross-talk between mitochondria and NADPH oxidases (Nox2) was shown in nitroglycerin-induced tolerance involving the mitochondrial permeability transition pore and ATP-sensitive potassium channels. The use of these redox signaling pathways as pharmacological targets is briefly discussed.

Keywords: Redox regulation; Oxidative protein modification; Nitric oxide; Superoxide; Peroxynitrite; Mitochondrion; NADPH oxidase

Redox signaling (cross-talk) from and to mitochondria involves mitochondrial pores and reactive oxygen species by Andreas Daiber (pp. 897-906).

Keywords: Redox regulation; Oxidative protein modification; Nitric oxide; Superoxide; Peroxynitrite; Mitochondrion; NADPH oxidase

Redox signaling (cross-talk) from and to mitochondria involves mitochondrial pores and reactive oxygen species by Andreas Daiber (pp. 897-906).

Keywords: Redox regulation; Oxidative protein modification; Nitric oxide; Superoxide; Peroxynitrite; Mitochondrion; NADPH oxidase

Calcium uptake mechanisms of mitochondria by Jaime Santo-Domingo; Nicolas Demaurex (pp. 907-912).

The ability of mitochondria to capture Ca²⁺ ions has important functional implications for cells, because mitochondria shape cellular Ca²⁺ signals by acting as a Ca²⁺ buffer and respond to Ca²⁺ elevations either by increasing the cell energy supply or by triggering the cell death program of apoptosis. A mitochondrial Ca²⁺ channel known as the uniporter drives the rapid and massive entry of Ca²⁺ ions into mitochondria. The uniporter operates at high, micromolar cytosolic Ca²⁺ concentrations that are only reached transiently in cells, near Ca²⁺ release channels. Mitochondria can also take up Ca²⁺ at low, nanomolar concentrations, but this high affinity mode of Ca²⁺ uptake is not well characterized. Recently, leucine-zipper-EF hand-containing transmembrane region (Letm1) was proposed to be an electrogenic 1:1 mitochondrial Ca²⁺/H⁺ antiporter that drives the uptake of Ca²⁺ into mitochondria at nanomolar cytosolic Ca²⁺ concentrations. In this article, we will review the properties of the Ca²⁺ import systems of mitochondria and discuss how Ca²⁺ uptake via an electrogenic 1:1 Ca²⁺/H⁺ antiport challenges our current thinking of the mitochondrial Ca²⁺ uptake mechanism.

Keywords: Calcium signalling; Ion channel; Cell metabolism; Apoptosis

Calcium uptake mechanisms of mitochondria by Jaime Santo-Domingo; Nicolas Demaurex (pp. 907-912).

Keywords: Calcium signalling; Ion channel; Cell metabolism; Apoptosis

Calcium uptake mechanisms of mitochondria by Jaime Santo-Domingo; Nicolas Demaurex (pp. 907-912).

Keywords: Calcium signalling; Ion channel; Cell metabolism; Apoptosis

Regulation of mitochondrial fission by intracellular Ca²⁺ in rat ventricular myocytes by Jennifer Hom; Tianzheng Yu; Yisang Yoon; George Porter; Shey-Shing Sheu (pp. 913-921).

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca²⁺) is one signal that has been shown to both regulate mitochondrial fission in various cell types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca²⁺ plays an important role in adult cardiac muscle cells for excitation–metabolism coupling, little is known about whether Ca²⁺ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca²⁺ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca²⁺ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca²⁺ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca²⁺-mediated ROS generation in cardiac myocytes. These results suggest that Ca²⁺, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.

Keywords: Mitochondria; Mitochondrial fusion and fission; Calcium; Ventricular myocytes; Reactive oxygen species; Cardiac muscle

Regulation of mitochondrial fission by intracellular Ca²⁺ in rat ventricular myocytes by Jennifer Hom; Tianzheng Yu; Yisang Yoon; George Porter; Shey-Shing Sheu (pp. 913-921).

Keywords: Mitochondria; Mitochondrial fusion and fission; Calcium; Ventricular myocytes; Reactive oxygen species; Cardiac muscle

Regulation of mitochondrial fission by intracellular Ca²⁺ in rat ventricular myocytes by Jennifer Hom; Tianzheng Yu; Yisang Yoon; George Porter; Shey-Shing Sheu (pp. 913-921).

Keywords: Mitochondria; Mitochondrial fusion and fission; Calcium; Ventricular myocytes; Reactive oxygen species; Cardiac muscle

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria by Zsofia Komary; Laszlo Tretter; Vera Adam-Vizi (pp. 922-928).

The effect of Ca²⁺ applied in high concentrations (50 and 300µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H₂O₂ release from mitochondria was induced by Ca²⁺ applied in 50µM, but not in 300µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150sec after exposure to Ca²⁺. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca²⁺ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca²⁺ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca²⁺ given in 50µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H₂O₂ was significantly higher than that observed in a medium containing ADP and Ca²⁺ no longer increased but rather decreased the rate of H₂O₂ release. With 300µM Ca²⁺ only a loss but no tendency of a recovery of ΔΨm was detected and H₂O₂ release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca²⁺ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H₂O₂ release is induced by calcium, but only in ≤ 100µM concentration, when after a transient Ca²⁺-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca²⁺-induced depolarization, Ca²⁺ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300µM) are unrelated to mitochondrial permeability transition.

Keywords: Mitochondria; Brain; Oxidative stress; Calcium; Membrane potential; Reactive oxygen species

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria by Zsofia Komary; Laszlo Tretter; Vera Adam-Vizi (pp. 922-928).

Keywords: Mitochondria; Brain; Oxidative stress; Calcium; Membrane potential; Reactive oxygen species

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria by Zsofia Komary; Laszlo Tretter; Vera Adam-Vizi (pp. 922-928).

Keywords: Mitochondria; Brain; Oxidative stress; Calcium; Membrane potential; Reactive oxygen species

Mitochondrial fatty acid oxidation and oxidative stress: Lack of reverse electron transfer-associated production of reactive oxygen species by Schonfeld Peter Schönfeld; Mariusz R. Więckowski; Magdalena Lebiedzińska; Lech Wojtczak (pp. 929-938).

Reverse electron transfer (RET) from succinate to NAD⁺ is known to be accompanied by high generation of reactive oxygen species (ROS). In contrast, oxidation of fatty acids by mitochondria, despite being a powerful source of FADH₂, does not lead to RET-associated high ROS generation. Here we show that oxidation of carnitine esters of medium- and long-chain fatty acids by rat heart mitochondria is accompanied by neither high level of NADH/NAD⁺ nor intramitochondrial reduction of acetoacetate to β-hydroxybutyrate, comparable to those accompanying succinate oxidation, although it produces the same or higher energization of mitochondria as evidenced by high transmembrane potential. Also in contrast to the oxidation of succinate, where conversion of the pH difference between the mitochondrial matrix and the medium into the transmembrane electric potential by addition of nigericin results in a decrease of ROS generation, the same treatment during oxidation of octanoylcarnitine produces a large increase of ROS. Analysis of respiratory chain complexes by Blue Native polyacrylamide gel electrophoresis revealed bands that could tentatively point to supercomplex formation between complexes II and I and complexes II and III. However, no such association could be found between complex I and the electron transferring flavoprotein that participates in fatty acid oxidation. It is speculated that structural association between respective respiratory chain components may facilitate effective reverse electron transfer.

Keywords: Abbreviations; ETF; electron transferring flavoprotein; FCCP; carbonylcyanide-; p; -trifluoromethoxyphenylhydrazone; RET; reverse electron transfer; ROS; reactive oxygen species; Δ; p; electrochemical proton gradient (protonmotive force); Δ; Ψ; _m; mitochondrial transmembrane potentialFatty acid oxidation; Reverse electron transfer; Reactive oxygen species (ROS); Mitochondrion; Respiratory chain complex

What are the sources of hydrogen peroxide production by heart mitochondria? by Vera G. Grivennikova; Alexandra V. Kareyeva; Andrei D. Vinogradov (pp. 939-944).

Coupled rat heart mitochondria produce externally hydrogen peroxide at the rates which correspond to about 0.8 and 0.3% of the total oxygen consumption at State 4 with succinate and glutamate plus malate as the respiratory substrates, respectively. Stimulation of the respiratory activities by ADP (State 4–State 3 transition) decreases the succinate- and glutamate plus malate-supported H₂O₂ production 8- and 1.3-times, respectively. NH₄⁺ strongly stimulates hydrogen peroxide formation with either substrate without any effect on State 4 and/or State 3 respiration. Rotenone-treated, alamethicin-permeabilized mitochondria catalyze NADH-supported H₂O₂ production at a rate about 10-fold higher than that seen in intact mitochondria under optimal (State 4 succinate-supported respiration in the presence of ammonium chloride) conditions. NADH-supported hydrogen peroxide production by the rotenone-treated mitochondria devoid of a permeability barrier for H₂O₂ diffusion by alamethicin treatment are only partially (∼50%) sensitive to the Complex I NADH binding site-specific inhibitor, NADH-OH. The residual activity is strongly (∼6-fold) stimulated by ammonium chloride. NAD⁺ inhibits both Complex I-mediated and ammonium-stimulated H₂O₂ production. In the absence of stimulatory ammonium about half of the total NADH-supported hydrogen peroxide production is catalyzed by Complex I. In the presence of ammonium about 90% of the total hydrogen peroxide production is catalyzed by matrix located, ammonium-dependent enzyme(s).

Keywords: Hydrogen peroxide production; Respiratory activity; Complex I; Matrix proteins; Ammonium; Mitochondria

What are the sources of hydrogen peroxide production by heart mitochondria? by Vera G. Grivennikova; Alexandra V. Kareyeva; Andrei D. Vinogradov (pp. 939-944).

Keywords: Hydrogen peroxide production; Respiratory activity; Complex I; Matrix proteins; Ammonium; Mitochondria

What are the sources of hydrogen peroxide production by heart mitochondria? by Vera G. Grivennikova; Alexandra V. Kareyeva; Andrei D. Vinogradov (pp. 939-944).

Keywords: Hydrogen peroxide production; Respiratory activity; Complex I; Matrix proteins; Ammonium; Mitochondria

The p13 protein of human T cell leukemia virus type 1 (HTLV-1) modulates mitochondrial membrane potential and calcium uptake by Roberta Biasiotto; Paola Aguiari; Rosario Rizzuto; Paolo Pinton; Donna M. D'Agostino; Vincenzo Ciminale (pp. 945-951).

Human T cell leukemia virus type 1 (HTLV-1) encodes p13, an 87-amino-acid protein that accumulates in the inner mitochondrial membrane. Recent studies performed using synthetic p13 and isolated mitochondria demonstrated that the protein triggers an inward potassium (K⁺) current and inner membrane depolarization. The present study investigated the effects of p13 on mitochondrial inner membrane potential (Δ ψ) in living cells. Using the potential-dependent probe tetramethyl rhodamine methyl ester (TMRM), we observed that p13 induced dose-dependent mitochondrial depolarization in HeLa cells. This effect was abolished upon mutation of 4 arginines in p13's α-helical domain that were previously shown to be essential for its activity in in vitro assays. As Δ ψ is known to control mitochondrial calcium (Ca²⁺) uptake, we next analyzed the effect of p13 on Ca²⁺ homeostasis. Experiments carried out in HeLa cells expressing p13 and organelle-targeted aequorins revealed that the protein specifically reduced mitochondrial Ca²⁺ uptake. These observations suggest that p13 might control key processes regulated through Ca²⁺ signaling such as activation and death of T cells, the major targets of HTLV-1 infection.

Keywords: Mitochondria; Viroporin; Membrane potential; Calcium homeostasis; HTLV-1; Potassium channel

Oxidative stress-dependent p66Shc phosphorylation in skin fibroblasts of children with mitochondrial disorders by Magdalena Lebiedzinska; Agnieszka Karkucinska-Wieckowska; Carlotta Giorgi; Elzbieta Karczmarewicz; Ewa Pronicka; Paolo Pinton; Jerzy Duszynski; Maciej Pronicki; Mariusz R. Wieckowski (pp. 952-960).

p66Shc, the growth factor adaptor protein, can have a substantial impact on mitochondrial metabolism through regulation of cellular response to oxidative stress. We investigated relationships between the extent of p66Shc phosphorylation at Ser36, mitochondrial dysfunctions and an antioxidant defense reactions in fibroblasts derived from five patients with various mitochondrial disorders (two with mitochondrial DNA mutations and three with methylglutaconic aciduria and genetic defects localized, most probably, in nuclear genes). We found that in all these fibroblasts, the extent of p66Shc phosphorylation at Ser36 was significantly increased. This correlated with a substantially decreased level of mitochondrial superoxide dismutase (SOD2) in these cells. This suggest that SOD2 is under control of the Ser36 phosphorylation status of p66Shc protein. As a consequence, an intracellular oxidative stress and accumulation of damages caused by oxygen free radicals are observed in the cells.

Keywords: p66Shc; Ser36-P-p66Shc; Mitochondrial disorder; ROS; Antioxidant enzyme

Mitochondrial energy metabolism and ageing by Ivana Bratic; Aleksandra Trifunovic (pp. 961-967).

Ageing can be defined as “a progressive, generalized impairment of function, resulting in an increased vulnerability to environmental challenge and a growing risk of disease and death”. Ageing is likely a multifactorial process caused by accumulated damage to a variety of cellular components. During the last 20years, gerontological studies have revealed different molecular pathways involved in the ageing process and pointed out mitochondria as one of the key regulators of longevity. Increasing age in mammals correlates with increased levels of mitochondrial DNA (mtDNA) mutations and a deteriorating respiratory chain function. Experimental evidence in the mouse has linked increased levels of somatic mtDNA mutations to a variety of ageing phenotypes, such as osteoporosis, hair loss, graying of the hair, weight reduction and decreased fertility. A mosaic respiratory chain deficiency in a subset of cells in various tissues, such as heart, skeletal muscle, colonic crypts and neurons, is typically found in aged humans. It has been known for a long time that respiratory chain-deficient cells are more prone to undergo apoptosis and an increased cell loss is therefore likely of importance in the age-associated mitochondrial dysfunction. In this review, we would like to point out the link between the mitochondrial energy balance and ageing, as well as a possible connection between the mitochondrial metabolism and molecular pathways important for the lifespan extension.

Keywords: Mitochondrial respiratory chain; The mitochondrial theory of ageing; Uncoupling to survive; The rate of living hypothesis

Mitochondrial energy metabolism and ageing by Ivana Bratic; Aleksandra Trifunovic (pp. 961-967).

Keywords: Mitochondrial respiratory chain; The mitochondrial theory of ageing; Uncoupling to survive; The rate of living hypothesis

Mitochondrial energy metabolism and ageing by Ivana Bratic; Aleksandra Trifunovic (pp. 961-967).

Keywords: Mitochondrial respiratory chain; The mitochondrial theory of ageing; Uncoupling to survive; The rate of living hypothesis

Cold tolerance of UCP1-ablated mice: A skeletal muscle mitochondria switch toward lipid oxidation with marked UCP3 up-regulation not associated with increased basal, fatty acid- or ROS-induced uncoupling or enhanced GDP effects by Irina G. Shabalina; Joris Hoeks; Tatiana V. Kramarova; Patrick Schrauwen; Barbara Cannon; Jan Nedergaard (pp. 968-980).

Mice lacking the thermogenic mitochondrial membrane protein UCP1 (uncoupling protein 1) - and thus all heat production from brown adipose tissue - can still adapt to a cold environment (4°C) if successively transferred to the cold. The mechanism behind this adaptation has not been clarified. To examine possible adaptive processes in the skeletal muscle, we isolated mitochondria from the hind limb muscles of cold-acclimated wild-type and UCP1(–/–) mice and examined their bioenergetic chracteristics. We observed a switch in metabolism, from carbohydrate towards lipid catabolism, and an increased total mitochondrial complement, with an increased total ATP production capacity. The UCP1(–/–) muscle mitochondria did not display a changed state-4 respiration rate (no uncoupling) and were less sensitive to the uncoupling effect of fatty acids than the wild-type mitochondria. The content of UCP3 was increased 3-4 fold, but despite this, endogenous superoxide could not invoke a higher proton leak, and the small inhibitory effect of GDP was unaltered, indicating that it was not mediated by UCP3. Double mutant mice (UCP1(–/–) plus superoxide dismutase 2-overexpression) were not more cold sensitive than UCP1(–/–), bringing into question an involvement of reactive oxygen species (ROS) in activation of any alternative thermogenic mechanism. We conclude that there is no evidence for an involvement of UCP3 in basal, fatty-acid- or superoxide-stimulated oxygen consumption or in GDP sensitivity. The adaptations observed did not imply any direct alternative process for nonshivering thermogenesis but the adaptations observed would be congruent with adaptation to chronically enhanced muscle activity caused by incessant shivering in these mice.

Keywords: Abbreviations; ANT; adenine nucleotide translocase; BSA; bovine serum albumin; COX1; cytochrome c oxidase subunit 1; CPT; carnitine palmitoyl transferase; EDTA; ethylenediamine tetraacetic acid; FA; fatty acid; FCCP; carbonyl cyanide; p; -(trifluoromethoxy)phenylhydrazone; GDP; guanosine 5'-diphosphate; HNE; 4-hydroxy nonenal; ROS; reactive oxygen species; SOD; superoxide dismutase; UCP; Uncoupling proteinUncoupling protein; Skeletal muscle mitochondria; Cold acclimation; Thermogenesis; Oxidative stress; Proton leak

Nitration of tyrosine 74 prevents human cytochrome c to play a key role in apoptosis signaling by blocking caspase-9 activation by Garcia-Heredia José M. García-Heredia; Diaz-Moreno Irene Díaz-Moreno; Pedro M. Nieto; Orzaez Mar Orzáez; Stella Kocanis; Miguel Teixeira; Perez-Paya Enrique Pérez-Payá; Diaz-Quintana Antonio Díaz-Quintana; Miguel A. De la Rosa (pp. 981-993).

Tyrosine nitration is one of the most common post-transcriptional modifications of proteins, so affecting their structure and function. Human cytochrome c, with five tyrosine residues, is an excellent case study as it is a well-known protein playing a double physiological role in different cell compartments. On one hand, it acts as electron carrier within the mitochondrial respiratory electron transport chain, and on the other hand, it serves as a cytoplasmic apoptosis-triggering agent. In a previous paper, we reported the effect of nitration on physicochemical and kinetic features of monotyrosine cytochrome c mutants. Here, we analyse the nitration-induced changes in secondary structure, thermal stability, haem environment, alkaline transition and molecular dynamics of three of such monotyrosine mutants – the so-called h-Y67, h-Y74 and h-Y97 – which have four tyrosines replaced by phenylalanines and just keep the tyrosine residue giving its number to the mutant. The resulting data, along with the functional analyses of the three mutants, indicate that it is the specific nitration of solvent-exposed Tyr74 which enhances the peroxidase activity and blocks the ability of C c to activate caspase-9, thereby preventing the apoptosis signaling pathway.

Keywords: Abbreviations; Ac-LEHD-AFC; N; -acetyl-Leu-Glu-His-Asp-(7-amino-4-trifluoromethyl coumarin); Apaf-1-xl; apoptosis protease-activating factor-1-x-long; BSA; bovine serum albumin; C; c; cytochrome; c; CD; circular dichroism; CLSTR; CLuSTeR CDPro database; DSF; differential scanning fluorimetry; DTT; dithiothreitol; EDC; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; H; ₂; DCF; reduced 2'7'-dichlorofluorescein; HEPES; N; -(2-hydroxyethyl)piperazine-; N; ′-ethanesulfonic acid; MD; molecular dynamics; PC9; pro-caspase 9; PMSF; phenylmethanesulphonylfluoride; RMSD; root mean square deviation; RMSF; root mean square fluctuations; RNOS; reactive nitrogen/oxygen species; T; _m; midpoint melting temperatureAlkaline transition; Apoptosis; Cytochrome; c; Peroxidase activity; Post-translational modification; Tyrosine nitration

Plasticity of the mitoproteome to nitrogen sources (nitrate and ammonium) in Chlamydomonas reinhardtii: The logic of Aox1 gene localization by Gerin Stéphanie Gérin; Grégory Mathy; Arnaud Blomme; Fabrice Franck; Francis E. Sluse (pp. 994-1003).

Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes. In Chlamydomonas, previous studies and the present one showed that the mitochondrial AOX is up-regulated in nitrate-grown cells in comparison with ammonium-grown cells. In this work, we have performed a comparative proteomic analysis of the soluble mitochondrial proteome of Chlamydomonas cells growth either on nitrate or ammonium. Our results highlight important proteomics modifications mostly related to primary metabolism in cells grown on nitrate. We could note an up-regulation of some TCA cycle enzymes and a down-regulation of cytochrome c₁ together with an up-regulation ofl-arginine and purine catabolism enzymes and of ROS scavenging systems. Hence, in nitrate-grown cells, AOX may play a dual role: (1) lowering the ubiquinone pool reduction level and (2) permitting the export of mitochondrial reducing power under the form of malate for nitrate and nitrite reduction. This role of AOX in the mitochondrial plasticity makes logical the localization of Aox1 in a nitrate assimilation gene cluster.

Keywords: Abbreviations; NR; nitrate reductase; NiR; nitrite reductase; GS; glutamine synthase; GOGAT; glutamate/2-oxoglutarate aminotransferase; AOX; alternative oxidase; ROS; reactive oxygen species; RNS; reactive nitrogen species; 2D-DIGE; two-dimensional differential in-gel electrophoresis; TCA cycle; tricarboxylic acid cycle; IDH; isocitrate dehydrogenase; CoA; coenzyme a; OXPHOS apparatus; oxidative phosphorylation apparatus; HCP; hybrid-cluster protein; P450nor; P450 NO reductase; GDH; glutamate dehydrogenase; HRGP; hydroxyproline-rich glycoprotein; GP; glutathione peroxidase; GR; glutathione reductaseAlternative oxidase; Nitrogen source; Comparative proteomics; Mitochondria; Bioenergetics; Metabolic flexibility

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L by Ilka Wittig; Bjoern Meyer; Heinrich Heide; Mirco Steger; Lea Bleier; Zibiernisha Wumaier; Michael Karas; Schagger Hermann Schägger (pp. 1004-1011).

Here we study ATP synthase from human ρ⁰ (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF₁ inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF₁ still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in ρ⁰ cells and not to the dimeric form. This supports previous suggestions that IF₁ plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.

Keywords: Oligomeric ATP synthase; Assembly; Supramolecular organization; Oxidative phosphorylation; Human mitochondria; Depletion of mitochondrial DNA

Estradiol-induced protection against ischemia-induced heart mitochondrial damage and caspase activation is mediated by protein kinase G by Ramune Morkuniene; Odeta Arandarcikaite; Laima Ivanoviene; Vilmante Borutaite (pp. 1012-1017).

We have previously reported that estradiol can protect heart mitochondria from the ischemia-induced mitochondrial permeability transition pore-related release of cytochrome c and subsequent apoptosis. In this study we investigated whether the effect of 17-beta-estradiol on ischemia-induced mitochondrial dysfunctions and apoptosis is mediated by activation of signaling protein kinases in a Langendorff-perfused rat heart model of stop-flow ischemia. We found that pre-perfusion of non-ischemic hearts with 100nM estradiol increased the resistance of subsequently isolated mitochondria to the calcium-induced opening of mitochondrial permeability transition pore and this was mediated by protein kinase G. Loading of the hearts with estradiol prevented ischemia-induced loss of cytochrome c from mitochondria and respiratory inhibition and these effects were reversed in the presence of the inhibitor of Akt kinase, NO synthase inhibitor L-NAME, guanylyl cyclase inhibitor ODQ and protein kinase G inhibitor KT5823. Estradiol prevented ischemia-induced activation of caspases and this was also reversed by KT5823. These findings suggest that estradiol may protect the heart against ischemia-induced injury activating the signaling cascade which involves Akt kinase, NO synthase, guanylyl cyclase and protein kinase G, and results in blockage of mitochondrial permeability transition pore-induced release of cytochrome c from mitochondria, respiratory inhibition and activation of caspases.

Keywords: Estrogens; Ischemia; Mitochondria; Cytochrome; c; Protein kinase G

The regulation of OXPHOS by extramitochondrial calcium by Frank N. Gellerich; Zemfira Gizatullina; Sonata Trumbeckaite; Huu P. Nguyen; Thilo Pallas; Odeta Arandarcikaite; Stephan Vielhaber; Enn Seppet; Frank Striggow (pp. 1018-1027).

Despite extensive research, the regulation of mitochondrial function is still not understood completely. Ample evidence shows that cytosolic Ca²⁺ has a strategic task in co-ordinating the cellular work load and the regeneration of ATP by mitochondria. Currently, the paradigmatic view is that Ca_cyt²⁺ taken up by the Ca²⁺ uniporter activates the matrix enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and isocitrate dehydrogenase. However, we have recently found that Ca²⁺ regulates the glutamate-dependent state 3 respiration by the supply of glutamate to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier. Since this activation is not affected by ruthenium red, glutamate transport into mitochondria is controlled exclusively by extramitochondrial Ca²⁺. Therefore, this discovery shows that besides intramitochondrial also extramitochondrial Ca²⁺ regulates oxidative phosphorylation. This new mechanism acts as a mitochondrial “gas pedal”, supplying the OXPHOS with substrate on demand. These results are in line with recent findings of Satrustegui and Palmieri showing that aralar as part of the malate–aspartate shuttle is involved in the Ca²⁺-dependent transport of reducing hydrogen equivalents (from NADH) into mitochondria. This review summarises results and evidence as well as hypothetical interpretations of data supporting the view that at the surface of mitochondria different regulatory Ca²⁺-binding sites exist and can contribute to cellular energy homeostasis. Moreover, on the basis of our own data, we propose that these surface Ca²⁺-binding sites may act as targets for neurotoxic proteins such as mutated huntingtin and others. The binding of these proteins to Ca²⁺-binding sites can impair the regulation by Ca²⁺, causing energetic depression and neurodegeneration.

Keywords: Abbreviations; AK; adenylate kinase; AOA; aminooxyacetate; CAG; DNA code for glutamine; CK; creatine kinase; COX; cytochrome-c-oxidase; CsA; cyclosporin A; DHAP; dihydroxyacetone phosphate; EGTA; ethylene glycol tetraacetic acid; FAD-GPDH; FAD-dependent (mitochondrial) glycerol-3-phosphate dehydrogenase; GP; glycerol-3-phosphate; GPS; glycerol-3-phosphate shuttle; HD; Huntington's disease; htt; _expQ; huntingtin with expanded CAG repeats; ICDH; NADH-isocitrate dehydrogenase; IF; ₁; inhibitor protein; IMS; intermembrane space; α-KGDH; α-ketoglutarate dehydrogenase; α-KG; α-ketoglutarate; MAS; malate–aspartate shuttle; PT; permeability transition; PTP; permeability transition pore; PDH; pyruvate dehydrogenase; PDHC; pyruvate dehydrogenase complex; State 3; _glu/mal; maximum respiration for the substrates glutamate plus malate; VDAC; voltage dependent anion channel; WT; wild typeOxidative phosphorylation; Regulation; Intra; mitochondrial calcium; Extra; mitochondrial calcium; Glutamate respiration; Aralar; Pyruvate dehydrogenase; α-Ketoglutarate dehydrogenase; Isocitrate dehydrogenase; ATP-Mg/Pi carrier; FAD-glycerol-3-phosphate dehydrogenase; Ca; ²⁺; uniporter; F; ₀; F; ₁; ATPase; Porin; Permeability transition pore; Transgenic Huntington rat, R6/2 mice

Alterations in the mitochondrial regulatory pathways constituted by the nuclear co-factors PGC-1α or PGC-1β and mitofusin 2 in skeletal muscle in type 2 diabetes by Antonio Zorzano; Hernandez-Alvarez María Isabel Hernández-Alvarez; Palacin Manuel Palacín; Geltrude Mingrone (pp. 1028-1033).

Muscle mitochondrial metabolism is regulated by a number of factors, many of which are responsible for the transcription of nuclear genes encoding mitochondrial proteins such as PPARδ, PGC-1α or PGC-1β. Recent evidence indicates that proteins participating in mitochondrial dynamics also regulate mitochondrial metabolism. Thus, in cultured cells the mitochondrial fusion protein mitofusin 2 (Mfn2) stimulates respiration, substrate oxidation and the expression of subunits involved in respiratory complexes. Mitochondrial dysfunction has been reported in skeletal muscle of type 2 diabetic patients. Reduced mitochondrial mass and defective activity has been proposed to explain this dysfunction. Alterations in mitochondrial metabolism may be crucial to account for some of the pathophysiological traits that characterize type 2 diabetes. Skeletal muscle of type 2 diabetic patients shows reduced expression of PGC-1α, PGC-1β, and Mfn2. In addition, a differential response to bilio-pancreatic diversion-induced weight loss in non-diabetic and type 2 diabetic patients has been reported. While non-diabetic morbidly obese subjects showed an increased expression of genes encoding Mfn2, PGC-1α, PGC-1β, PPARδ or SIRT1 in response to bariatric surgery-induced weight loss, no effect was detected in type 2 diabetic patients. These observations suggest the existence of a heritable component responsible for the abnormal control of the expression of genes encoding for modulators of mitochondrial biogenesis/metabolism, and which may participate in the development of the disease.

Keywords: Mitochondrial regulatory pathway; Skeletal muscle; Type 2 diabetes; Mitofusin; Insulin resistance

Detection and manipulation of mitochondrial reactive oxygen species in mammalian cells by Marleen Forkink; Jan A.M. Smeitink; Roland Brock; Peter H.G.M. Willems; Werner J.H. Koopman (pp. 1034-1044).

Reactive oxygen species (ROS) are formed upon incomplete reduction of molecular oxygen (O₂) as an inevitable consequence of mitochondrial metabolism. Because ROS can damage biomolecules, cells contain elaborate antioxidant defense systems to prevent oxidative stress. In addition to their damaging effect, ROS can also operate as intracellular signaling molecules. Given the fact that mitochondrial ROS appear to be only generated at specific sites and that particular ROS species display a unique chemistry and have specific molecular targets, mitochondria-derived ROS might constitute local regulatory signals. The latter would allow individual mitochondria to auto-regulate their metabolism, shape and motility, enabling them to respond autonomously to the metabolic requirements of the cell. In this review we first summarize how mitochondrial ROS can be generated and removed in the living cell. Then we discuss experimental strategies for (local) detection of ROS by combining chemical or proteinaceous reporter molecules with quantitative live cell microscopy. Finally, approaches involving targeted pro- and antioxidants are presented, which allow the local manipulation of ROS levels.

Keywords: Abbreviations; α-KGDH; α-ketoglutarate dehydrogenase; MIM; mitochondrial inner membrane; MOM; mitochondrial outer membrane; RET; reverse electron transfer; ROI; region of interest; t; _1/2; half-life; TPP; ⁺; triphenylphosphoniumHydroethidine; Redox-sensitive GFP; cpYFP; Lipid peroxidation; MitoQ

The many faces of the mitochondrial TIM23 complex by Dejana Mokranjac; Walter Neupert (pp. 1045-1054).

The TIM23 complex in the inner membrane of mitochondria mediates import of essentially all matrix proteins and a large number of inner membrane proteins. Here we present an overview on the latest insights into the structure and function of this remarkable molecular machine.

Keywords: TIM23 complex; Protein translocase; Mitochondria; Protein sorting; Targeting signals

The many faces of the mitochondrial TIM23 complex by Dejana Mokranjac; Walter Neupert (pp. 1045-1054).

Keywords: TIM23 complex; Protein translocase; Mitochondria; Protein sorting; Targeting signals

The many faces of the mitochondrial TIM23 complex by Dejana Mokranjac; Walter Neupert (pp. 1045-1054).

Keywords: TIM23 complex; Protein translocase; Mitochondria; Protein sorting; Targeting signals

Role of calcineurin, hnRNPA2 and Akt in mitochondrial respiratory stress-mediated transcription activation of nuclear gene targets by Manti Guha; Weigang Tang; Neal Sondheimer; Narayan G. Avadhani (pp. 1055-1065).

Pathophysiological conditions causing mitochondrial dysfunction and altered transmembrane potential (∆ψm) initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a variety of mammalian cells. An increase in the cytosolic Ca²⁺ [Ca²⁺]_c as part of this signaling cascade activates Ca²⁺ responsive phosphatase, calcineurin (Cn). Activation of IGF1R accompanied by increased glycolysis, invasiveness, and resistance to apoptosis is a phenotypic hallmark of C2C12 skeletal muscle cells subjected to this stress. The signaling is associated with activation and increased nuclear translocation of a number of transcription factors including a novel NFκB (cRel:p50) pathway, NFAT, CREB and C/EBPδ. This culminates in the upregulation of a number of nuclear genes including Cathepsin L, RyR1, Glut4 and Akt1. We observed that stress regulated transcription activation of nuclear genes involves a cooperative interplay between NFκB (cRel:p50), C/EBPδ, CREB, and NFAT. Our results show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein, hnRNPA2 as a transcriptional coactivator. We report here that mitochondrial stress leads to induced expression and activation of serine threonine kinase Akt1. Interestingly, we observe that Akt1 phosphorylates hnRNPA2 under mitochondrial stress conditions, which is a crucial step for the recruitment of this coactivator to the stress target promoters and culmination in mitochondrial stress-mediated transcription activation of target genes. We propose that mitochondrial stress plays an important role in tumor progression and emergence of invasive phenotypes.

Keywords: Mitochondrial respiratory stress; Retrograde signaling; Calcineurin activation; Akt1 activation; hnRNPA2 as a coactivator

RNA turnover in human mitochondria: More questions than answers? by Lukasz S. Borowski; Roman J. Szczesny; Lien K. Brzezniak; Piotr P. Stepien (pp. 1066-1070).

Protein complexes responsible for RNA degradation play important role in three key aspects of RNA metabolism: they control stability of physiologically functional transcripts, remove the unnecessary RNA processing intermediates and destroy aberrantly formed RNAs. In mitochondria the post-transcriptional events seem to play a major role in regulation of gene expression, therefore RNA turnover is of particular importance. Despite many years of research, the details of this process are still a challenge. This review summarizes emerging landscape of interplay between the Suv3p helicase (SUPV3L1, Suv3), poly(A) polymerase and polynucleotide phosphorylase in controlling RNA degradation in human mitochondria.

Keywords: Abbreviations; mtDNA; mitochondrial DNA; mtRNA; mitochondrial RNA; IMS; intermembrane space; PNPase; polynucleotide phosphorylase; mtPAP; mitochondrial poly(A) polymerase; hSuv3p; human Suv3 helicaseMitochondrion; RNA degradation; RNA surveillance; Polynucleotide phosphorylase (PNPase); Poly(A) polymerase (PAP); Suv3 helicase (SUPV3L1, hSuv3p)

RNA turnover in human mitochondria: More questions than answers? by Lukasz S. Borowski; Roman J. Szczesny; Lien K. Brzezniak; Piotr P. Stepien (pp. 1066-1070).

ATP-dependent proteases in biogenesis and maintenance of plant mitochondria by Hanna Janska; Janusz Piechota; Malgorzata Kwasniak (pp. 1071-1075).

ATP-dependent proteases from three families have been identified experimentally in Arabidopsis mitochondria: four FtsH proteases (AtFtsH3, AtFtsH4, AtFtsH10, and AtFtsH11), two Lon proteases (AtLon1 and AtLon4), and one Clp protease (AtClpP2 with regulatory subunit AtClpX). In this review we discuss their submitochondrial localization, expression profiles and proposed functions, with special emphasis on their impact on plant growth and development. The best characterized plant mitochondrial ATP-dependent proteases are AtLon1 and AtFtsH4. It has been proposed that AtLon1 is necessary for proper mitochondrial biogenesis during seedling establishment, whereas AtFtsH4 is involved in maintaining mitochondrial homeostasis late in rosette development under short-day photoperiod.

Keywords: ATP-dependent protease; AAA protease; FtsH; Clp; Lon; Oxidative phosphorylation; Plant mitochondria; Arabidopsis thaliana

ATP-dependent proteases in biogenesis and maintenance of plant mitochondria by Hanna Janska; Janusz Piechota; Malgorzata Kwasniak (pp. 1071-1075).

Keywords: ATP-dependent protease; AAA protease; FtsH; Clp; Lon; Oxidative phosphorylation; Plant mitochondria; Arabidopsis thaliana

ATP-dependent proteases in biogenesis and maintenance of plant mitochondria by Hanna Janska; Janusz Piechota; Malgorzata Kwasniak (pp. 1071-1075).

Keywords: ATP-dependent protease; AAA protease; FtsH; Clp; Lon; Oxidative phosphorylation; Plant mitochondria; Arabidopsis thaliana

The organellar peptidasome, PreP: A journey from Arabidopsis to Alzheimer's disease by Elzbieta Glaser; Nyosha Alikhani (pp. 1076-1080).

The novel peptidasome, called presequence protease, PreP, was originally identified and characterized in Arabidopsis thaliana as a mitochondrial matrix and chloroplast stroma localized metalloprotease. PreP has a function as the organellar peptide clearing protease and is responsible for degrading free targeting peptides and also other unstructured peptides up to 65 amino acid residues that might be toxic to organellar functions. PreP contains an inverted Zn-binding motif and belongs to the pitrilysin protease family. The crystal structure of AtPreP refined at 2.1Å demonstrated a unique totally enclosed large cavity of 10000Å³ that opens and closes in response to peptide binding, revealing a novel catalytic mechanism for proteolysis. Homologues of PreP have been found in yeast and human mitochondria. Interestingly, the human PreP, hPreP, is the protease that is responsible for clearing the human brain mitochondria from the toxic amyloid-β peptide (Aβ) associated with Alzheimer's disease (AD). Accumulation of Aβ has been shown in the brain mitochondria from AD patients and mutant transgenic mice overexpressing Aβ. Here, we present a review of our present knowledge on structural and functional characteristics of PreP and discuss its mitochondrial Aβ-degrading activity in the human brain mitochondria in relation to AD.

Keywords: Mitochondria; Presequence protease; Peptide degradation; Amyloid-β peptide; Alzheimer's disease; Targeting peptide

The organellar peptidasome, PreP: A journey from Arabidopsis to Alzheimer's disease by Elzbieta Glaser; Nyosha Alikhani (pp. 1076-1080).

Keywords: Mitochondria; Presequence protease; Peptide degradation; Amyloid-β peptide; Alzheimer's disease; Targeting peptide

The organellar peptidasome, PreP: A journey from Arabidopsis to Alzheimer's disease by Elzbieta Glaser; Nyosha Alikhani (pp. 1076-1080).

Keywords: Mitochondria; Presequence protease; Peptide degradation; Amyloid-β peptide; Alzheimer's disease; Targeting peptide

Human mitochondrial mRNAs—like members of all families, similar but different by Richard J. Temperley; Mateusz Wydro; Robert N. Lightowlers; Zofia M. Chrzanowska-Lightowlers (pp. 1081-1085).

The messenger RNAs containing the thirteen protein coding sequences of the human mitochondrial genome have frequently been regarded as a single functional category, alike in arrangement and hence in mode of expression. The “generic” mitochondrial mRNA is perceived as having (i) an arrangement within the polycistronic unit that permits its liberation following mt-tRNA processing, (ii) no 5′ cap structure or introns, (iii) essentially no untranslated regions, and (iv) a poly(A) tail of approximately fifty nucleotides that is required in part to complete the termination codon. Closer inspection reveals that only two molecules fit this pattern. This article examines the extent to which human mitochondrial mRNA species differ from one another.

Keywords: Abbreviations; mt-mRNA; mitochondrial messenger RNA; mtDNA; mitochondrial genome; ORF; open reading frame; UTR; untranslated region; nt; nucleotideMitochondria; RNA; Poly(A); Processing; Transcript; Translation; Termination

Human mitochondrial mRNAs—like members of all families, similar but different by Richard J. Temperley; Mateusz Wydro; Robert N. Lightowlers; Zofia M. Chrzanowska-Lightowlers (pp. 1081-1085).

Maintenance and expression of the S. cerevisiae mitochondrial genome—From genetics to evolution and systems biology by Kamil A. Lipinski; Aneta Kaniak-Golik; Pawel Golik (pp. 1086-1098).

As a legacy of their endosymbiotic eubacterial origin, mitochondria possess a residual genome, encoding only a few proteins and dependent on a variety of factors encoded by the nuclear genome for its maintenance and expression. As a facultative anaerobe with well understood genetics and molecular biology, Saccharomyces cerevisiae is the model system of choice for studying nucleo-mitochondrial genetic interactions. Maintenance of the mitochondrial genome is controlled by a set of nuclear-coded factors forming intricately interconnected circuits responsible for replication, recombination, repair and transmission to buds. Expression of the yeast mitochondrial genome is regulated mostly at the post-transcriptional level, and involves many general and gene-specific factors regulating splicing, RNA processing and stability and translation. A very interesting aspect of the yeast mitochondrial system is the relationship between genome maintenance and gene expression. Deletions of genes involved in many different aspects of mitochondrial gene expression, notably translation, result in an irreversible loss of functional mtDNA. The mitochondrial genetic system viewed from the systems biology perspective is therefore very fragile and lacks robustness compared to the remaining systems of the cell. This lack of robustness could be a legacy of the reductive evolution of the mitochondrial genome, but explanations involving selective advantages of increased evolvability have also been postulated.

Keywords: Mitochondria; Yeast; Gene expression; RNA processing; Systems biology; Evolution

Maintenance and expression of the S. cerevisiae mitochondrial genome—From genetics to evolution and systems biology by Kamil A. Lipinski; Aneta Kaniak-Golik; Pawel Golik (pp. 1086-1098).

Keywords: Mitochondria; Yeast; Gene expression; RNA processing; Systems biology; Evolution

Maintenance and expression of the S. cerevisiae mitochondrial genome—From genetics to evolution and systems biology by Kamil A. Lipinski; Aneta Kaniak-Golik; Pawel Golik (pp. 1086-1098).

Keywords: Mitochondria; Yeast; Gene expression; RNA processing; Systems biology; Evolution

Evolution and disease converge in the mitochondrion by D. Mishmar; I. Zhidkov (pp. 1099-1104).

Mitochondrial DNA (mtDNA) mutations are long known to cause diseases but also underlie tremendous population divergence in humans. It was assumed that the two types of mutations differ in one major trait: functionality. However, evidence from disease association studies, cell culture and animal models support the functionality of common mtDNA genetic variants, leading to the hypothesis that disease-causing mutations and mtDNA genetic variants share considerable common features. Here we provide evidence showing that the two types of mutations obey the rules of evolution, including random genetic drift and natural selection. This similarity does not only converge at the principle level; rather, disease-causing mutations could recapitulate the ancestral DNA sequence state. Thus, the very same mutations could either mark ancient evolutionary changes or cause disease.

Keywords: Disease; Evolution; Mitochondria; Mitochondrial DNA (mtDNA); Mutation rate; Variant; Haplogroup

Evolution and disease converge in the mitochondrion by D. Mishmar; I. Zhidkov (pp. 1099-1104).

Keywords: Disease; Evolution; Mitochondria; Mitochondrial DNA (mtDNA); Mutation rate; Variant; Haplogroup

Evolution and disease converge in the mitochondrion by D. Mishmar; I. Zhidkov (pp. 1099-1104).

Keywords: Disease; Evolution; Mitochondria; Mitochondrial DNA (mtDNA); Mutation rate; Variant; Haplogroup

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase by Roza Kucharczyk; Nahia Ezkurdia; Elodie Couplan; Vincent Procaccio; Sharon H. Ackerman; Marc Blondel; Jean-Paul di Rago (pp. 1105-1112).

Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F₁F_O-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F₁F_O complex to work in the reverse mode, i.e. F₁-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F₁, Atp9p-ring, unassembled α-F₁ subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.

Keywords: ATP synthase; Mitochondrial disease; Leigh syndrome; NARP syndrome; Yeast; Mitochondrial DNA

Cyclophilin D in mitochondrial pathophysiology by Valentina Giorgio; Maria Eugenia Soriano; Emy Basso; Elena Bisetto; Giovanna Lippe; Michael A. Forte; Paolo Bernardi (pp. 1113-1118).

Cyclophilins are a family of peptidyl-prolyl cis–trans isomerases whose enzymatic activity can be inhibited by cyclosporin A. Sixteen cyclophilins have been identified in humans, and cyclophilin D is a unique isoform that is imported into the mitochondrial matrix. Here we shall (i) review the best characterized functions of cyclophilin D in mitochondria, i.e. regulation of the permeability transition pore, an inner membrane channel that plays an important role in the execution of cell death; (ii) highlight new regulatory interactions that are emerging in the literature, including the modulation of the mitochondrial F1FO ATP synthase through an interaction with the lateral stalk of the enzyme complex; and (iii) discuss diseases where cyclophilin D plays a pathogenetic role that makes it a suitable target for pharmacologic intervention.

Keywords: Abbreviations; ANT; adenine nucleotide translocator; Cs; cyclosporin; CyP; cyclophilin; FKBP; FK506-binding proteins; PPIase; peptidyl-prolyl; cis–trans; isomerase; PTP; permeability transition poreCyclophilin; Mitochondria; ATP synthase; Permeability transition; Cyclosporin A

Cyclophilin D in mitochondrial pathophysiology by Valentina Giorgio; Maria Eugenia Soriano; Emy Basso; Elena Bisetto; Giovanna Lippe; Michael A. Forte; Paolo Bernardi (pp. 1113-1118).

Genotype–phenotype correlations in Leber hereditary optic neuropathy by Katarzyna Tońska; Agata Kodroń; Ewa Bartnik (pp. 1119-1123).

Leber hereditary optic neuropathy (LHON), acute or subacute vision loss due to retinal ganglion cell death which in the long run leads to optic nerve atrophy is one of the most widely studied maternally inherited diseases caused by mutations in mitochondrial DNA. Although three common mutations, 11778G>A, 14484T>C or 3460G>A are responsible for over 90% of cases and affect genes encoding complex I subunits of the respiratory chain, their influence on bioenergetic properties of the cell is marginal and cannot fully explain the pathology of the disease. The following chain of events was proposed, based on biochemical and anatomical properties of retinal ganglion cells whose axons form the optic nerve: mitochondrial DNA mutations increase reactive oxygen species production in these sensitive cells, leading to caspase-independent apoptosis. As LHON is characterized by low penetrance and sex bias (men are affected about 5 times more frequently than women) the participation of the other factors—genetic and environmental—beside mtDNA mutations was studied. Mitochondrial haplogroups and smoking are some of the factors involved in the complex etiology of this disease.

Keywords: Leber hereditary optic neuropathy; Optic nerve; Mitochondrial DNA mutation; Apoptosis; ROS

Genotype–phenotype correlations in Leber hereditary optic neuropathy by Katarzyna Tońska; Agata Kodroń; Ewa Bartnik (pp. 1119-1123).

Keywords: Leber hereditary optic neuropathy; Optic nerve; Mitochondrial DNA mutation; Apoptosis; ROS

Genotype–phenotype correlations in Leber hereditary optic neuropathy by Katarzyna Tońska; Agata Kodroń; Ewa Bartnik (pp. 1119-1123).

Keywords: Leber hereditary optic neuropathy; Optic nerve; Mitochondrial DNA mutation; Apoptosis; ROS

Knockdown of F₁ epsilon subunit decreases mitochondrial content of ATP synthase and leads to accumulation of subunit c by Havličkova Vendula Havlíčková; Kaplanova Vilma Kaplanová; Nůskova Hana Nůsková; Zdeněk Drahota; Josef Houštěk (pp. 1124-1129).

The subunit ε of mitochondrial ATP synthase is the only F₁ subunit without a homolog in bacteria and chloroplasts and represents the least characterized F₁ subunit of the mammalian enzyme. Silencing of the ATP5E gene in HEK293 cells resulted in downregulation of the activity and content of the mitochondrial ATP synthase complex and of ADP-stimulated respiration to approximately 40% of the control. The decreased content of the ε subunit was paralleled by a decrease in the F₁ subunits α and β and in the F_o subunits a and d while the content of the subunit c was not affected. The subunit c was present in the full-size ATP synthase complex and in subcomplexes of 200–400kDa that neither contained the F₁ subunits, nor the F_o subunits. The results indicate that the ε subunit is essential for the assembly of F₁ and plays an important role in the incorporation of the hydrophobic subunit c into the F₁-c oligomer rotor of the mitochondrial ATP synthase complex.

Keywords: Abbreviations; DDM; dodecyl maltoside; F; ₁; catalytic part of ATP synthase; F; _o; membrane-embedded part of ATP synthaseMitochondria; ATP synthase; Epsilon subunit; c subunit; Biogenesis

Mitochondrial dysfunction in autism spectrum disorders: Cause or effect? by Luigi Palmieri; Antonio M. Persico (pp. 1130-1137).

Autism Spectrum Disorders encompass severe developmental disorders characterized by variable degrees of impairment in language, communication and social skills, as well as by repetitive and stereotypic patterns of behaviour. Substantial percentages of autistic patients display peripheral markers of mitochondrial energy metabolism dysfunction, such as (a) elevated lactate, pyruvate, and alanine levels in blood, urine and/or cerebrospinal fluid, (b) serum carnitine deficiency, and/or (c) enhanced oxidative stress. These biochemical abnormalities are accompanied by highly heterogeneous clinical presentations, which generally (but by no means always) encompass neurological and systemic symptoms relatively unusual in idiopathic autistic disorder. In some patients, these abnormalities have been successfully explained by the presence of specific mutations or rearrangements in their mitochondrial or nuclear DNA. However, in the majority of cases, abnormal energy metabolism cannot be immediately linked to specific genetic or genomic defects. Recent evidence from post-mortem studies of autistic brains points toward abnormalities in mitochondrial function as possible downstream consequences of dysreactive immunity and altered calcium (Ca²⁺) signalling.

Keywords: Abbreviations; AGC; aspartate/glutamate carrier; ASD; autism spectrum disorders; CK; creatine kinase; CNS; central nervous system; CNV; copy number variant; GSH; reduced glutathione; GSSG; oxidized glutathione; MDA; malonyldialdehyde; OXPHOS; oxidative phosphorylation; ROS; reactive oxigen species; SAH; H-adenosylhomocysteine; SAM; S-adenosylmethionineAutism; Mitochondria; Aspartate-glutamate carrier; Calcium signalling; Oxidative stress; Immune dysfunction

Mitochondrial dysfunction in autism spectrum disorders: Cause or effect? by Luigi Palmieri; Antonio M. Persico (pp. 1130-1137).

Diminished NADPH transhydrogenase activity and mitochondrial redox regulation in human failing myocardium by Freya L. Sheeran; Rydstrom Jan Rydström; Mikhail I. Shakhparonov; Nikolay B. Pestov; Salvatore Pepe (pp. 1138-1148).

Although the functional role of nicotinamide nucleotide transhydrogenase (Nnt) remains to be fully elucidated, there is strong evidence that Nnt plays a critical part in mitochondrial metabolism by maintaining a high NADPH-dependant GSH/GSSG ratio, and thus the control of cellular oxidative stress. Using real-time PCR, spectrophotometric and western blotting techniques, we sought to determine the presence, abundance and activity level of Nnt in human heart tissues and to discern whether these are altered in chronic severe heart failure. Left ventricular levels of the NNT gene and protein expression did not differ significantly between the non-failing donor (NF) and heart failure (HF) group. Notably, compared to NF, Nnt activity rates in the HF group were 18% lower, which coincided with significantly higher levels of oxidized glutathione, lower glutathione reductase activity, lower NADPH and a lower GSH/GSSG ratio. In the failing human heart a partial loss of Nnt activity adversely impacts NADPH-dependent enzymes and the capacity to maintain membrane potential, thus contributing to a decline in bioenergetic capacity, redox regulation and antioxidant defense, exacerbating oxidative damage to cellular proteins.

Keywords: NADPH transhydrogenase; Mitochondria; Glutathione; Heart failure; Oxidative stress; Myocardium; Human

Assembly factors and ATP-dependent proteases in cytochrome c oxidase biogenesis by Lukas Stiburek; Jiri Zeman (pp. 1149-1158).

Eukaryotic cytochrome c oxidase (CcO), the terminal enzyme of the energy-transducing mitochondrial electron transport chain is a hetero-oligomeric, heme–copper oxidase complex composed of both mitochondrially and nuclear-encoded subunits. It is embedded in the inner mitochondrial membrane where it couples the transfer of electrons from reduced cytochrome c to molecular oxygen with vectorial proton translocation across the membrane. The biogenesis of CcO is a complicated sequential process that requires numerous specific accessory proteins, so-called assembly factors, which include translational activators, translocases, molecular chaperones, copper metallochaperones and heme a biosynthetic enzymes. Besides these CcO-specific protein factors, the correct biogenesis of CcO requires an even greater number of proteins with much broader substrate specificities. Indeed, growing evidence indicates that mitochondrial ATP-dependent proteases might play an important role in CcO biogenesis. Out of the four identified energy-dependent mitochondrial proteases, three were shown to be directly involved in proteolysis of CcO subunits. In addition to their well-established protein-quality control function these oligomeric proteolytic complexes with chaperone-like activities may function as molecular chaperones promoting productive folding and assembly of subunit proteins. In this review, we summarize the current knowledge of the functional involvement of eukaryotic CcO-specific assembly factors and highlight the possible significance for CcO biogenesis of mitochondrial ATP-dependent proteases.

Keywords: Mitochondria; Cytochrome; c; oxidase; Assembly factor; SCO1; SCO2; SURF1; TACO1; OXA1L; ATP-dependent protease; YME1L; LON; i; -AAA; m; -AAA

Assembly factors and ATP-dependent proteases in cytochrome c oxidase biogenesis by Lukas Stiburek; Jiri Zeman (pp. 1149-1158).

Keywords: Mitochondria; Cytochrome; c; oxidase; Assembly factor; SCO1; SCO2; SURF1; TACO1; OXA1L; ATP-dependent protease; YME1L; LON; i; -AAA; m; -AAA

Assembly factors and ATP-dependent proteases in cytochrome c oxidase biogenesis by Lukas Stiburek; Jiri Zeman (pp. 1149-1158).

Keywords: Mitochondria; Cytochrome; c; oxidase; Assembly factor; SCO1; SCO2; SURF1; TACO1; OXA1L; ATP-dependent protease; YME1L; LON; i; -AAA; m; -AAA

Mitochondrial DNA deletions in mice in men: Substantia nigra is much less affected in the mouse by Xinhong Guo; Elena Kudryavtseva; Natalya Bodyak; Alexander Nicholas; Igor Dombrovsky; Deye Yang; Yevgenya Kraytsberg; David K. Simon; Konstantin Khrapko (pp. 1159-1162).

Mitochondrial DNA (mtDNA) deletions have been reported to accumulate to high levels in substantia nigra of older humans, and these mutations are suspected of causing age-related degeneration in this area. We have compared levels of mtDNA deletions in humans and mice and report here that levels of deletions in the mouse are very significantly lower than in humans. While human mtDNA from substantia nigra contained more than 5% of deleted molecules, mouse substantia nigra contained less than 0.5%. These results imply that mtDNA deletions are unlikely to play any significant role in of murine substantia nigra aging and further call for caution in using mouse models in studies of the role of mtDNA deletions in aging and neurodegeneration. On a more general note, these results support the view that critical targets of the various aging processes may differ significantly between distant species.

Keywords: Aging; Mutations; Brain; Human; Mice; Mitochondrial DNA

Complex III-dependent superoxide production of brain mitochondria contributes to seizure-related ROS formation by Dominika Malinska; Bogusz Kulawiak; Alexei P. Kudin; Richard Kovacs; Christine Huchzermeyer; Oliver Kann; Adam Szewczyk; Wolfram S. Kunz (pp. 1163-1170).

Brain seizure activity is characterised by intense activation of mitochondrial oxidative phosphorylation. This stimulation of oxidative phosphorylation is in the low magnesium model of seizure-like events accompanied by substantial increase in formation of reactive oxygen species (ROS). However, it has remained unclear which ROS-generating sites can be attributed to this phenomenon. Here, we report stimulatory effects of calcium ions and uncouplers, mimicking mitochondrial activation, on ROS generation of isolated rat and mouse brain mitochondria. Since these stimulatory effects were visible with superoxide sensitive dyes, but with hydrogen peroxide sensitive dyes only in the additional presence of SOD, we conclude that the complex redox properties of the ‘Qo’ center at respiratory chain complex III are very likely responsible for these observations. In accordance with this hypothesis redox titrations of the superoxide production of antimycin-inhibited submitochondrial particles with the succinate/fumarate redox couple confirmed for brain tissue a bell-shaped dependency with a maximal superoxide production rate at +10mV (pH=7.4). This reflects the complex redox properties of a semiquinone species which is the direct electron donor for oxygen reduction in complex III-dependent superoxide production. Therefore, we conclude that under conditions of increased energy load the complex III site can contribute to superoxide production of brain mitochondria, which might be relevant for epilepsy-related seizure activity.

Keywords: Abbreviations; ROS; reactive oxygen species; FMN; flavine mononucleotide; SOD; superoxide dismutase; SMP; submitochondrial particles; SQ; semiquinoneReactive oxygen species; Brain mitochondria; Epilepsy

Hypoxia and mitochondrial oxidative metabolism by Giancarlo Solaini; Alessandra Baracca; Giorgio Lenaz; Gianluca Sgarbi (pp. 1171-1177).

It is now clear that mitochondrial defects are associated with a large variety of clinical phenotypes. This is the result of the mitochondria's central role in energy production, reactive oxygen species homeostasis, and cell death. These processes are interdependent and may occur under various stressing conditions, among which low oxygen levels (hypoxia) are certainly prominent. Cells exposed to hypoxia respond acutely with endogenous metabolites and proteins promptly regulating metabolic pathways, but if low oxygen levels are prolonged, cells activate adapting mechanisms, the master switch being the hypoxia-inducible factor 1 (HIF-1). Activation of this factor is strictly bound to the mitochondrial function, which in turn is related with the oxygen level. Therefore in hypoxia, mitochondria act as [O₂] sensors, convey signals to HIF-1directly or indirectly, and contribute to the cell redox potential, ion homeostasis, and energy production. Although over the last two decades cellular responses to low oxygen tension have been studied extensively, mechanisms underlying these functions are still indefinite. Here we review current knowledge of the mitochondrial role in hypoxia, focusing mainly on their role in cellular energy and reactive oxygen species homeostasis in relation with HIF-1 stabilization. In addition, we address the involvement of HIF-1 and the inhibitor protein of F₁F₀ ATPase in the hypoxia-induced mitochondrial autophagy.

Keywords: Abbreviations; HIF-1; hypoxia-inducible factor 1; HIF; hypoxia-inducible factor; IF; ₁; ATP synthase natural inhibitor protein; F; ₁; F; ₀; ATPase; ATP synthase; COX; cytochrome; c; oxidase; Δ; Ψ; _m; electrical membrane potential of mitochondria; mtDNA; mitochondrial DNA; AMPK; AMP-activated protein kinase; PHD; prolyl hydroxylase; PDK1; pyruvate dehydrogenase kinase 1; BNIP3; Bcl-2/adenovirus E1B 19; kDa interacting protein 3; IEX-1; immediate early response gene X-1; NOS; nitric oxide synthaseMitochondrion; Oxidative phosphorylation; Hypoxia; ROS; Autophagy; F; ₁; F; ₀; ATPase; IF; ₁

Hypoxia and mitochondrial oxidative metabolism by Giancarlo Solaini; Alessandra Baracca; Giorgio Lenaz; Gianluca Sgarbi (pp. 1171-1177).

Gender-specific role of mitochondria in the vulnerability of 6-hydroxydopamine-treated mesencephalic neurons by Magdalena Misiak; Cordian Beyer; Susanne Arnold (pp. 1178-1188).

Many neurodegenerative diseases, such as Morbus Parkinson, exhibit a gender-dependency showing a higher incidence in men than women. Most of the neurodegenerative disorders involve either causally or consequently a dysfunction of mitochondria. Therefore, neuronal mitochondria may demonstrate a gender-specificity with respect to structural and functional characteristics of these organelles during toxic and degenerative processes. The application of 6-OHDA (6-hydroxydopamine) in vitro and in vivo represents a well-accepted experimental model of Parkinson's disease causing Parkinsonian symptoms. Besides the known effects of 6-OHDA on mitochondria and neuronal survivability, we aimed to demonstrate that the mitochondrial neurotoxin affects the morphology and survival of primary dopaminergic and non-dopaminergic neurons in the mesencephalon in a gender-specific manner by influencing the transcription of mitochondrial genes, ATP and reactive oxygen species production. Our data suggest that cell death in response to 6-OHDA is primarily caused due to increased oxidative stress which is more pronounced in male than in female mesencephalic neurons.

Keywords: Abbreviations; 18S rRNA; 18S ribosomal RNA; 6-OHDA; 6-hydroxy-dopamine; ATP; adenosine 5'-triphosphate; BSA; bovine serum albumin; cDNA; first strand complementary DNA; COX; cytochrome c oxidase; C; _t; threshold cycle; E; 17β-estradiol/estrogen; FCS; fetal calf serum; H; ₂; DCFDA AM; 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester); Hprt; hypoxanthine guanine phosphoribosyl transferase; LDH; lactate dehydrogenase; NBM; neurobasal medium; PBS; phosphate-buffered saline; PD; Parkinson's disease; PI; propidium iodide; qRT-PCR; quantitative real time PCR; ROS; reactive oxygen species; TH; tyrosine hydroxylaseParkinson's disease; Gender; Neuron; Mitochondria; Oxidative stress; Estrogen

Gender-specific role of mitochondria in the vulnerability of 6-hydroxydopamine-treated mesencephalic neurons by Magdalena Misiak; Cordian Beyer; Susanne Arnold (pp. 1178-1188).

Stearoyl-CoA desaturase and insulin signaling — What is the molecular switch? by Pawel Dobrzyn; Magdalena Jazurek; Agnieszka Dobrzyn (pp. 1189-1194).

Increasing evidence suggests that stearoyl-CoA desaturase (SCD), the rate-limiting enzyme of monounsaturated fatty acid biosynthesis, is an important factor in the pathogenesis of lipid-induced insulin resistance. Mice with a targeted disruption of the SCD1 gene have improved glucose tolerance compared to wild-type mice, despite lower fasting plasma insulin levels. Increased SCD activity has been found in insulin-resistant humans and animals, whereas SCD1 deficiency attenuates both diet- and genetically-induced impairment of insulin action. Phosphorylation of serine and threonine residues on insulin receptor, insulin receptor substrates (IRS1 and IRS2), and on Akt has been shown to be the major step in insulin signaling that is altered due to the lack of SCD1. In this review we discuss perturbations in cell signaling and lipid metabolism cascades in insulin-sensitive tissues due to SCD1 deficiency. In particular, we address the role of cellular signaling molecules including free fatty acids, ceramides, fatty acyl-CoAs, AMP-activated protein kinase, protein tyrosine phosphatase 1B as well as of membrane fluidity. While the precise mechanism of SCD action on insulin signaling remains to be clarified, current findings on SCD point to a very promising novel target for the treatment of insulin resistance.

Keywords: Abbreviations; FFA; free fatty acid; FA-CoA; fatty acyl-CoA; PTP-1B; protein tyrosine phosphatase 1B; IR; insulin receptor; IRS; insulin receptor substrate; GLUT4; glucose transporter 4; AMPK; AMP-activated protein kinase; CPT1; carnitine palmitoyltransferase 1; PPAR; peroxisome proliferator-activated receptor; SCD; stearoyl-CoA desaturase; MUFA; monounsaturated fatty acidInsulin resistance; Lipid metabolism; Ceramide; Protein tyrosine phosphatase 1B

Stearoyl-CoA desaturase and insulin signaling — What is the molecular switch? by Pawel Dobrzyn; Magdalena Jazurek; Agnieszka Dobrzyn (pp. 1189-1194).

Mitochondrial fatty acid synthesis and respiration by J. Kalervo Hiltunen; Kaija J. Autio; Melissa S. Schonauer; V.A. Samuli Kursu; Carol L. Dieckmann; Alexander J. Kastaniotis (pp. 1195-1202).

Recent studies have revealed that mitochondria are able to synthesize fatty acids in a malonyl-CoA/acyl carrier protein (ACP)-dependent manner. This pathway resembles bacterial fatty acid synthesis (FAS) type II, which uses discrete, nuclearly encoded proteins. Experimental evidence, obtained mainly through using yeast as a model system, indicates that this pathway is essential for mitochondrial respiratory function. Curiously, the deficiency in mitochondrial FAS cannot be complemented by inclusion of fatty acids in the culture medium or by products of the cytosolic FAS complex. Defects in mitochondrial FAS in yeast result in the inability to grow on nonfermentable carbon sources, the loss of mitochondrial cytochromes a/a3 and b, mitochondrial RNA processing defects, and loss of cellular lipoic acid. Eukaryotic FAS II generates octanoyl-ACP, a substrate for mitochondrial lipoic acid synthase. Endogenous lipoic acid synthesis challenges the hypothesis that lipoic acid can be provided as an exogenously supplied vitamin. Purified eukaryotic FAS II enzymes are catalytically active in vitro using substrates with an acyl chain length of up to 16 carbon atoms. However, with the exception of 3-hydroxymyristoyl-ACP, a component of respiratory complex I in higher eukaryotes, the fate of long-chain fatty acids synthesized by the mitochondrial FAS pathway remains an enigma. The linkage of FAS II genes to published animal models for human disease supports the hypothesis that mitochondrial FAS dysfunction leads to the development of disorders in mammals.

Keywords: Mitochondria; RNA processing; Respiration; Metabolism; Lipids; Lipoic acid

Mitochondrial fatty acid synthesis and respiration by J. Kalervo Hiltunen; Kaija J. Autio; Melissa S. Schonauer; V.A. Samuli Kursu; Carol L. Dieckmann; Alexander J. Kastaniotis (pp. 1195-1202).

Keywords: Mitochondria; RNA processing; Respiration; Metabolism; Lipids; Lipoic acid

Mitochondrial fatty acid synthesis and respiration by J. Kalervo Hiltunen; Kaija J. Autio; Melissa S. Schonauer; V.A. Samuli Kursu; Carol L. Dieckmann; Alexander J. Kastaniotis (pp. 1195-1202).

Keywords: Mitochondria; RNA processing; Respiration; Metabolism; Lipids; Lipoic acid

Cationic carriers of genetic material and cell death: A mitochondrial tale by A. Christy Hunter; S. Moein Moghimi (pp. 1203-1209).

Central to gene therapy technology has been the use of cationic polymers as vectors for DNA and RNA (polyfectins). These have been presumed to be safer than viral systems which, for example, have been found to switch on oncogenes. Two key polycations that have been intensively researched for use as synthetic vectors are poly(ethylenimine) and poly(l-lysine). A frequent stumbling block with these polyfectins is that long-term gene expression in cell lines has not been achieved. Recently it has transpired that both of these polycations can induce mitochondrially mediated apoptosis. It is the aim of this review to discuss the mechanisms behind the observed polycation toxicity including roles for little studied cellular organelles in the process such as the lysosome and endoplasmic reticulum.

Keywords: Mitochondria; Polycation; Apoptosis; Lysosome; Endolysosomotrophic agent; Gene therapy; Poly(ethylenimine); Poly(; l; -lysine); Bid; Gene transfection; Surfactant

Cationic carriers of genetic material and cell death: A mitochondrial tale by A. Christy Hunter; S. Moein Moghimi (pp. 1203-1209).

Keywords: Mitochondria; Polycation; Apoptosis; Lysosome; Endolysosomotrophic agent; Gene therapy; Poly(ethylenimine); Poly(; l; -lysine); Bid; Gene transfection; Surfactant

Cationic carriers of genetic material and cell death: A mitochondrial tale by A. Christy Hunter; S. Moein Moghimi (pp. 1203-1209).

Keywords: Mitochondria; Polycation; Apoptosis; Lysosome; Endolysosomotrophic agent; Gene therapy; Poly(ethylenimine); Poly(; l; -lysine); Bid; Gene transfection; Surfactant

Mitochondria generated nitric oxide protects against permeability transition via formation of membrane protein S-nitrosothiols by Ana Catarina R. Leite; Helena C.F. Oliveira; Fabiane L. Utino; Rafael Garcia; Luciane C. Alberici; Mariana P. Fernandes; Roger F. Castilho; Aníbal E. Vercesi (pp. 1210-1216).

Mitochondria generated nitric oxide (NO) regulates several cell functions including energy metabolism, cell cycling, and cell death. Here we report that the NO synthase inhibitors (L-NAME, L-NNA and L-NMMA) administered either in vitro or in vivo induce Ca²⁺-dependent mitochondrial permeability transition (MPT) in rat liver mitochondria via a mechanism independent on changes in the energy state of the organelle. MPT was determined by the occurrence of cyclosporin A sensitive mitochondrial membrane potential disruption followed by mitochondrial swelling and Ca²⁺ release. In in vitro experiments, the effect of NOS inhibitors was dose-dependent (1 to 50µM). In addition to cyclosporin A, L-NAME-induced MPT was sensitive to Mg²⁺ plus ATP, EGTA, and to a lower degree, to catalase and dithiothreitol. In contrast to L-NAME, its isomer D-NAME did not induce MPT. L-NAME-induced MPT was associated with a significant decrease in both the rate of NO generation and the content of mitochondrial S-nitrosothiol. Acute and chronic in vivo treatment with L-NAME also promoted MPT and decreased the content of mitochondrial S-nitrosothiol. SNAP (a NO donor) prevented L-NAME mediated MPT and reversed the decrease in the rate of NO generation and in the content of S-nitrosothiol. We propose that S-nitrosylation of critical membrane protein thiols by NO protects against MPT.

Keywords: Abbreviations; Alam; alamethicin; CsA; cyclosporin A; DAF-FM; (4-amino-5-methylamino-2′,7′-difluorofluorescein) diacetate; D-NAME; N; ^G; -Nitro-; d; -arginine methyl ester hydrochloride; DTT; dithiothreitol; EGTA; ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid tetrasodium salt; GSH; glutathione; L-NAME; N; ^G; -nitro-; l; -arginine methyl ester hydrochloride; L-NMMA; N; ^G; -methyl-; l; -arginine acetate salt; L-NNA; N; ⁵; -(Nitroamidino)-; l; -2,5-diaminopentanoic acid; MPT; mitochondrial permeability transition; NO; nitric oxide; NOS; nitric oxide synthase; PTP; permeability transition pore; RLM; rat liver mitochondria; RNS; reactive nitrogen species; ROS; reactive oxygen species; SNAP; S-nitroso-N-acetyl-; dl; -penicillamine; SOD; superoxide dismutaseMitochondrial permeability transition; Mitochondrial nitric oxide synthase; S-nitrosothiol; Protein S-nitrosylation; Nitric oxide synthase inhibitor

Cholesterol and peroxidized cardiolipin in mitochondrial membrane properties, permeabilization and cell death by Joan Montero; Montserrat Mari; Anna Colell; Albert Morales; Basanez Gorka Basañez; Carmen Garcia-Ruiz; Fernandez-Checa Jose C. Fernández-Checa (pp. 1217-1224).

Mitochondria are known to actively regulate cell death with the final phenotype of demise being determined by the metabolic and energetic status of the cell. Mitochondrial membrane permeabilization (MMP) is a critical event in cell death, as it regulates the degree of mitochondrial dysfunction and the release of intermembrane proteins that function in the activation and assembly of caspases. In addition to the crucial role of proapoptotic members of the Bcl-2 family, the lipid composition of the mitochondrial membranes is increasingly recognized to modulate MMP and hence cell death. The unphysiological accumulation of cholesterol in mitochondrial membranes regulates their physical properties, facilitating or impairing MMP during Bax and death ligand-induced cell death depending on the level of mitochondrial GSH (mGSH), which in turn regulates the oxidation status of cardiolipin. Cholesterol-mediated mGSH depletion stimulates TNF-induced reactive oxygen species and subsequent cardiolipin peroxidation, which destabilizes the lipid bilayer and potentiates Bax-induced membrane permeabilization. These data suggest that the balance of mitochondrial cholesterol to peroxidized cardiolipin regulates mitochondrial membrane properties and permeabilization, emerging as a rheostat in cell death.

Keywords: Cholesterol; Cardiolipin; Reactive oxygen species; Mitochondrial GSH; Cell death

The pivotal roles of mitochondria in cancer: Warburg and beyond and encouraging prospects for effective therapies by Saroj P. Mathupala; Young H. Ko; Peter L. Pedersen (pp. 1225-1230).

Tumors usurp established metabolic steps used by normal tissues for glucose utilization and ATP production that rely heavily on mitochondria and employ a route that, although involving mitochondria, includes a much greater dependency on glycolysis. First described by Otto Warburg almost nine decades ago , this aberrant phenotype becomes more pronounced with increased tumor malignancy . Thus, while maintaining their capacity for respiration, tumors “turn more parasitic” by enhancing their ability to scavenge glucose from their surroundings. With excess glucose at hand, tumors shunt their metabolic flux more toward glycolysis than do their normal cells of origin, a strategy that allows for their survival when oxygen is limiting while providing them a mechanism to poison their extra-cellular environment with acid, thus paving the way for invasion and metastasis. Significantly, tumors harness a crucial enzyme to regulate and support this destructive path—to entrap and channel glucose toward glycolysis. This enzyme is an isoform of hexokinase, referred to as hexokinase type II, and also in abbreviated form as HK-2 or HK II. Due to many-faceted molecular features at genetic, epigenetic, transcriptional, and enzymatic levels, including sub-cellular localization to mitochondria, HK-2 facilitates and promotes the high glycolytic tumor phenotype . Thus, HK-2 represents a pivotal model gene or enzyme that tumors “select for” during tumorigenesis in order to facilitate their destructive path. In this review, we examine the roles played by mitochondrial bound HK-2 within the context of the highly choreographed metabolic roulette of malignant tumors. Recent studies that outline how the aberrant glycolytic flux can be subverted toward a more “normal” metabolic phenotype, and how the glycolytic flux affects the tumor microenvironment to facilitate tumor dissemination are also described, including how these very features can be harnessed in new metabolic targeting strategies to selectively debilitate tumors.

Keywords: Cancer; Mitochondria; Warburg effect; Hexokinase 2; VDAC; 3-bromopyruvate

The pivotal roles of mitochondria in cancer: Warburg and beyond and encouraging prospects for effective therapies by Saroj P. Mathupala; Young H. Ko; Peter L. Pedersen (pp. 1225-1230).

Keywords: Cancer; Mitochondria; Warburg effect; Hexokinase 2; VDAC; 3-bromopyruvate

The pivotal roles of mitochondria in cancer: Warburg and beyond and encouraging prospects for effective therapies by Saroj P. Mathupala; Young H. Ko; Peter L. Pedersen (pp. 1225-1230).

Keywords: Cancer; Mitochondria; Warburg effect; Hexokinase 2; VDAC; 3-bromopyruvate

MAC and Bcl-2 family proteins conspire in a deadly plot by Laurent M. Dejean; Shin-Young Ryu; Sonia Martinez-Caballero; Oscar Teijido; Pablo M. Peixoto; Kathleen W. Kinnally (pp. 1231-1238).

Apoptosis is an elemental form of programmed cell death; it is fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Apoptosis is also involved in many pathologies including cancer, neurodegenerative diseases, aging, and infarcts. This cell death program is tightly regulated by Bcl-2 family proteins by controlling the formation of the mitochondrial apoptosis-induced channel or MAC. Assembly of MAC corresponds to permeabilization of the mitochondrial outer membrane, which is the so called commitment step of apoptosis. MAC provides the pathway through the mitochondrial outer membrane for the release of cytochrome c and other pro-apoptotic factors from the intermembrane space. While overexpression of anti-apoptotic Bcl-2 eliminates MAC activity, oligomers of the pro-apoptotic members Bax and/or Bak are essential structural component(s) of MAC. Assembly of MAC from Bax or Bak was monitored in real time by directly patch-clamping mitochondria with micropipettes containing the sentinel tBid, a direct activator of Bax and Bak. Herein, a variety of high affinity inhibitors of MAC (iMAC) that may prove to be crucial tools in mechanistic studies have recently been identified. This review focuses on characterization of MAC activity, its regulation by Bcl-2 family proteins, and a discussion of how MAC can be pharmacologically turned on or off depending on the pathology to be treated.

Keywords: Mitochondrial apoptosis-induced channel; MAC; Apoptosis; Cytochrome; c; Patch-clamp; Bcl-2

MAC and Bcl-2 family proteins conspire in a deadly plot by Laurent M. Dejean; Shin-Young Ryu; Sonia Martinez-Caballero; Oscar Teijido; Pablo M. Peixoto; Kathleen W. Kinnally (pp. 1231-1238).

Keywords: Mitochondrial apoptosis-induced channel; MAC; Apoptosis; Cytochrome; c; Patch-clamp; Bcl-2

MAC and Bcl-2 family proteins conspire in a deadly plot by Laurent M. Dejean; Shin-Young Ryu; Sonia Martinez-Caballero; Oscar Teijido; Pablo M. Peixoto; Kathleen W. Kinnally (pp. 1231-1238).

Keywords: Mitochondrial apoptosis-induced channel; MAC; Apoptosis; Cytochrome; c; Patch-clamp; Bcl-2

Ceramide channels and their role in mitochondria-mediated apoptosis by Marco Colombini (pp. 1239-1244).

A key, decision-making step in apoptosis is the release of proteins from the mitochondrial intermembrane space. Ceramide can self-assemble in the mitochondrial outer membrane to form large stable channels capable of releasing said proteins. Ceramide levels measured in mitochondria early in apoptosis are sufficient to form ceramide channels in the outer membrane. The channels are in dynamic equilibrium with non-conducting forms of ceramide in the membrane. This equilibrium can be strongly influenced by other sphingolipids and Bcl-2 family proteins. The properties of ceramide channels formed in a defined system, planar phospholipid membranes, demonstrate that proteins are not required for channel formation. In addition, experiments in the defined system reveal structural information. The results indicated that the channels are barrel-like structures whose staves are ceramide columns that span the membrane. Ceramide channels are good candidates for the protein release pathway that initiates the execution phase of apoptosis.

Keywords: Abbreviation; MOM; mitochondrial outer membraneChannel; Outer membrane; Mitochondrion; Bcl-2; Pore; Self-assembly

Ceramide channels and their role in mitochondria-mediated apoptosis by Marco Colombini (pp. 1239-1244).

Keywords: Abbreviation; MOM; mitochondrial outer membraneChannel; Outer membrane; Mitochondrion; Bcl-2; Pore; Self-assembly

Ceramide channels and their role in mitochondria-mediated apoptosis by Marco Colombini (pp. 1239-1244).

Keywords: Abbreviation; MOM; mitochondrial outer membraneChannel; Outer membrane; Mitochondrion; Bcl-2; Pore; Self-assembly

The dopamine-D2-receptor agonist ropinirole dose-dependently blocks the Ca²⁺-triggered permeability transition of mitochondria by Suhel Parvez; Kirstin Winkler-Stuck; Silvia Hertel; Schonfeld Peter Schönfeld; Detlef Siemen (pp. 1245-1250).

Ropinirole, an agonist of the post-synaptic dopamine D2-receptor, exerts neuroprotective activity. The mechanism is still under discussion. Assuming that this neuroprotection might be associated with inhibition of the apoptotic cascade underlying cell death, we examined a possible effect of ropinirole on the permeability transition pore (mtPTP) in the mitochondrial inner membrane. Using isolated rat liver mitochondria, the effect of ropinirole was studied on Ca²⁺-triggered large amplitude swelling, membrane depolarization and cytochrome c release. In addition, the effect of ropinirole on oxidation of added, membrane-impermeable NADH was investigated. The results revealed doubtlessly, that ropinirole can inhibit permeability transition. In patch-clamp experiments on mitoplasts, we show directly that ropinirole interacts with the mtPTP. Thus, ropinirole reversibly inhibits the opening of mtPTP with an IC₅₀ of 3.4µM and a Hill coefficient of 1.3. In both systems (i.e. energized mitochondria and mitoplasts) the inhibitory effect on permeability transition was attenuated by increasing concentrations of inorganic phosphate. In addition, we showed with antimycin A-treated mitochondria that ropinirole failed to suppress respiratory chain-linked reactive oxygen species release. In conclusion, our data suggest that the neuroprotective activity of ropinirole is due to the blockade of the Ca²⁺-triggered permeability transition.

Keywords: Abbreviations; CsA; cyclosporin A; Cyt; c; cytochrome; c; FCCP; carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; IC; ₅₀; inhibitor concentration causing 50% inhibition; mtPTP; mitochondrial permeability transition pore; PD; Parkinson's disease; RBM; rat brain mitochondria; RLM; rat liver mitochondria; ROS; reactive oxygen species; Pi; inorganic phosphate; Po; open probability; Δ; ψ; _m; mitochondrial membrane potentialParkinson's disease; Permeability transition; Apoptosis; Mitochondria; Ropinirole; Neuroprotection

Role of Kv1.3 mitochondrial potassium channel in apoptotic signalling in lymphocytes by Erich Gulbins; Nicola Sassi; Grassme Heike Grassmè; Mario Zoratti; Szabo Ildikò Szabò (pp. 1251-1259).

Mitochondria have been shown to play a pivotal role in apoptotic signalling in various cell types. We have recently reported that in lymphocytes the voltage-gated potassium channel Kv1.3, known to reside in the plasma membrane, is active also in the inner mitochondrial membrane. Upon induction of apoptosis, outer-membrane inserted Bax binds to and inhibits Kv1.3 resulting in hyperpolarization, an increase in reactive oxygen species production and cytochrome c release. In cells lacking Kv1.3 these events do not take place. Here, we present new data which further corroborates an important role of this channel in the sequence of events leading to Bax-induced cytochrome c release. Recombinant Kv1.3, when pre-incubated with Bax, prevents the actions of Bax at the level of mitochondria. Furthermore, we report the presence of Kv1.3 protein in mitochondria from PC3 and MCF-7 cancer cells, suggesting that this channel might play a role in the apoptotic signalling not only in lymphocytes but also in other cells.

Keywords: Abbreviations; CHO; Chinese hamster ovary cell line; CTLL-2; interleukin-2 dependent murine cytotoxic T lymphocyte; FITC; fluorescein isothiocyanate; GST; glutathione S-transferase; IMM; inner mitochondrial membrane; IP3R; inositol triphosphate receptor; MCF-7; human breast adenocarcinoma cell line; MPT; mitochondrial permeability transition; MgTx; Margatoxin; OMM; outer mitochondrial membrane; PC3; human prostate cancer cell line; PMCA; plasma membrane calcium ATP-ase; PTP; permeability transition pore; ROS; reactive oxygen species; SERCA; sarcoplasmic/endoplasmic reticulum calcium ATP-ase; ShK; Stichodactyla; toxin; TNF; tumor necrosis factor α; VDAC; voltage-dependent anion channelPotassium channel; Mitochondria; Apoptosis

Role of Kv1.3 mitochondrial potassium channel in apoptotic signalling in lymphocytes by Erich Gulbins; Nicola Sassi; Grassme Heike Grassmè; Mario Zoratti; Szabo Ildikò Szabò (pp. 1251-1259).

An investigation of the occurrence and properties of the mitochondrial intermediate-conductance Ca²⁺-activated K⁺ channel mtK_Ca3.1 by Nicola Sassi; Umberto De Marchi; Bernard Fioretti; Lucia Biasutto; Erich Gulbins; Fabio Franciolini; Szabo Ildikò Szabò; Mario Zoratti (pp. 1260-1267).

The mitochondrial intermediate-conductance Ca²⁺-activated K⁺ channel mtK_Ca3.1 has recently been discovered in the HCT116 colon tumor-derived cell line, which expresses relatively high levels of this protein also in the plasma membrane. Electrophysiological recordings revealed that the channel can exhibit different conductance states and kinetic modes, which we tentatively ascribe to post-translational modifications. To verify whether the localization of this channel in mitochondria might be a peculiarity of these cells or a more widespread feature we have checked for the presence of mtK_Ca3.1 in a few other cell lines using biochemical and electrophysiological approaches. It turned out to be present at least in some of the cells investigated. Functional assays explored the possibility that mtK_Ca3.1 might be involved in cell proliferation or play a role similar to that of the Shaker-type K_V1.3 channel in lymphocytes, which interacts with outer mitochondrial membrane-inserted Bax thereby promoting apoptosis (Szabò, I. et al., Proc. Natl. Acad Sci. USA 105 (2008) 14861–14866). A specific K_Ca3.1 inhibitor however did not have any detectable effect on cell proliferation or death.

Keywords: Abbreviations; BK; _Ca; big conductance Ca; ²⁺; -activated K; ⁺; -channel (K; _Ca; 1.1); Clotrimazole; 1-[(2-chlorophenyl)-diphenyl-methyl]imidazole; CsA; Cyclosporin A; DC-EBIO; 5,6-dichloro-1-ethyl-1,3-dihydro-2; H; -benzimidazol-2-one; DMEM; Dulbecco's modified Eagle medium; ER; endoplasmatic reticulum; GST; glutathione-S-transferase; HBSS; Hank's balanced saline solution; HCT116; human colon tumor 116; Hepes; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IK; _Ca; intermediate-conductance Ca; ²⁺; -activated K; ⁺; channel (K; _Ca; 3.1); IMM; inner mitochondrial membrane; I/R; ischemia/reperfusion; K; _ATP; ATP-sensitive K; ⁺; channel; MAM; mitochondria-associated membranes; MDR; multiple drug resistance; MEF; mouse embryonic fibroblast; MgTx; margatoxin; MPTP; mitochondrial permeability transition pore; MTT; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OMM; outer mitochondrial membrane; PACS; phosphofurin acidic cluster sorting; PBS; phosphate buffered saline; PKA; protein kinase A; PKC; protein kinase C; PM; plasma membrane; PMCA; plasma membrane Ca; ²⁺; -ATPase; RISK; reperfusion injury salvage kinases; RLM; rat liver mitochondria; ROS; reactive oxygen species; SERCA; sarcoplasmic/endoplasmic reticulum Ca; ²⁺; -ATPase; ShK; stichodactyla toxin; TASK-3; TWIK-related acid-sensitive K; ⁺; channel (K; _2P; 9.1); TRAM-34; [1-(2-chlorophenyl)diphenyl)methyl]-1; H; -pyrazole; TES; N; -tris(hydroxymethyl)methyl-2-aminoethanesulfonic acidInner mitochondrial membrane; IK(Ca); HeLa cells; Patch-clamp; Calcium; TRAM-34

Characterization of human VDAC isoforms: A peculiar function for VDAC3? by Vito De Pinto; Francesca Guarino; Andrea Guarnera; Angela Messina; Simona Reina; Flora M. Tomasello; Vanessa Palermo; Cristina Mazzoni (pp. 1268-1275).

VDACs are a family of pore-forming proteins mainly located in the mitochondrial outer membrane. In mammals three isoforms exist. In this work we review the information available about them with the addition of new results. We have compared the human VDACs transformed in a yeast strain lacking the endogenous porin. VDAC1 and 2 are able to complement the lack of porin in mitochondrial respiration and modulation of ROS. VDAC3 has a limited ability to support the mitochondrial respiration and has no influence in the control of ROS production. The over-expression of VDAC isoforms in wild type yeast strain led to a dramatic sensitivity to oxidative stress, especially for VDAC3, and a shorter lifespan in respiratory conditions. Real-time PCR comparison of the isoforms indicated that in HeLa cells VDAC1 is 10 times more abundant than VDAC2 and 100 times than VDAC3. The over-expression of any single isoform caused a 10 times increase of the transcripts of VDAC2 and VDAC3, while VDAC1 is not changed by the over-expression of the other isoforms. Models of VDAC2 and VDAC3 isoform structure showed that they could be made of a 19-strand β-barrel and an N-terminal sequence with variable features. In this work we show for the first time a functional characterization of VDAC3 in a cellular context.

Keywords: VDAC isoform; Saccharomyces cerevisiae; Mitochondria; VDAC model; ROS; Real-time PCR

Communication between mitochondria and nucleus: Putative role for VDAC in reduction/oxidation mechanism by Hanna Galganska; Andonis Karachitos; Malgorzata Wojtkowska; Olgierd Stobienia; Malgorzata Budzinska; Hanna Kmita (pp. 1276-1280).

Voltage dependent anion channel (VDAC) was identified in 1976 and since that time has been extensively studied. It is well known that VDAC transports metabolites across the outer mitochondrial membrane. The simple transport function is indispensable for proper mitochondria functions and, consequently for cell activity, and makes VDAC crucial for a range of cellular processes including ATP rationing, Ca²⁺ homeostasis and apoptosis execution. Here, we review recent data obtained for Saccharomyces cerevisiae cells used as a model system concerning the putative role of VDAC in communication between mitochondria and the nucleus. The S. cerevisiae VDAC isoform known as VDAC1 (termed here YVDAC) mediates the cytosol reduction/oxidation (redox) state that contributes to regulation of expression and activity of cellular proteins including proteins that participate in protein import into mitochondria and antioxidant enzymes. Simultaneously, copper-and-zinc-containing superoxide dismutase (CuZnSOD) plays an important role in controlling YVDAC activity and expression levels. Thus, it is proposed that VDAC constitutes an important component of a regulatory mechanism based on the cytosol redox state.

Keywords: VDAC; Superoxide anion; Cytosol redox state; Expression of mitochondrial proteins

Apoptosis is regulated by the VDAC1 N-terminal region and by VDAC oligomerization: release of cytochrome c, AIF and Smac/Diablo by Varda Shoshan-Barmatz; Nurit Keinan; Salah Abu-Hamad; Dalia Tyomkin; Lior Aram (pp. 1281-1291).

Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H₂O₂ or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2.

Keywords: Abbreviations; AIF; apoptosis-inducing factor; ANT; adenine nucleotide translocase; AzRu; Azido ruthenium; BRET2; bioluminescence resonance energy transfer; Cyto; c; cytochrome; c; DBC; DeepBlueC coelentrazine; DFDNB; 1,5-difluoro-2,4-dinitrobenzene; DIDS; 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; EGS; ethylene glycol-bis (succinimidylsuccinate); HK-I; hexokinase-I; OMM and IMM; outer and inner mitochondrial membranes; PDL; poly-; d; -lysine; STS; staurosporine; RuR; Ruthenium Red; Smac/Diablo; Second mitochondria activator of caspases; VDAC; voltage-dependent anion channelAIF; Apoptosis; Cytochrome; c; Mitochondria; Oligomerization; VDAC

Structure and evolution of mitochondrial outer membrane proteins of β-barrel topology by Kornelius Zeth (pp. 1292-1299).

Gram-negative bacteria are the ancestors of mitochondrial organelles. Consequently, both entities contain two surrounding lipid bilayers known as the inner and outer membranes. While protein synthesis in bacteria is accomplished in the cytoplasm, mitochondria import 90–99% of their protein ensemble from the cytosol in the opposite direction. Three protein families including Sam50, VDAC and Tom40 together with Mdm10 compose the set of integral β-barrel proteins embedded in the mitochondrial outer membrane in S. cerevisiae (MOM). The 16-stranded Sam50 protein forms part of the sorting and assembly machinery (SAM) and shows a clear evolutionary relationship to members of the bacterial Omp85 family. By contrast, the evolution of VDAC and Tom40, both adopting the same fold cannot be traced to any bacterial precursor. This finding is in agreement with the specific function of Tom40 in the TOM complex not existent in the enslaved bacterial precursor cell. Models of Tom40 and Sam50 have been developed using X-ray structures of related proteins. These models are analyzed with respect to properties such as conservation and charge distribution yielding features related to their individual functions.

Keywords: Abbreviations; MOM; mitochondrial outer membrane; TOM; translocase of the outer membrane; SAM; sorting and assembly machineryVDAC; Tom40; Sam50; Membrane protein structure

Structure and evolution of mitochondrial outer membrane proteins of β-barrel topology by Kornelius Zeth (pp. 1292-1299).

Keywords: Abbreviations; MOM; mitochondrial outer membrane; TOM; translocase of the outer membrane; SAM; sorting and assembly machineryVDAC; Tom40; Sam50; Membrane protein structure

Structure and evolution of mitochondrial outer membrane proteins of β-barrel topology by Kornelius Zeth (pp. 1292-1299).

Keywords: Abbreviations; MOM; mitochondrial outer membrane; TOM; translocase of the outer membrane; SAM; sorting and assembly machineryVDAC; Tom40; Sam50; Membrane protein structure

Modulation of intracellular chloride channels by ATP and Mg²⁺ by Viera Kominkova; Lubica Malekova; Zuzana Tomaskova; Peter Slezak; Adam Szewczyk; Karol Ondrias (pp. 1300-1312).

We report the effects of ATP and Mg²⁺ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels ( n=112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5–2mmol/l) modulated and/or inhibited the chloride channel activities ( n=38/112) in a concentration-dependent manner. The inhibition effect was irreversible ( n=5/93) or reversible ( n=15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels ( n=6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5–2mmol/l) without the presence of Mg²⁺ decreased the chloride channel current ( n=12/14), whereas Mg²⁺ significantly reversed the effect ( n=4/4). We suggest that ATP-intracellular chloride channel interactions and Mg²⁺ modulation of these interactions may regulate different physiological and pathological processes.

Keywords: Abbreviations; BLM; bilayer lipid membrane; P; -open; open probability of single channel; E; _rev; reversal potential; AMP-PNP; adenosine 5′-(β,γ-imido) triphosphate; DIDS; 4, 4′-diisothiocyanatostilbene-2, 2′-disulfonic acid disodium salt; PTP; permeability transition poreMitochondrial membrane; Lysosomes; Mitochondrial chloride channel; ATP; Mg; ²⁺; Bilayer lipid membrane

Modulation of intracellular chloride channels by ATP and Mg²⁺ by Viera Kominkova; Lubica Malekova; Zuzana Tomaskova; Peter Slezak; Adam Szewczyk; Karol Ondrias (pp. 1300-1312).

Simultaneous in vivo recording of prompt and delayed fluorescence and 820-nm reflection changes during drying and after rehydration of the resurrection plant Haberlea rhodopensis by Reto J. Strasser; Merope Tsimilli-Michael; Sheng Qiang; Vasilij Goltsev (pp. 1313-1326).

A new instrument (M-PEA), which measures simultaneously kinetics of prompt fluorescence (PF), delayed fluorescence (DF) and modulated light reflection at 820nm (MR), was used to screen dark-adapted leaves of the resurrection plant Haberlea rhodopensis during their progressive drying, down to 1% relative water content (RWC), and after their re-watering. This is the first investigation using M-PEA, which employs alternations of actinic light (627-nm peak, 5000μmol photons m−²s−¹) and dark intervals, where PF-MR and DF kinetics are respectively recorded, with the added advantages: (a) all kinetics are recorded with high time resolution (starting from 0.01ms), (b) the dark intervals' duration can be as short as 0.1ms, (c) actinic illumination can be interrupted at different times during the PF transient (recorded up to 300s), with the earliest interruption at 0.3ms. Analysis of the simultaneous measurements at different water-content-states of H. rhodopensis leaves allowed the comparison and correlation of complementary information on the structure/function of the photosynthetic machinery, which is not destroyed but only inactivated (reversibly) at different degrees; the comparison and correlation helped also to test current interpretations of each signal and advance their understanding. Our results suggest that the desiccation tolerance of the photosynthetic machinery in H. rhodopensis is mainly based on mechanism(s) that lead to inactivation of photosystem II reaction centres (transformation to heat sinks), triggered already by a small RWC decrease.

Keywords: Abbreviations; Chl; chlorophyll; DT; drying-time (of leaves); DF; delayed fluorescence; Fd; ferredoxin; FNR; ferredoxin-NADP; ⁺; reductase; F; _M; and; F; ₀; maximum and minimum fluorescence intensity, respectively; J and I; intermediate steps in the Chl; a; fluorescence rise (OJIP) appearing between; F; ₀; and; F; _M; at about 2 and 30; ms, respectively; LES; “light emitting state” (responsible for DF emission by PSII); MR; modulated reflection (here at 820; nm for the determination of P700 and PC oxido-reduction “modulated” refers to the light beam used); P700; Chl of PSI RC; PC; plastocyanin; PF; prompt fluorescence; OEC; oxygen-evolving complex; Pheo; pheophytine; PQ; plastoquinone; PQH; ₂; plastoquinol; PS; photosystem; Q; _A; and Q; _B; primary and secondary quinone electron acceptors of PS II; RC; reaction centre; RWC; relative water content (of leaves); Tyr; tyrosine. For abbreviations used in the JIP-test, see Table A1 in the Appendix Haberlea rhodopensis; Delayed fluorescence; JIP-test; Prompt fluorescence; Reflection changes at 820; nm; Resurrection plants

4Pi microscopy reveals an impaired three-dimensional mitochondrial network of pancreatic islet β-cells, an experimental model of type-2 diabetes by Dlaskova Andrea Dlasková; Tomáš Špaček; Šantorova Jitka Šantorová; Plecita-Hlavata Lydie Plecitá-Hlavatá; Berkova Zuzana Berková; František Saudek; Mark Lessard; Joerg Bewersdorf; Petr Ježek (pp. 1327-1341).

Insulin production in pancreatic β-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the β-cells' glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic β-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an ∼7-fold improved axial resolution (∼100nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary β-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redox-sensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that β-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of β-cell condition, before their transplantations.

Keywords: Abbreviations; EM; electron microscopy; mRoGFP; mitochondrial matrix targeted redox-sensitive type 1 GFP; STED; stimulated emission depletion; T2DM; type-2 diabetes mellitus3D morphology of mitochondrial network; 4Pi microscopy; 3D image analysis; Pancreatic β-cell; Type-2 diabetes; Morphological diagnostic

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BBA - Bioenergetics (v.1797, #6-7)