BBA - Bioenergetics (v.1757, #9-10)

Supercomplexes and subcomplexes of mitochondrial oxidative phosphorylation by Ilka Wittig; Rosalba Carrozzo; Filippo M. Santorelli; Hermann Schägger (1066-1072).
Dimerization or oligomerization of ATP synthase has been proposed to play an important role for mitochondrial cristae formation and to be involved in regulating ATP synthase activity. We found comparable oligomycin-sensitive ATPase activity for monomeric and oligomeric ATP synthase suggesting that oligomerization/monomerization dynamics are not directly involved in regulating ATP synthase activity. Binding of the natural IF1 inhibitor protein has been shown to induce dimerization of F1-subcomplexes. This suggested that binding of IF1 might also dimerize holo ATP synthase, and possibly link dimerization and inhibition. Analyzing mitochondria of human rho zero cells that contain mitochondria but lack mitochondrial DNA, we identified three subcomplexes of ATP synthase: (i) F1 catalytic domain, (ii) F1-domain with bound IF1, and (iii) F1-c subcomplex with bound IF1 and a ring of subunits c. Since both IF1 containing subcomplexes were present in monomeric state and exhibited considerably reduced ATPase activity as compared to the third subcomplex lacking IF1, we postulate that inhibition and induction of dimerization of F1-subcomplexes by IF1 are independent events. F1-subcomplexes were also found in mitochondria of patients with specific mitochondrial disorders, and turned out to be useful for the clinical differentiation between various types of mitochondrial biosynthesis disorders. Supramolecular associations of respiratory complexes, the “respirasomes”, seem not to be the largest assemblies in the structural organization of the respiratory chain, as suggested by differential solubilization of mitochondria and electron microscopic analyses of whole mitochondria. We present a model for a higher supramolecular association of respirasomes into a “respiratory string”.
Keywords: Dimeric ATP synthase; Oligomeric ATP synthase; Supercomplex; Supramolecular organization; Respiratory chain; Respirasome;

Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially inactivated state, which can be activated by removal of tightly bound oxaloacetate (E.B. Kearney, et al., Biochem. Biophys. Res. Commun. 49 1115–1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the “malate-like intermediate” found in Shewanella Flavocytochrome c crystallized with fumarate (P. Taylor, et al., Nat. Struct. Biol. 6 1108–1112) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Y.O. Belikova, et al., Biochim. Biophys. Acta 936 1–9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate.
Keywords: Membrane protein; Protein crystallography; X-ray structure; Electron transport; Redox enzyme; Succinate dehydrogenase;

The menaquinol-oxidizing cytochrome bc complex from Thermus thermophilus: Protein domains and subunits by Daniela Mooser; Oliver Maneg; Fraser MacMillan; Francesco Malatesta; Tewfik Soulimane; Bernd Ludwig (1084-1095).
A recently resolved respiratory complex III, isolated from the extreme thermophile Thermus thermophilus, is discussed in terms of cofactor and subunit composition, and with respect to the origin of its protein modules. The four polypeptides, encoded by a single operon, share general homologies to canonical complexes both of the bc and b 6 f type, but exhibit some unexpected features as well. Evidence for high thermostability of the isolated protein and for its quinol substrate specificity is derived from EPR and kinetic measurements. A functional integration of this complex into an aerobic electron transfer scheme, connecting known dehydrogenase activities to the terminal oxidase branches of Thermus is outlined, as well as the specific principles of redox protein interactions prevailing at high temperature. Findings from this enzyme are linked to present knowledge on other menaquinol oxidizing bc complexes.
Keywords: Thermus thermophilus; Respiratory chain; Cytochrome bc; Complex III; Menaquinone; EPR; Electron transfer; Stopped flow kinetic;

Electron tunneling chains of mitochondria by Christopher C. Moser; Tammer A. Farid; Sarah E. Chobot; P. Leslie Dutton (1096-1109).
The single, simple concept that natural selection adjusts distances between redox cofactors goes a long way towards encompassing natural electron transfer protein design. Distances are short or long as required to direct or insulate promiscuously tunneling single electrons. Along a chain, distances are usually 14 Å or less. Shorter distances are needed to allow climbing of added energetic barriers at paired-electron catalytic centers in which substrate and the required number of cofactors form a compact cluster. When there is a short-circuit danger, distances between shorting centers are relatively long. Distances much longer than 14Å will support only very slow electron tunneling, but could act as high impedance signals useful in regulation. Tunneling simulations of the respiratory complexes provide clear illustrations of this simple engineering.
Keywords: Mitochondria; Cytochrome c oxidase cytochrome bc1; Succinate dehydrogenase; NADH-ubiquinone oxidoreductase; Electron and nuclear tunneling; Oxidoreduction catalytic transformation Energy conversion;

Proton pumping mechanism of bovine heart cytochrome c oxidase by Shinya Yoshikawa; Kazumasa Muramoto; Kyoko Shinzawa-Itoh; Hiroshi Aoyama; Tomitake Tsukihara; Kunitoshi Shimokata; Yukie Katayama; Hideo Shimada (1110-1116).
X-ray structures of bovine heart cytochrome c oxidase at 1.8/1.9 Å resolution in the oxidized/reduced states exhibit a redox coupled conformational change of an aspartate located near the intermembrane surface of the enzyme. The alteration of the microenvironment of the carboxyl group of this aspartate residue indicates the occurrence of deprotonation upon reduction of the enzyme. The residue is connected with the matrix surface of the enzyme by a hydrogen-bond network that includes heme a via its propionate and formyl groups. These X-ray structures provide evidence that proton pumping occurs through the hydrogen bond network and is driven by the low spin heme. The function of the aspartate is confirmed by mutation of the aspartate to asparagine. Although the amino acid residues of the hydrogen bond network and the structures of the low spin heme peripheral groups are not completely conserved amongst members of the heme-copper terminal oxidase superfamily, the existence of low spin heme and the hydrogen bond network suggests that the low spin heme provides the driving element of the proton-pumping process.
Keywords: Cytochrome c oxidase; Heme protein; Membrane protein; Proton pump; O2 reduction; Electron transfer; X-ray crystallography; Site-directed mutagenesis;

The K-pathway revisited: A computational study on cytochrome c oxidase by Anne Tuukkanen; Michael I. Verkhovsky; Liisa Laakkonen; Mårten Wikström (1117-1121).
Cytochrome c oxidase contains two established proton-conducting structures, the D- and K-pathways. The role of the K-pathway appears to be to conduct the first two protons to be used in water formation, which are taken up on reduction of the oxidized enzyme. Previous computational work has suggested that Lys(I)-319 is neutral over a large pH range and in various redox states. We have constructed oxidase models in different redox states using quantum-chemically derived charge parameters for the redox metal centers. The protonation behaviour of titratable sites in the two-subunit enzyme was defined by continuum electrostatics. The calculations reported here show substantial protonation of Lys(I)-319 at neutral pH once the stable X-ray crystallographic water molecule found immediately next to it is treated explicitly. The immediate structure of the Lys(I)-319 environment is independent of redox state, but the pK a value of this residue changes with the redox state of the binuclear heme a 3 /CuB site whenever that change is electrically uncompensated. Lys(I)-319 is also found to interact electrostatically with the conserved residue Glu(II)-62 in subunit II. These results are discussed in relation to the role of the K-pathway in oxidase function.
Keywords: Proton transfer; Electrostatic; Protein structure;

Single-electron photoreduction of the PM intermediate of cytochrome c oxidase by Sergey A. Siletsky; Dan Han; Sue Brand; Joel E. Morgan; Marian Fabian; Lois Geren; Francis Millett; Bill Durham; Alexander A. Konstantinov; Robert B. Gennis (1122-1132).
The PM  → F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The PM state of the oxidase was generated by exposing the oxidized enzyme to CO plus O2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a3 with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is ∼ 5-fold more rapid than the rate of electron transfer from heme a to heme a3 in the F → O transition, but is significantly slower than formation of the F state from the PR intermediate in the reaction of the fully reduced enzyme with O2 to form state F (70–90 μs). The ∼ 0.3 ms PM  → F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the PM  → F transition is generated much later, with a time constant of 1.3 ms. In addition, the PM  → F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with τ values of 0.18 and 0.85 ms.
Keywords: Bioenergetic; Oxidase; Photoreduction; Electrogenic; Electron transfer; Proton transfer;

In cytochrome c oxidase, oxido-reductions of heme a/CuA and heme a 3 /CuB are cooperatively linked to proton transfer at acid/base groups in the enzyme. H+/e cooperative linkage at Fe a3 /CuB is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H+/e linkage at heme a (and CuA). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H2O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/CuA and heme a 3/CuB in the soluble oxidase; (iii) H+ release in the external phase (i.e. H+ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H+/e linkage at heme a/CuA and heme a 3 /CuB with acid/base clusters, C1 and C2 respectively, and protonmotive steps of the reduction of O2 to water are involved in proton pumping.
Keywords: Cytochrome c oxidase; Proton pump; Cooperative H+/e linkage;

Nitric oxide and the respiratory enzyme by Maurizio Brunori; Elena Forte; Marzia Arese; Daniela Mastronicola; Alessandro Giuffrè; Paolo Sarti (1144-1154).
Available information on the molecular mechanisms by which nitric oxide (NO) controls the activity of the respiratory enzyme (cytochrome-c-oxidase) is reviewed. We report that, depending on absolute electron flux, NO at physiological concentrations reversibly inhibits cytochrome-c-oxidase by two alternative reaction pathways, yielding either a nitrosyl- or a nitrite-heme a 3 derivative. We address a number of hypotheses, envisaging physiological and/or pathological effects of the reactions between NO and cytochrome-c-oxidase.
Keywords: Mitochondrial physiology; Reaction mechanism; Free radical; Cytochrome c oxidase; Electron transfer; Inhibition of respiration;

The spatial arrangement and chemical reactivity of the activation-dependent thiol in the mitochondrial Complex I was studied using the membrane penetrating N-ethylmaleimide (NEM) and non-penetrating anionic 5,5′-dithiobis-(2-nitrobenzoate) (DTNB) as the specific inhibitors of the enzyme in mitochondria and inside-out submitochondrial particles (SMP). Both NEM and DTNB rapidly inhibited the de-activated Complex I in SMP. In mitochondria NEM caused rapid inhibition of Complex I, whereas the enzyme activity was insensitive to DTNB. In the presence of the channel-forming antibiotic alamethicin, mitochondrial Complex I became sensitive to DTNB. Neither active nor de-activated Complex I in SMP was inhibited by oxidized glutathione (10 mM, pH 8.0, 75 min). The data suggest that the active/de-active transition sulfhydryl group of Complex I which is sensitive to inhibition by NEM is located at the inner membrane–matrix interface. These data include the sidedness dependency of inhibition, effect of pH, ionic strength, and membrane bilayer modification on enzyme reactivity towards DTNB and its neutral analogue.
Keywords: NADH:ubiquinone oxidoreductase; Respiratory Complex I; Active/de-active transition; Sulfhydryl group; Mitochondria;

In ATP synthase, proton translocation through the Fo subcomplex and ATP synthesis/hydrolysis in the F1 subcomplex are coupled by subunit rotation. The static, non-rotating portions of F1 and Fo are attached to each other via the peripheral “stator stalk”, which has to withstand elastic strain during subunit rotation. In Escherichia coli, the stator stalk consists of subunits b 2δ; in other organisms, it has three or four different subunits. Recent advances in this area include affinity measurements between individual components of the stator stalk as well as a detailed analysis of the interaction between subunit δ (or its mitochondrial counterpart, the oligomycin-sensitivity conferring protein, OSCP) and F1. The current status of our knowledge of the structure of the stator stalk and of the interactions between its subunits will be discussed in this review.
Keywords: ATP synthase; F1-ATPase; Stator; Stator stalk; Peripheral stalk;

Mitochondrial and nuclear DNAs contribute to encode the whole mitochondrial protein complement. The two genomes possess highly divergent features and properties, but the forces influencing their evolution, even if different, require strong coordination. The gene content of mitochondrial genome in all Metazoa is in a frozen state with only few exceptions and thus mitochondrial genome plasticity especially concerns some molecular features, i.e. base composition, codon usage, evolutionary rates. In contrast the high plasticity of nuclear genomes is particularly evident at the macroscopic level, since its redundancy represents the main feature able to introduce genetic material for evolutionary innovations. In this context, genes involved in oxidative phosphorylation (OXPHOS) represent a classical example of the different evolutionary behaviour of mitochondrial and nuclear genomes. The simple DNA sequence of Cytochrome c oxidase I (encoded by the mitochondrial genome) seems to be able to distinguish intra- and inter-species relations between organisms (DNA Barcode). Some OXPHOS subunits (cytochrome c, subunit c of ATP synthase and MLRQ) are encoded by several nuclear duplicated genes which still represent the trace of an ancient segmental/genome duplication event at the origin of vertebrates.
Keywords: Evolution; Mitochondrial genome; Nuclear genome; Oxidative phosphorylation; DNA Barcode; Cytochrome c; Subunit c of ATP synthase; Synteny;

Mitochondrial DNA transcription and diseases: Past, present and future by Julio Montoya; Manuel J. López-Pérez; Eduardo Ruiz-Pesini (1179-1189).
The transcription of mitochondrial DNA has been studied for 30 years. However, many of the earlier observations are still unsolved. In this review we will recall the basis of mitochondrial DNA transcription, established more than twenty years ago, will include some of the recent progress in the understanding of this process and will suggest hypotheses for some of the unexplained topics. Moreover, we will show some examples of mitochondrial pathology due to altered transcription and RNA metabolism.
Keywords: Mitochondrial DNA; Mitochondrial transcription; Mitochondrial diseases; Transcriptional machinery;

Modeling human mitochondrial diseases in flies by Álvaro Sánchez-Martínez; Ningguang Luo; Paula Clemente; Cristina Adán; Rosana Hernández-Sierra; Pilar Ochoa; Miguel Ángel Fernández-Moreno; Laurie S. Kaguni; Rafael Garesse (1190-1198).
Human mitochondrial diseases are associated with a wide range of clinical symptoms, and those that result from mutations in mitochondrial DNA affect at least 1 in 8500 individuals. The development of animal models that reproduce the variety of symptoms associated with this group of complex human disorders is a major focus of current research. Drosophila represents an attractive model, in large part because of its short life cycle, the availability of a number of powerful techniques to alter gene structure and regulation, and the presence of orthologs of many human disease genes. We describe here Drosophila models of mitochondrial DNA depletion, deafness, encephalopathy, Freidreich's ataxia, and diseases due to mitochondrial DNA mutations. We also describe several genetic approaches for gene manipulation in flies, including the recently developed method of targeted mutagenesis by recombinational knock-in.
Keywords: Drosophila; Mitochondria; Mitochondrial diseases; Gene targeting; Oxidative phosphorylation;

MTERF3, the most conserved member of the mTERF-family, is a modular factor involved in mitochondrial protein synthesis by Marina Roberti; Francesco Bruni; Paola Loguercio Polosa; Caterina Manzari; Maria Nicola Gadaleta; Palmiro Cantatore (1199-1206).
The MTERF-family is a wide family of proteins identified in Metazoa and plants which includes the known mitochondrial transcription termination factors. With the aim to shed light on the function of MTERF-family members in Drosophila, we performed the cloning and characterization of D-MTERF3, a component of the most conserved group of this family. D-MTERF3 is a mitochondrial protein of 323 amino acids. Sequence analysis in seven different organisms showed that the protein contains five conserved “mTERF-motifs”, three of which include a leucine zipper-like domain. D-MTERF3 knock-down, obtained by RNAi in D.Mel-2 cells, did not affect mitochondrial replication and transcription. On the contrary, it decreased to a variable extent the rate of labelling of about half of the mitochondrial polypeptides, with ND1 being the most affected by D-MTERF3 depletion. These results indicate that D-MTERF3 is involved in mitochondrial translation. This role, likely based on protein–protein interactions, may be exerted either through a direct interaction with the translation machinery or by bridging the mitochondrial transcription and translation apparatus.
Keywords: mitochondria; mTERF; MTERF3; Drosophila; mitochondrial protein synthesis;

Comparison of promoters controlling on the sunflower mitochondrial genome the transcription of two copies of the same native trnK gene reveals some differences in their structure by Antonio Placido; Fabrizio Damiano; Marta Sciancalepore; Caterina De Benedetto; Guglielmo Rainaldi; Raffaele Gallerani (1207-1216).
Two copies of native trnK (UUU) gene are encoded on the sunflower mitochondrial DNA. They lie within two 12-kb direct repeats, presumably generated by a duplication event. During an investigation aimed at detecting DNA regions activating the trnK1 and trnK2 genes, three distinct promoters have been identified. Their locations were deduced using standard procedures (RT-PCR, RNA capping and 5′RACE) usually employed for the detection of transcription initiation sites (TISs). Promoters P3 and P2 control two independent partially overlapping transcription units containing the trnK2 and ccb206 genes, respectively. Promoter P1 has been mapped about 5200 bp upstream of the trnK1 gene which is part of a transcription unit also containing exons c, d and e of the nad2 gene, 5′ to the tRNA gene. Most probably this promoter is not alone in controlling this transcription unit because this DNA region could be cotranscribed, at least partially, starting from other two promoters located upstream of the trnC and trnN genes, respectively. These genes have been previously mapped in a 5′ region adjacent to the cluster containing nad2 exons c, d and e and the trnK1 gene. The comparative analysis of promoters P3 and P1 suggests that the difference between them could be related to the duplication event generating the second copy of trnK gene. The availability of a high number of new promoters belonging to dicot mitochondrial genomes makes possible to note some of their specific features.
Keywords: Higher plant; tRNA gene; Mitochondrial transcription; Promoter structure;

Enolase takes part in a macromolecular complex associated to mitochondria in yeast by Irina Brandina; James Graham; Christelle Lemaitre-Guillier; Nina Entelis; Igor Krasheninnikov; Lee Sweetlove; Ivan Tarassov; Robert P. Martin (1217-1228).
In eucaryotes, glycolytic enzymes are classically regarded as being localised in the cytosol. Recently, we have shown that part of the cellular pool of the glycolytic enzyme, enolase, is tightly associated with the mitochondrial surface in the yeast Saccharomyces cerevisiae (N. Entelis, I. Brandina, P. Kamenski, I.A. Krasheninnikov, R.P. Martin and I. Tarassov, A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae, Genes Dev. 20 (2006) 1609-1620). Here, using enzymatic assays, we show that all glycolytic enzymes are associated with mitochondria in yeast, to extents similar to those previously reported for Arabidopsis cells. Using separation of mitochondrial complexes by blue-native/SDS-PAGE and coimmunoprecipitation of mitochondrial proteins with anti-enolase antibodies, we found that enolase takes part in a large macromolecular complex associated to mitochondria. The identified components included additional glycolytic enzymes, mitochondrial membrane carriers, and enzymes of the TCA cycle. We suggest a possible role of the enolase complex in the channeling of pyruvate, the terminal product of glycolysis, towards the TCA cycle within mitochondria. Moreover, we show that the mitochondrial enolase-containing complex also contains the cytosolic tRNACUU Lys, which is mitochondrially-imported, and its presumed import carrier, the precursor of the mitochondrial lysyl-tRNA synthetase. This suggests an unsuspected novel function for this complex in tRNA mitochondrial import.
Keywords: Enolase; Mitochondria; Saccharomyces cerevisiae; tRNA import;

Transport catalysis by Martin Klingenberg (1229-1236).
Carrier linked solute transport through biomembranes is analysed with the viewpoint of catalysis. Different from enzymes, in carriers the unchanged substrate induces optimum fit in the transition state. The enhanced intrinsic binding energy pays for the energy required of the global conformation changes, thus decreasing the activation energy barrier. This “induced transition fit” (ITF) explains several phenomena of carrier transport, e.g., high or low affinity substrate requirements for unidirectional versus exchange, external energy requirement for “low affinity” transport, the existence of side specific inhibitors to ground states of the carrier, the requirement of external energy in active transport to supplement catalytic energy in addition to generate electrochemical gradients.
Keywords: Transport; Catalytic energy; Induced fit; Active transport; Electrochemical gradient;

The conserved substrate binding site of mitochondrial carriers by Edmund R.S. Kunji; Alan J. Robinson (1237-1248).
Mitochondrial carriers transport nucleotides, co-factors and metabolic intermediates across the inner mitochondrial membrane permeability barrier. They belong to a family of transporters unique to eukaryotes and they differ in structure and transport mechanism from other secondary transporters. The main structural fold consists of a barrel of six transmembrane α-helices closed at the matrix side by a salt-bridge network at the bottom of the cavity. The significant sequence conservation in the mitochondrial carrier family suggests that specific recognition of substrates is coupled to a common mechanism of transport. We have identified a common substrate binding site comprising residues that are highly conserved and, as demonstrated by mutagenesis, are essential for function. The binding site explains substrate selectivity, ion coupling and the effects of the membrane potential on transport. The main contact points in the site are related by threefold symmetry like the common structural fold. The substrate is bound at the midpoint of the membrane and may function as a pivot point for the movements of the transmembrane α-helices as the carrier changes conformation. The trigger for the translocation event is likely to be the substrate-induced perturbation of the salt bridge network at the bottom of the cavity.
Keywords: Mitochondrial carrier; Substrate binding; Transporter; Bioinformatics; Structure;

Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins by Ferdinando Palmieri; Gennaro Agrimi; Emanuela Blanco; Alessandra Castegna; Maria A. Di Noia; Vito Iacobazzi; Francesco M. Lasorsa; Carlo M.T. Marobbio; Luigi Palmieri; Pasquale Scarcia; Simona Todisco; Angelo Vozza; John Walker (1249-1262).
The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane α-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.
Keywords: Mitochondria; Mitochondrial carrier; Proteomics; Saccharomyces cerevisiae; Transport; Transporter;

There are 67 proteins in the human mitochondrial transport protein family. They have been identified from among the proteins of the RefSeq database on the basis of sequence similarity to proteins that have been functionally identified as mitochondrial transport proteins. They have also been identified by matching their predicted structure to the high resolution structure of the bovine ADP/ATP T1 transporter subunit/carboxyatractyloside complex. 74 nonsynonymous single nucleotide polymorphisms (nsSNP) have been identified in their gene sequences. These nsSNPs are present in genes of 30 of the proteins. No nsSNP has been found in 24 of the protein genes and no search has as yet been carried out on the rest (13) of them. The largest number of nsSNPs are in the ADP/ATP T3 transporter, the uncoupling protein 3 L, and the phosphate transporter genes with 7, 6, and 6, respectively. nsSNPs are located in groups along the protein sequence suggesting that certain protein domains are too critical for transport function to tolerate mutations. This interpretation has been validated with mutation and function studies of the phosphate transporter. Human diseases have been identified with replacement mutations in seven of these proteins. Their genes are not abnormally susceptible to mutations since they have the smallest number of nsSNPs. Disease causing mutations have also been observed as: substitution, silent (may affect stability of messages), frameshift (protein truncation or elongation), splicing (exon skipping), residue deletion. Disease causing mutations have only been identified in few transporter genes because others do not yield dramatic symptoms or are essential and thus lethal. Mutations in other transporter genes may also only have a major impact through their combination with other genes and their nsSNPs.
Keywords: Human; Mitochondria; Transport; Protein; Carrier;

Previous examination of the accessibility of a panel of single-Cys mutants in transmembrane domain III (TMDIII) of the yeast mitochondrial citrate transport protein to the hydrophilic, cysteine-specific methanethiosulfonate reagent MTSES enabled identification of the water-accessible surface of this TMD. Further studies on the effect of citrate on MTS reagent accessibility, indicated eight sites within TMD III at which citrate conferred temperature-independent protection, thus providing strong evidence for participation of these residues in the formation of a portion of the substrate translocation pathway. Unexpectedly, citrate did not protect against inhibition of the Leu120Cys variant, despite its location on a water- and citrate-accessible surface of the TMDIII helix. This led to the hypothesis that in the 3-dimensional CTP structure, TMDIV packs against TMDIII in a manner such that the Leu120 side-chain folds behind the side-chain of Gln182. The present investigations addressed this hypothesis by examining the properties of the Gln182Cys single mutant and the Leu120Cys/Gln182Ala double mutant. We observed that in contrast to our findings with the Leu120Cys mutant, citrate did protect the Gln182Cys variant against MTSES-mediated inhibition. Importantly, truncation of the Gln182 side-chain to Ala enabled citrate to protect the Leu120Cys double mutant against inhibition. In combination these data support the idea that the Gln182 side-chain lines the transport path and sterically blocks access of citrate to the Leu120 side-chain. In a parallel series of investigations, we constructed 24 single-Cys substitution mutants that were chosen based on their hypothesized importance in substrate binding and/or translocation. We observed that substitution of Cys for residues E34, K37, K83, R87, Y148, D236, K239, T240, R276, and R279 resulted in ≥ 98% inactivation of CTP function, suggesting an essential structural and/or mechanistic role for these native residues. Superposition of this functional data onto a detailed 3-dimensional homology model of the CTP structure indicates that the side-chains of each of these residues project into the putative transport pathway. We hypothesize that a subset of these residues, in combination with four previously identified essential residues, define the citrate binding site(s) within the CTP.
Keywords: Citrate; Transporter; Mitochondria; Structure; Carrier;

Arabidopsis mitochondria have two basic amino acid transporters with partially overlapping specificities and differential expression in seedling development by Luigi Palmieri; Christopher D. Todd; Roberto Arrigoni; Mary Elizabeth Hoyos; Antonella Santoro; Joseph C. Polacco; Ferdinando Palmieri (1277-1283).
To shed light on the metabolic role of two mitochondrial transporters for basic amino acids in Arabidopsis, we compared their functional properties in liposomes and expression during germination. Recombinant and purified BAC2, as previously reported for BAC1, transported various basic l-amino acids upon reconstitution in phospholipid vesicles. Both displayed highest affinity for arginine with similar K m and V max. However, BAC2 transported citrulline for which BAC1 had little or no affinity. Furthermore, BAC2 was less stereospecific than BAC1, transporting d-arginine and d-lysine at significant rates, and displayed a striking alkaline pH optimum (pH 8.0) whereas BAC1 activity was unaltered from pH 7.0 to 9.0. By semi-quantitative RT-PCR BAC1 transcript levels were found to be higher than those of BAC2 in germinated seeds. However, BAC2 expression transiently increased 2 days after germination. Disruption of the Arabidopsis arginase structural genes (ARGAH1 or ARGAH2) accentuated the increases of transcript levels of BAC1 at germination and of BAC2 2 days after germination and from 6 days on. Early expression of BAC1 and BAC2 is consistent with the delivery of arginine, released from seed reserves, to mitochondrial arginase and the export of ornithine. Increase of BAC2 transcript levels later in seedling development is consistent with roles in NO, polyamine or proline metabolism—processes involving arginine, citrulline and/or ornithine.
Keywords: Basic amino acid; Transport; Mitochondria; Arginase; Nitrogen mobilization; Germination; Nitric oxide synthase;

UCP2, UCP3, avUCP, what do they do when proton transport is not stimulated? Possible relevance to pyruvate and glutamine metabolism by François Criscuolo; Julien Mozo; Corinne Hurtaud; Tobias Nübel; Frédéric Bouillaud (1284-1291).
Uncoupling proteins (UCPs) are specialized members of the mitochondrial transporter family. They allow passive proton transport through the mitochondrial inner membrane. This activity leads to uncoupling of mitochondrial respiration and to energy waste, which is well documented with UCP1 in brown adipose tissue. The uncoupling activity of the new UCPs (discovered after 1997), such as UCP2 and UCP3 in mammals or avUCP in birds, is more difficult to characterize. However, extensive data support the idea that the new UCPs are involved in the control of reactive oxygen species (ROS) generation. This fits with the hypothesis that mild uncoupling caused by the UCPs prevents ROS production. Activators and inhibitors regulate the proton transport activity of the UCPs. In the absence of activators of proton transport, the UCP allows the permeation of other ions. We suggest that this activity has physiological significance and, for example, UCP3 expressed in glycolytic muscle fibres may be a passive pyruvate transporter ensuring equilibrium between glycolysis and oxidative phosphorylation. Induction of UCP2 expression by glutamine strengthens the proposal that new UCPs could act to determine the choice of mitochondrial substrate. This would obviously have an impact on mitochondrial bioenergetics and ROS production.
Keywords: Mitochondria; Uncoupling protein; Reactive oxygen species; Translation; Glycolysis; Krebs cycle;

Fatty acid activation of the uncoupling proteins requires the presence of the central matrix loop from UCP1 by Jesús Jiménez-Jiménez; Amalia Ledesma; Pilar Zaragoza; M. Mar González-Barroso; Eduardo Rial (1292-1296).
Noradrenaline signals the initiation of brown fat thermogenesis and the fatty acids liberated by the hormone-stimulated lipolysis act as second messengers to activate the uncoupling protein UCP1. UCP1 is a mitochondrial transporter that catalyses the re-entry of protons to the mitochondrial matrix thus allowing a regulated discharge of the proton gradient. The high affinity of UCP1 for fatty acids is a distinct feature of this uncoupling protein. The uncoupling proteins belong to a protein superfamily formed by the mitochondrial metabolite carriers. Members of this family present a tripartite structure where a domain containing two transmembrane helices, linked by a long hydrophilic loop, is repeated three times. Using protein chimeras, where the repeats had been swapped between UCP1 and UCP3, it has been shown that the central third of UCP1 is necessary and sufficient for the response of the protein to fatty acids. We have extended those studies and in the present report we have generated protein chimeras where different regions of the second repeat of UCP1 have been sequentially replaced with their UCP2 counterparts. The resulting chimeras present a progressive degradation of the characteristic bioenergetic properties of UCP1. We demonstrate that the presence of the second matrix loop is necessary for the high affinity activation of UCP1 by fatty acids.
Keywords: Uncoupling protein; Transporter; Mutagenesis; Mitochondria; Fatty acid;

Mitochondrial membrane permeability transition and cell death by Yoshihide Tsujimoto; Takashi Nakagawa; Shigeomi Shimizu (1297-1300).
Mitochondria are important organelles for energy production, Ca2+ homeostasis, and cell death. In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received much attention. In apoptotic and necrotic death, an increase of mitochondrial membrane permeability is considered to be one of the key events, although the detailed mechanism remains to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca2+-dependent increase in the permeability of the mitochondrial membrane that leads to loss of Δψ, mitochondrial swelling, and rupture of the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel, which is termed the permeability transition pore (PTP) and putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (Cyp D: a mitochondrial peptidyl prolyl-cis, trans-isomerase), and other molecule(s). Our studies of mice lacking Cyp D have revealed that it is essential for occurrence of the MPT and that the Cyp D-dependent MPT regulates some forms of necrotic cell death, but not apoptotic death. We have also shown that two anti-apoptotic proteins, Bcl-2 and Bcl-xL, block the MPT by directly inhibition of VDAC activity. Here we summarize a role of the MPT in cell death.
Keywords: Mitochondria; Permeability transition; Cell death;

Mitochondria as the target of the pro-apoptotic protein Bax by Emine Er; Lisa Oliver; Pierre-François Cartron; Philippe Juin; Stephen Manon; François M. Vallette (1301-1311).
During apoptosis, engagement of the mitochondrial pathway involves the permeabilization of the outer mitochondrial membrane (OMM), which leads to the release of cytochrome c and other apoptogenic proteins such as Smac/DIABLO, AIF, EndoG, Omi/HtraA2 and DDP/TIMM8a. OMM permeabilization depends on activation, translocation and oligomerization of multidomain Bcl-2 family proteins such as Bax or Bak. Factors involved in Bax conformational change and the function(s) of the distinct domains controlling the addressing and the insertion of Bax into mitochondria are described in this review. We also discuss our current knowledge on Bax oligomerization and on the molecular mechanisms underlying the different models accounting for OMM permeabilization during apoptosis.
Keywords: Apoptosis; Bax; Activation; Conformational change; Addressing/targeting; Permeabilization; Mitochondrion;

Therapeutic peptides: Targeting the mitochondrion to modulate apoptosis by Etienne Jacotot; Aurélien Deniaud; Annie Borgne-Sanchez; Zahia Touat; Jean-Paul Briand; Morgane Le Bras; Catherine Brenner (1312-1323).
For many years, medical drug discovery has extensively exploited peptides as lead compounds. Currently, novel structures of therapeutic peptides are derived from active pre-existing peptides or from high-throughput screening, and optimized following a rational drug design approach. Molecules of interest may prove their ability to influence the disease outcome in animal models and must respond to a set of criteria based on toxicity studies, ease of administration, the cost of their synthesis, and logistic for clinical use to validate it as a good candidate in a therapeutic perspective. This applies to the potential use of peptides to target one central intracellular organelle, the mitochondrion, to modulate (i.e. activate or prevent) apoptosis. Putative mitochondrial protein targets and the strategies already elaborated to correct the defects linked to these proteins (overexpression, inactivation, mutation…, etc.) are described, and recent advances that led or may lead to the conception of therapeutic peptides via a specific action on these mitochondrial targets in the future are discussed.
Keywords: Peptides; Mitochondrion; Permeability transition; Apoptosis; Bcl-2;

Participation of ATP/ADP antiporter in oleate- and oleate hydroperoxide-induced uncoupling suppressed by GDP and carboxyatractylate by Lyudmila S. Khailova; Elena A. Prikhodko; Vera I. Dedukhova; Elena N. Mokhova; Vasily N. Popov; Vladimir P. Skulachev (1324-1329).
In experiments on isolated kidney and liver mitochondria, it is shown that oleate hydroperoxide induces a much smaller increase in the controlled respiration rate and ΔΨ decrease than the same concentrations of oleate. Palmitate appears to be less efficient than oleate but more efficient than oleate hydroperoxide. In all cases, GDP and CAtr cause some recoupling, CAtr being more effective. Addition of 0.2 mM GDP before CAtr does not prevent further ΔΨ increase by subsequent CAtr addition. On the other hand, GDP added after CAtr is without any effect. GDP partially prevents the ΔΨ lowering by ADP at the State 4 — State 3 transition if small amounts of CAtr are present. The data are consistent with the suggestion of F. Goglia and V.P. Skulachev (FASEB J. 17, 1585–1591, 2003) that fatty acid anions are translocated by mitochondrial anion carriers much better than their hydroperoxides. As to GDP recoupling, it cannot be regarded as a specific probe for uncoupling by UCPs since it can be mediated by the ATP/ADP antiporter.
Keywords: Mitochondria; Uncoupling; Fatty acid; Peroxide; Uncoupling protein; ATP/ADP antiporter;

Long chain free fatty acids (FFA) exert, according to their actual concentration, different effects on the energy conserving system of mitochondria. Sub-micromolar concentrations of arachidonic acid (AA) rescue ΔpH-dependent depression of the proton pumping activity of the bc1 complex. This effect appears to be due to a direct interaction of AA with the proton-input mouth of the pump. At micromolar concentrations FFA increase the proton conductance of the inner membrane acting as protonophores. FFA can act as natural uncouplers, causing a mild uncoupling, which prevents reactive oxygen species production in the respiratory resting state. When Ca2+-loaded mitochondria are exposed to micromolar concentrations of FFA, the permeability of the inner membrane increases, resulting in matrix swelling, rupture of the outer membrane and release of intermembrane pro-apoptotic proteins. The characteristics of AA-induced swelling appear markedly different in mitochondria isolated from heart or liver. While in the latter it presents the canonical features of the classical permeability transition (PT), in heart mitochondria substantial differences are observed concerning CsA sensitivity, ΔΨ dependence, reversibility by BSA and specificity for the activating divalent cation. In heart mitochondria, the AA-dependent increase of the inner membrane permeability is affected by ANT ligands such as adenine nucleotides and atractyloside. AA apparently causes a Ca2+-mediated conversion of ANT from a translocator to a channel system. Upon diamide treatment of heart mitochondria, the Ca2+/AA-induced CsA insensitive channel is converted into the classical PT pore. The relevance of these observations in terms of tissue-specific components of the putative PTP and heart ischemic and post-ischemic process is discussed.
Keywords: Mitochondria; Free fatty acid; Arachidonic acid; Permeability transition; Uncoupling; bc1 complex;

Expression of transforming K-Ras oncogene affects mitochondrial function and morphology in mouse fibroblasts by Ferdinando Chiaradonna; Daniela Gaglio; Marco Vanoni; Lilia Alberghina (1338-1356).
K-ras transformed fibroblasts have been shown to have a stronger dependence from glycolysis, reduced oxidative phosphorylation ability and a fragility towards glucose depletion compared to their immortalized, normal counterparts. In this paper, using RNA profiling assays and metabolic perturbations, we report changes in expression of genes encoding mitochondrial proteins and alterations in mitochondrial morphology that correlate with mitochondrial functionality. In fact, unlike normal cells, transformed cells show reduced ATP content and inability to modify mitochondria morphology upon glucose depletion. Being reverted by GEF-DN expression, such morphological and functional changes are directly connected to Ras activation. Taken together with reported partial mitochondrial uncoupling and more sustained apoptosis of transformed cells, our results indicate that activation of the Ras pathway strikingly impacts on energy and signaling-related aspects of mitochondria functionality, that in turn may affect the terminal phenotype of transformed cells.
Keywords: Transformed cell; Ras; Glucose metabolism; Mitochondrial morphology; Energy metabolism; Apoptosis;

“Tissue” transglutaminase contributes to the formation of disulphide bridges in proteins of mitochondrial respiratory complexes by Pier Giorgio Mastroberardino; Maria Grazia Farrace; Irene Viti; Flaminia Pavone; Gian Maria Fimia; Gennaro Melino; Carlo Rodolfo; Mauro Piacentini (1357-1365).
In this study we provide the first in vivo evidences showing that, under physiological conditions, “tissue” transglutaminase (TG2) might acts as a protein disulphide isomerase (PDI) and through this activity contributes to the correct assembly of the respiratory chain complexes. Mice lacking TG2 exhibit mitochondrial energy production impairment, evidenced by decreased ATP levels after physical challenge. This defect is phenotypically reflected in a dramatic decrease of motor behaviour of the animals. We propose that the molecular mechanism, underlying such a phenotype, resides in a defective disulphide bonds formation in ATP synthase (complex V), NADH-ubiquinone oxidoreductase (complex I), succinate-ubiquinone oxidoreductase (complex II) and cytochrome c oxidase (complex IV). In addition, TG2-PDI might control the respiratory chain by modulating the formation of the prohibitin complexes. These data elucidate a new pathway that directly links the TG2-PDI enzymatic activity with the regulation of mitochondrial respiratory chain function.
Keywords: Respiratory chain complex; ATP synthase; HSP60; Prohibitin; Transglutaminase 2 knock out mice; Protein Disulphide Isomerase;

Ysp2 mediates death of yeast induced by amiodarone or intracellular acidification by Sviatoslav Sokolov; Dmitry Knorre; Ekaterina Smirnova; Olga Markova; Andrey Pozniakovsky; Vladimir Skulachev; Fedor Severin (1366-1370).
Recently we have found that the drug amiodarone induces apoptosis in yeast, which is mediated by reactive oxygen species (ROS). Here we have used this finding as a tool to screen for genes involved in the death program. We have described a novel mitochondrial protein, Ysp2, acting in the amiodarone-induced death cascade. After amiodarone addition both the control and amiodarone-resistant ysp2-deleted cells formed ROS, but the mutant (unlike the control) did not undergo the mitochondrial thread-to-grain transition. To test whether the action of Ysp2 is amiodarone-specific we tried to induce PCD by other agents. We have found that acetic acid-induced PCD also depends on Ysp2. We also demonstrate that, like acetic acid, propionic acid or nigericin triggered intracellular acidification causing ROS-dependent death. We suggest that intracellular acidification results in the protonation of superoxide anion (O2 •) to form HO2, one of the most aggressive ROS, which in turn induces Ysp2-mediated PCD.
Keywords: Apoptosis; Programmed cell death; Acetate; ROS; superoxide; pH; Acid stress;

Necrosis has long been described as a consequence of physico-chemical stress and thus accidental and uncontrolled. Recently, it is becoming clear that necrotic cell death is as well controlled and programmed as caspase-dependent apoptosis, and that it may be an important cell death mode that is both pathologically and physiologically relevant. Necrotic cell death is not the result of one well-described signalling cascade but is the consequence of extensive crosstalk between several biochemical and molecular events at different cellular levels. Recent data indicate that serine/threonine kinase RIP1, which contains a death domain, may act as a central initiator. Calcium and reactive oxygen species (ROS) are main players during the propagation and execution phases of necrotic cell death, directly or indirectly provoking damage to proteins, lipids and DNA, which culminates in disruption of organelle and cell integrity. Necrotically dying cells initiate pro-inflammatory signalling cascades by actively releasing inflammatory cytokines and by spilling their contents when they lyse. Unravelling the signalling cascades contributing to necrotic cell death will permit us to develop tools to specifically interfere with necrosis at certain levels of signalling. Necrosis occurs in both physiological and pathophysiological processes, and is capable of killing tumour cells that have developed strategies to evade apoptosis. Thus detailed knowledge of necrosis may be exploited in therapeutic strategies.
Keywords: Calcium; Inflammation; Mitochondria; Phospholipases; Proteases; ROS;

Mitochondrial DNA involvement in human longevity by Aurelia Santoro; Stefano Salvioli; Nicola Raule; Miriam Capri; Federica Sevini; Silvana Valensin; Daniela Monti; Dina Bellizzi; Giuseppe Passarino; Giuseppina Rose; Giovanna De Benedictis; Claudio Franceschi (1388-1399).
The main message of this review can be summarized as follows: aging and longevity, as complex traits having a significant genetic component, likely depend on a number of nuclear gene variants interacting with mtDNA variability both inherited and somatic. We reviewed the data available in the literature with particular attention to human longevity, and argued that what we hypothesize for aging and longevity could have a more general relevance and be extended to other age-related complex traits such as Alzheimer's and Parkinson's diseases. The genetics which emerges for complex traits, including aging and longevity, is thus even more complicated than previously thought, as epistatic interactions between nuclear gene polymorphisms and mtDNA variability (both somatic and inherited) as well as between mtDNA somatic mutations (tissue specific) and mtDNA inherited variants (haplogroups and sub-haplogroups) must be considered as additional players capable of explaining a part of the aging and longevity phenotype. To test this hypothesis is one of the main challenge in the genetics of aging and longevity in the next future.
Keywords: Mitochondrial DNA; Longevity; mtDNA mutation; mtDNA haplogroup; Centenarian; Nuclear–mitochondrial interaction; Alzheimer's Disease;

Mitochondrial diseases and genetic defects of ATP synthase by Josef Houštěk; Andrea Pícková; Alena Vojtíšková; Tomáš Mráček; Petr Pecina; Pavel Ješina (1400-1405).
ATP synthase is a key enzyme of mitochondrial energy conversion. In mammals, it produces most of cellular ATP. Alteration of ATP synthase biogenesis may cause two types of isolated defects: qualitative when the enzyme is structurally modified and does not function properly, and quantitative when it is present in insufficient amounts. In both cases the cellular energy provision is impaired, and diminished use of mitochondrial ΔμH+ promotes ROS production by the mitochondrial respiratory chain. The primary genetic defects have so far been localized in mtDNA ATP6 gene and nuclear ATP12 gene, however, involvement of other nuclear genes is highly probable.
Keywords: Mitochondrial diseases; ATP synthase; Biogenesis; ATP6; ATP12; Energy provision; Reactive oxygen species;

Mitochondrial Complex I: Structural and functional aspects by Giorgio Lenaz; Romana Fato; Maria Luisa Genova; Christian Bergamini; Cristina Bianchi; Annalisa Biondi (1406-1420).
This review examines two aspects of the structure and function of mitochondrial Complex I (NADH Coenzyme Q oxidoreductase) that have become matter of recent debate. The supramolecular organization of Complex I and its structural relation with the remainder of the respiratory chain are uncertain. Although the random diffusion model [C.R. Hackenbrock, B. Chazotte, S.S. Gupte, The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport, J. Bioenerg. Biomembranes 18 (1986) 331–368] has been widely accepted, recent evidence suggests the presence of supramolecular aggregates. In particular, evidence for a Complex I–Complex III supercomplex stems from both structural and kinetic studies. Electron transfer in the supercomplex may occur by electron channelling through bound Coenzyme Q in equilibrium with the pool in the membrane lipids. The amount and nature of the lipids modify the aggregation state and there is evidence that lipid peroxidation induces supercomplex disaggregation. Another important aspect in Complex I is its capacity to reduce oxygen with formation of superoxide anion. The site of escape of the single electron is debated and either FMN, iron–sulphur clusters, and ubisemiquinone have been suggested. The finding in our laboratory that two classes of hydrophobic inhibitors have opposite effects on superoxide production favours an iron–sulphur cluster (presumably N2) is the direct oxygen reductant. The implications in human pathology of better knowledge on these aspects of Complex I structure and function are briefly discussed.
Keywords: Complex I; Supercomplex; Coenzyme Q; ROS; Rotenone; Stigmatellin;

Acetyl-l-carnitine feeding to unloaded rats triggers in soleus muscle the coordinated expression of genes involved in mitochondrial biogenesis by P. Cassano; A.G. Sciancalepore; V. Pesce; M. Flück; H. Hoppeler; M. Calvani; L. Mosconi; P. Cantatore; M.N. Gadaleta (1421-1428).
The expressional profile of mitochondrial transcripts and of genes involved in the mitochondrial biogenesis pathway induced by ALCAR daily supplementation in soleus muscle of control and unloaded 3-month-old rats has been analyzed. It has been found that ALCAR treatment is able to upregulate the expression level of mitochondrial transcripts (COX I, ATP6, ND6, 16 S rRNA) in both control and unloaded animals. Interestingly, ALCAR feeding to unloaded rats resulted in the increase of transcript level for master factors involved in mitochondrial biogenesis (PGC-1α, NRF-1, TFAM). It also prevented the unloading-induced downregulation of mRNA levels for kinases able to transduce metabolic (AMPK) and neuronal stimuli (CaMKIIβ) into mitochondrial biogenesis. No significant effect on the expressional level of such genes was found in control ALCAR-treated rats. In addition, ALCAR feeding was able to prevent the loss of mitochondrial protein content due to unloading condition. Correlation analysis revealed a strong coordination in the expression of genes involved in mitochondrial biogenesis only in ALCAR-treated suspended animals, supporting a differentiated effect of ALCAR treatment in relation to the loading state of the soleus muscle. In conclusions, we demonstrated the ability of ALCAR supplementation to promote only in soleus muscle of hindlimb suspended rats an orchestrated expression of genes involved in mitochondrial biogenesis, which might counteract the unloading-induced metabolic changes, preventing the loss of mitochondrial proteins.
Keywords: Mitochondrial biogenesis; Gene expression; Soleus muscle; Unloading; Acetyl-l-carnitine;

Mitochondrial dysfunction in hepatitis C virus infection by C. Piccoli; R. Scrima; A. D'Aprile; M. Ripoli; L. Lecce; D. Boffoli; N. Capitanio (1429-1437).
The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood though HCV induces a state of hepatic oxidative stress that is more pronounced than that present in many other inflammatory diseases. This mini-review will focus on recent findings revealing an unexpected role of mitochondria in providing a central role in the innate immunity and in addition will illustrate the application of stably transfected human-derived cell lines, inducibly expressing the entire HCV open reading frame for in vitro studies on mitochondria. Results obtained by a comparative analysis of the respiratory chain complexes activities along with mitochondrial morpho-functional confocal microscopy imaging show a detrimental effect of HCV proteins on the cell oxidative metabolism with specific inhibition of complex I activity, decrease of mtΔΨ, increased production of reactive oxygen species. A possible de-regulation of calcium recycling between the endoplasmic reticulum and the mitochondrial network is discussed to provide new insights in the pathogenesis of hepatitis C.
Keywords: HCV; Inducible gene expression; Mitochondria; Reactive oxygen specie; Complex I; Endoplasmic reticulum; Calcium;

Shotgun proteomics for the characterization of subunit composition of mitochondrial complex I by Gabriella Pocsfalvi; Manuela Cuccurullo; Gitta Schlosser; Giuseppina Cacace; Rosa Anna Siciliano; Maria Fiorella Mazzeo; Salvatore Scacco; Tiziana Cocco; Antonio Gnoni; Antonio Malorni; Sergio Papa (1438-1450).
Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.
Keywords: Complex I; Bovine heart mitochondria; Subunit composition; Shotgun proteomics; Mass spectrometry;