Portal de Periódicos da CAPES

OXPHOS mutations and neurodegeneration

2012; Springer Nature; Volume: 32; Issue: 1 Linguagem: Inglês

10.1038/emboj.2012.300

1460-2075

Werner J.H. Koopman, Felix Distelmaier, Jan Smeitink, Peter H.G.M. Willems,

RNA modifications and cancer

Review13 November 2012free access OXPHOS mutations and neurodegeneration Werner J H Koopman Corresponding Author Werner J H Koopman Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Felix Distelmaier Felix Distelmaier Department of General Pediatrics and Neonatology, University Children's Hospital, Heinrich-Heine-University, Düsseldorf, Germany Search for more papers by this author Jan AM Smeitink Jan AM Smeitink Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Peter HGM Willems Peter HGM Willems Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Werner J H Koopman Corresponding Author Werner J H Koopman Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Felix Distelmaier Felix Distelmaier Department of General Pediatrics and Neonatology, University Children's Hospital, Heinrich-Heine-University, Düsseldorf, Germany Search for more papers by this author Jan AM Smeitink Jan AM Smeitink Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Peter HGM Willems Peter HGM Willems Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Author Information Werner J H Koopman 1, Felix Distelmaier2, Jan AM Smeitink3 and Peter HGM Willems1 1Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands 2Department of General Pediatrics and Neonatology, University Children's Hospital, Heinrich-Heine-University, Düsseldorf, Germany 3Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands *Corresponding author. Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, 286 Biochemistry, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.:+31 24 3614589; Fax:+31 24 3616413; E-mail: [email protected] The EMBO Journal (2013)32:9-29https://doi.org/10.1038/emboj.2012.300 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mitochondrial oxidative phosphorylation (OXPHOS) sustains organelle function and plays a central role in cellular energy metabolism. The OXPHOS system consists of 5 multisubunit complexes (CI–CV) that are built up of 92 different structural proteins encoded by the nuclear (nDNA) and mitochondrial DNA (mtDNA). Biogenesis of a functional OXPHOS system further requires the assistance of nDNA-encoded OXPHOS assembly factors, of which 35 are currently identified. In humans, mutations in both structural and assembly genes and in genes involved in mtDNA maintenance, replication, transcription, and translation induce 'primary' OXPHOS disorders that are associated with neurodegenerative diseases including Leigh syndrome (LS), which is probably the most classical OXPHOS disease during early childhood. Here, we present the current insights regarding function, biogenesis, regulation, and supramolecular architecture of the OXPHOS system, as well as its genetic origin. Next, we provide an inventory of OXPHOS structural and assembly genes which, when mutated, induce human neurodegenerative disorders. Finally, we discuss the consequences of mutations in OXPHOS structural and assembly genes at the single cell level and how this information has advanced our understanding of the role of OXPHOS dysfunction in neurodegeneration. Introduction Nearly every activity of the cell is powered by the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). In order to maintain ATP homeostasis and, therefore, cell integrity and function, ATP must be continuously replenished. The energy required for this process comes from the stepwise oxidation of fuel molecules originating from three different carbon sources, i.e., monosaccharides, mainly glucose (GLC) but also fructose (FRC) and galactose (GAL), fatty acids (FAs) and amino acids. Following food uptake, these fuel molecules enter the body from the intestine, where they are produced upon the enzymatic breakdown of carbohydrates, triacylglycerols (TAGs) and proteins. Their distribution throughout the body occurs via the circulatory system and cells take up the required nutrients for energy production, biosynthesis and replenishment of intracellular glycogen stores (liver cells and skeletal muscle cells) and TAGs (fat cells). Liver cells convert excess GLC to TAGs, which they package in very low density lipoprotein (VLDL) particles for transport to the fat cells. In between feeding, the blood GLC level is maintained by the liver mobilizing its glycogen stores and producing GLC from lactate (LAC), glycerol and glucogenic amino acids. At the same time, fat cells mobilize their TAG stores to release FAs and glycerol. GLC is the only fuel molecule for red blood cells and, normally, brain cells and to limit its consumption, liver cells and skeletal muscle cells at rest primarily use FAs for the production of ATP. Mature red blood cells and skeletal muscle cells at work convert GLC to LAC, which they release in the circulation. This LAC is taken up mainly by the liver, which uses ATP derived from FAs to reconvert it to GLC. For GLC and glycerol, the stepwise oxidation process starts in the cytosol, where a series of enzymes catalyse their partial oxidation to pyruvate (PYR; Figure 1). During this process, the major part of the chemical bond energy of the fuel molecule is transferred in the form of electrons to the electron carrier nicotinamide adenine dinucleotide (NAD+) thus reducing to reduced nicotinamide adenine dinucleotide (NADH), whereas a smaller part is transferred in the form of a phosphoryl group to ADP. The latter process, referred to as substrate-level phosphorylation, uses a phosphorylated reactive intermediate as a donor. In the case of GLC, cytosolic oxidation yields two molecules each of PYR, ATP and NADH. Other contributions to the cytosolic PYR pool come from LAC and certain amino acids. Figure 1.Energy metabolism in a typical mammalian cell. To meet cellular energy demands, ATP is generated by the glycolysis pathway (blue), the tricarboxylic acid (TCA) cycle and the oxidative phosphorylation (OXPHOS) system. The main energy substrate glucose (GLC) enters the cell via GLC transporters (GLUTs) and is converted into pyruvate (PYR). Alternatively, surplus GLC can be stored as glycogen for later use or enter the pentose phosphate pathway (green). PYR can have two different fates: either it is converted into lactate (LAC) that leaves the cell, or it enters the mitochondrion (yellow) to form Acetyl coenzyme A (AcCoA). The latter is processed by the TCA cycle to yield NADH and FADH2, which are substrates of the OXPHOS system. In addition to GLC also fructose (FRC), galactose (GAL), fatty acids (FAs) and glutamine (GLN) can enter the ATP producing system (see text for details). 6PG, 6-phosphogluconate; 6PGL, 6-phosphogluconolactone; DHAP, dihydroxyacetone phosphate; FRU6P, fructose 6-phosphate; FRUBP, fructose 1,6-bisphosphate; GA3P, glyceraldehyde 3-phosphate; GLU, glutamate; G6PDH, glucose 6-phosphate dehydrogenase; G6PGDH, 6-phosphogluconate dehydrogenase; GT, glutamine transporter; GPI, phosphoglycose isomerase; HK, hexokinase; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; PDH, pyruvate dehydrogenase; PFK, phospofructokinase; RL5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; TA, transaldolase; TK, transketolase; TPI, triosephosphate isomerase. Download figure Download PowerPoint PYR oxidation involves the combined action of a series of enzymes located within the mitochondrial matrix. First, PYR is oxidatively decarboxylated by pyruvate dehydrogenase (PDH), yielding one molecule each of CO2, NADH and Acetyl coenzyme A (AcCoA). Next, AcCoA is oxidized by the enzymes of the tricarboxylic acid (TCA), producing two molecules of CO2, three molecules of NADH, one molecule of the reduced form of the electron carrier flavin adenine dinucleotide (FADH2) and one molecule of GTP, by substrate-level phosphorylation. The oxidation of FAs takes place entirely in the mitochondrial matrix by a process referred to as β oxidation. Also, this process occurs in a stepwise manner yielding one molecule each of AcCoA, NADH and FADH2 per step. The end product is either AcCoA (even-numbered FAs) or propionyl-CoA (odd-numbered FAs). The latter molecule can be converted into succinyl-CoA, which is an intermediate of the TCA cycle. Also, the oxidation of amino acids occurs entirely in the mitochondrial matrix. To this end, amino acids are first deaminated and then, depending on the type of amino acid, processed to PYR, AcCoA or an intermediate of the TCA cycle (α-ketoglutarate, succinyl-CoA, fumarate or oxaloacetate) (Lunt and Vander Heiden, 2011). Intermediates can be withdrawn from the above oxidation processes, e.g., for the synthesis of neurotransmitters and amino acids (Dienel, 2012). Furthermore, GLC can be metabolized through the pentose phosphate pathway (PPP; Figure 1), yielding reduced nicotinamide adenine dinucleotide phosphate (NADPH) for anabolic reactions and pentoses for the synthesis of nucleotides and aromatic amino acids. For the oxidation processes to continue, reoxidation of the reduced electron carriers (NADH and FADH2) is a prerequisite. This reoxidation can take place in the cytosol by the enzyme lactate dehydrogenase (LDH) and in the mitochondrial matrix by the combined action of the enzymes and electron carriers of the electron transport chain (ETC; Smeitink et al, 2001). During the LDH reaction, the NADH electrons are transferred to PYR, yielding LAC, whereas during the ETC reaction, the NADH and FADH2 electrons are transferred to molecular oxygen (O2), yielding H2O. The mitochondrial inner membrane (MIM) is impermeable to NADH, and under normal conditions of oxygen supply the electrons of cytosolic NADH are transferred across this membrane by shuttle systems such as the malate-aspartate shuttle and the glycerol-phosphate shuttle, yielding cytosolic NAD+ for continuation of glycolysis and mitochondrial NADH (malate-aspartate shuttle) or FADH2 (glycerol-phosphate shuttle) for reoxidation by the ETC. Together, the enzymes of the ETC convert the oxidation energy temporarily stored in NADH and FADH2 into an electrochemical proton gradient across the MIM that is used by a proton-transporting enzyme (F1Fo-ATP synthase) to produce ATP. This process is referred to as oxidative phosphorylation (OXPHOS). Here, it is important to realize that many other MIM transporters are driven by the electrochemical proton gradient and it is for that reason that a proper electrochemical proton gradient is essential for the maintenance of mitochondrial integrity and many other aspects of mitochondrial function (apoptosis, innate immunity, redox control, calcium homeostasis and several biosynthetic processes) (Kwong et al, 2007; Wang and Youle, 2009; Koopman et al, 2010, 2012; Arnoult et al, 2011; Mammucari et al, 2011). In addition, some energy of the electrochemical proton gradient is used for thermogenesis. The balance between cytosolic and mitochondrial ATP production depends on the type of cell and its physiological demands and environmental conditions (supply of fuel molecules and O2). Some cells depend completely on cytosolic ATP production and produce LAC to reoxidize NADH (mature red blood cells), others depend largely on the complete oxidation of GLU (brain cells) or FAs (liver cells) and use O2 as the final electron acceptor, again others oxidize mainly FAs at rest and GLU at a sudden burst of activity (skeletal muscle cells). In the latter case, LAC is produced because of a hampered supply of O2. In terms of ATP production, the maximum yield per molecule of GLU is 2 ATP in the case of oxidation to LAC and ∼30 ATP in the case of full oxidation to CO2 and H2O (Rich, 2003). Under pathological conditions, the mechanism of ATP production can change dramatically. For instance, most cancer cells oxidize GLU to LAC to produce ATP, even in the presence of O2 (Warburg effect) (Cairns et al, 2011). Other pathological conditions are caused by inborn errors of enzymes that convert energy from fuel molecules to NADH, FADH2 and ATP by substrate phosphorylation or from NADH and FADH2 to ATP by OXPHOS. Moreover, such errors can develop in time, e.g., as a consequence of insufficient control of reactive oxygen species (ROS) levels. Neurons are high consumers of ATP and because they have no glycogen stores they depend entirely on the uninterrupted supply of GLU through the extracellular fluid. For the same reasons, neurons preferentially oxidize GLC to CO2 and H2O providing the highest yield of ATP per GLU. Therefore, maintenance of mitochondrial integrity and function is of highest priority to these cells. Mitochondria are motile organelles that exhibit fusion and fission and display a dynamic internal structure (Benard and Rossignol, 2008). The balance between these processes determines net mitochondrial (ultra)structure and distribution, which is linked to mitochondrial (dys)function and metabolism during healthy and pathological conditions including neurodegeneration (Knott et al, 2008; Lizana et al, 2008; Willems et al, 2009; Dieteren et al, 2011; Campbell et al, 2012; Court and Coleman, 2012; Kageyama et al, 2012). In humans, a (progressive) decrease in mitochondrial function in general, and of the OXPHOS system in particular, has been linked to neurodegeneration during normal ageing and many other conditions including inborn errors of energy metabolism, amyotrophic lateral sclerosis (ALS), Parkinson disease (PD), Alzheimer disease (AD), Huntington disease (HD), certain forms of (brain) cancer, diabetes, epilepsy, obesity, cognitive impairment, psychosis and anxiety (Chandra and Singh, 2011; Martin, 2011; Anglin et al, 2012; Costa and Scorrano, 2012; Finsterer and Mahjoub, 2012; Nunnari and Suomalainen, 2012; Schapira, 2012). OXPHOS inhibition is also evoked by off-target (drug) effects, likely differentially affecting healthy individuals and patients with mitochondrial dysfunction (Wallace, 2008; Dimauro and Rustin, 2009; Cohen, 2010; Finsterer and Segall, 2010; Morán et al, 2012). For example, mice with fatal encephalomyopathy due to mitochondrial dysfunction were 2.5- to 3-fold more sensitive to the volatile anaesthetics isoflurane and halothane than wild-type (wt) mice (Quintana et al, 2012). Moreover, environmental toxins including rotenone and persistent organic pollutants (POPs) like the insecticide dichlorodiphenyltrichloroethane (DDT), the herbicide and industrial waste product 2,3,7,8-tetrachlorodibenzodioxin (TCCD) and the phenolic flame retardant tetrabromobisphenol A (TBBPA) directly or indirectly inhibit OXPHOS function (Lee et al, 2010; Schapira, 2010). During recent years, substantial progress has been made in understanding the role of mitochondrial dysfunction in neurodegeneration. We recently argued that understanding the cellular (patho)physiology of monogenic mitochondrial disorders, particularly those associated with (relatively rare) OXPHOS mutations, will not only enhance our understanding of mitochondrial (dys)function but is also therapeutically relevant for the many diseases in which OXPHOS function is disturbed (Koopman et al, 2012). Below we first provide a theoretical background regarding the OXPHOS system. This is followed by an inventory of OXPHOS genes that are, when mutated, associated with neurodegeneration in humans. Finally, we present the insights obtained from studying the consequences of mutations in OXPHOS structural and assembly genes in living cells. The mitochondrial OXPHOS system The OXPHOS system (Figure 2) consists of five MIM-embedded multisubunit complexes: complex I (CI or NADH:ubiquinone oxidoreductase; EC 1.6.5.3), complex II (CII or succinate:ubiquinone oxidoreductase; EC 1.3.5.1), complex III (CIII or ubiquinol:cytochrome c oxidoreductase; EC 1.10.2.2), complex IV (CIV or cytochrome-c oxidase; EC 1.9.3.1) and complex V (CV or FoF1-ATP-synthase; EC 3.6.1.34). These complexes are divided into two functional parts: (i) the four complexes (CI–CIV) of the ETC and (ii) CV that generates ATP (Distelmaier et al, 2009; Smeitink et al, 2001; Koopman et al, 2012). Genetically, 92 different genes encoding structural OXPHOS subunits have been identified (Figure 2). CII is exclusively derived from the nuclear DNA (nDNA), whereas the other OXPHOS complexes contain subunits that are encoded by nDNA and the mitochondrial DNA (mtDNA). In addition to the structural OXPHOS subunit genes, the mtDNA also contains genetic information for the 2 mitochondrial ribosomal RNAs (mt-rRNAs) and the 22 mitochondrial transfer RNAs (mt-tRNAs). All proteins involved in mtDNA repair, replication, transcription, translation and maintenance of the mitochondrial deoxynucleoside triphosphate (dNTP) pool, as well as mt-tRNA synthetases and mitochondrial ribosomal proteins, are nDNA encoded (Peralta et al, 2012). Biogenesis of a functional OXPHOS system further requires a large set (>75) of nDNA-encoded proteins (Supplementary Table 1). Figure 2.Genetic origin and functional interaction of the mitochondrial oxidative phosphorylation (OXPHOS) complexes. The mitochondrial OXPHOS system consists of five multisubunit complexes (CI–CV) that reside in the mitochondrial inner membrane (MIM). The MIM encloses the mitochondrial matrix and is surrounded by the mitochondrial outer membrane (MOM). An inter-membrane space (IMS) is located between the MIM and MOM. The subunits of CI, CIII, CIV and CV are encoded by the mitochondrial (mtDNA; red) and nuclear DNA (nDNA; blue), whereas CII exclusively consists of nDNA-encoded subunits (table at the top). OXPHOS biogenesis is mediated by nDNA-encoded assembly factors (green). The nDNA-encoded proteins are imported into the mitochondrial matrix via the TOM (translocator of the inner membrane) and TIM (translocator of the inner membrane) systems. At CI and CII, NADH and FADH2 are oxidized, respectively, and the released electrons are transported to CIII via Coenzyme Q10 (CoQ10; 'Q'). From thereon, electrons are transported to CIV via cytochrome-c (cyt-c; 'c') and donated to oxygen (O2). Together, CI–CIV constitute the electron transport chain (ETC). The energy derived from the electron transport is used to expel protons (H+) from the mitochondrial matrix across the MIM. This establishes an electrochemical proton-motive force, associated with an inside-negative mitochondrial membrane potential (Δψ) and increased matrix pH. The controlled backflow of H+ is used by CV to drive the production of ATP (see text for details). Download figure Download PowerPoint CI is the largest OXPHOS enzyme proposed to consist of 45 different subunits. Recent evidence suggests a number of 44 subunits since the NDUFA4 protein hitherto classified as a CI constituent appears to be a component of CIV (Balsa et al, 2012). Seven CI subunits (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6) are encoded by the mtDNA and the remainder by the nDNA (Figure 2; Supplementary Table 1). CI oxidizes NADH to NAD+ and donates the released electrons to the electron carrier coenzyme Q10 (CoQ10, a.k.a. ubiquinone). To perform its enzymatic reactions, CI only requires a set of 14 evolutionary conserved 'core subunits', consisting of the 7 mtDNA-encoded ND subunits and 7 nDNA-encoded subunits (NDUFV1, NDUFV2, NDUFS1, NDUFS3, NDUFS7, NDUFS8; Koopman et al, 2010; Hirst, 2011). The remaining subunits are denoted as 'accessory' or 'supernumerary'. Although the role of accessory subunits in CI biogenesis, stability and function still is incompletely understood, recent evidence in the aerobic yeast Yarrowia lipolytica suggests that they are important for CI stability (Angerer et al, 2011). Biogenesis of holo-CI is assisted by at least 11 assembly factors (NDUFAF1, NDUFAF2, NDUFAF3, NDUFAF4, C8orf38, C20orf7, ACAD9, FOXRED1, ECSIT, NUBPL and OXA1L). Details about the CI assembly mechanism are provided elsewhere (e.g., Vogel et al, 2007; Dieteren et al, 2008, 2011; Koopman et al, 2010; Mckenzie and Ryan, 2010; Perales-Clemente et al, 2010; Moreno-Lastres et al, 2012; Nouws et al, 2012). In mammals, fungi and bacteria CI displays an L-shaped form consisting of a hydrophilic (matrix-protruding) and a lipophilic (MIM-embedded) arm (Clason et al, 2010). During recent years, significant progress has been made in understanding the link between electron and H+ transport in CI (Sazanov and Hinchliffe, 2006; Efremov et al, 2010; Hunte et al, 2010; Efremov and Sazanov, 2011a, 2011b). In the proposed coupling mechanism, electrons extracted from NADH are transported by a chain of iron-sulphur (Fe-S) clusters (Xu and Møller, 2011) to CoQ10 (Hinchliffe and Sazanov, 2005; Hayashi and Stuchebrukhov, 2010). This transport is linked to H+ translocation due to long-range conformational changes within the complex (Onishi, 2010; Efremov and Sazanov, 2011a, 2011b). CII constitutes part of both the OXPHOS system and TCA cycle, oxidizes FADH2 to flavin adenine dinucleotide (FAD) and also transfers the released electrons to CoQ10 (Figure 2). CII is a heterotetrameric complex consisting of four nDNA-encoded subunits (SDHA, SDHB, SDHC and SDHD) and its assembly is assisted by two assembly factors (SDHAF1 and SDHAF2; Supplementary Table 1). Details about CII biogenesis are provided elsewhere (Rutter et al, 2010). Structurally, the SDHC and SDHC subunits are embedded in the MIM, whereas SDHA and SDHB protrude in the mitochondrial matrix (Brière et al, 2005). SDH-encoding genes are tumour suppressors, and their mutation predisposes carriers to carotid body paragangliomas and adrenal gland pheochromocytomas (Raimundo et al, 2011). In addition to CI and CII, also other enzymes can potentially donate electrons to CoQ10. These include: (i) the MIM-associated electron-transferring flavoprotein (ETF)-ubiquinone oxidoreductase, which transfers electrons generated during the flavin-linked oxidation step in the catabolism of FAs, (ii) s,n-glycerophosphate dehydrogenase and (iii) dihydroorotate dehydrogenase, present only in certain types of mitochondria (see Koopman et al, 2010 and the references therein). Electrons from CoQ10 are received by CIII and transported further to CIV by the electron carrier cytochrome-c (cyt-c). Similarly to CoQ10, cyt-c can receive electrons from an alternative source (especially in the liver) during oxidation of sulphur-containing amino acids by sulphite oxidase. However, this reaction usually occurs at a very low rate relative to other ETC inputs (see Koopman et al, 2010 and the references therein). CIII contains 11 subunits, one of which is encoded by the mtDNA (CYB). Its assembly is described elsewhere (Smith et al, 2012) and requires the action of two identified assembly factors (BCS1L and UQCC; Supplementary Table 1). At CIV, electrons are donated to molecular oxygen (O2) to form water. About 95% of the O2 we breathe is consumed by CIV (Ferguson-Miller et al, 2012). CIV consists of 14 subunits, 3 of which are mtDNA-encoded (CO1, CO2, and CO3), and its biogenesis is assisted by at least 18 assembly factors (Supplementary Table 1), as discussed in detail elsewhere (Mick et al, 2011). At three sites in the ETC (CI, CIII and CIV), the energy released by the electron transport is used to drive the trans-MIM efflux of protons (H+) from the mitochondrial matrix. As a consequence, a trans-MIM proton motive force (PMF or Δpm) is established, which consists of an (inside negative) electric charge (Δψ) and (inside more alkaline) pH (ΔpH) difference across the MIM (Mailloux and Harper, 2012; Figure 2). At CV, the energy released by the controlled backflow of H+ is coupled to the formation of ATP from ADP and inorganic phosphate (Pi). Experimental evidence in eukaryotes revealed that each ATP produced requires the CV-mediated backflow of 2.7 protons (Watt et al, 2010). CV is built up of 19 subunits, 2 of which are encoded by the mtDNA (ATP6 and ATP8), and its assembly requires 4 nDNA-encoded proteins (Supplementary Table 1). CV is a molecular machine composed of two mechanical rotary motors (Fo and F1), which interconvert the chemical energy of ATP hydrolysis and H+ electrochemical potential via a mechanical rotational mechanism (e.g., Okuno et al, 2011; Watanabe et al, 2011; Jonckheere et al, 2012a). This means that CV can either dissipate Δpm to generate ATP, or use ATP to fuel the trans-MIM efflux of H+. The latter condition sustains Δpm and is known as the 'reverse-mode' of CV (Chinopoulos and Adam-Vizi, 2010). In addition to ATP generation, the Δψ and/or ΔpH gradient is also required for mitochondrial fusion, the import of mitochondrial preproteins and the exchange of metabolite and ions with the cytosol (Figure 3), as reviewed previously (Garlid and Paucek, 2003; Kaasik et al, 2007; O'Rourke, 2007; Klingenberg, 2008; Palmieri, 2008; Koopman et al, 2010; Becker et al, 2012). Figure 3.Integration of the OXPHOS system and mitochondrial metabolism. The five OXPHOS complexes, depicted on the lower left of the figure (see also Figure 2), maintain the inside-negative mitochondrial membrane potential (Δψ) and generate reactive oxygen species (ROS; red) in the form of superoxide (O2·−) and hydrogen peroxide (H2O2). ROS can also be generated by the TCA cycle enzyme α-ketoglutarate dehydrogenase (αKGDH), under conditions of elevated NADH/NAD+ ratio. ROS are removed by several antioxidant systems (green). In addition to fuelling ATP generation by CV, a sufficiently negative Δψ is also crucial for import of nDNA-encoded mitochondrial preproteins (PreP) via the TIM system. Moreover, metabolite and ion exchange across the mitochondrial inner membrane (MIM; right part of the figure) is driven by Δψ (orange) or its associated pH gradient (ΔpH; blue) (see text for details). ANT, adenine nucleotide translocase; GR, glutathione reductase; GPX, glutathione peroxidase; GSH, glutathione; HCa, proton/calcium transporter; IF, flavin site in CI; IQ, CoQ10-binding site in CI; KH, potassium/proton transporter; NaCa, sodium/calcium transporter; NaH, sodium/proton transporter; Pi, inorganic phosphate/proton transporter; PYR, pyruvate/proton transporter; Qo, CoQ10-binding site in CIII; SOD2, superoxide dismutase 2; TRXR, thioredoxin reductase; TIM, translocator of the inner membrane; UCP, uncoupling protein; UNI, mitochondrial calcium uniporter. Download figure Download PowerPoint Supramolecular architecture of the OXPHOS system In bovine heart mitochondria, the unit stoichiometry of the OXPHOS system equalled 1/1.3/3/6.7/0.5 for CI/CII/CIII/CIV/CV and 2–5 units of the adenine nucleotide translocase (ANT; Lenaz and Genova, 2007), which mediates the trans-MIM exchange of ADP and ATP (Figure 3). Analysis of various rat tissues (Benard et al, 2006) revealed different molecular CII/CoQ10/CIII/cyt-c/CIV ratios in heart (1:24:3:12:8), kidney (1:73:3:18:7), muscle (1:58:3:11:7), brain (1:58:3:35:8) and liver (1:135:3:9:7). This suggests that the amount of CoQ10 and cyt-c display tissue-dependent differences, whereas CII, CIII and CIV do not. Statistical analysis predicted that different tissues display different sensitivities to a pathological OXPHOS defect, with brain being more sensitive than liver and kidney tissue but less sensitive than skeletal muscle and heart tissue (Benard et al, 2006). Experimental evidence suggests that CI assembly/stability depends on its interaction with other OXPHOS complexes (Schägger et al, 2004). In addition, CIII is required to maintain CI (Acín-Pérez et al, 2004) and deficiency of CIV reduces CI function (Suthammarak et al, 2009). Moreover, in human patient cells the presence of a truncated CIV subunit destabilized not only CIV but also other ETC complexes, leading to their rapid clearance by mitochondrial quality control systems (Hornig-Do et al, 2012). These observations, supported by other experimental evidence (reviewed in Boekema and Braun, 2007; Wittig and Schägger, 2009; Dudkina et al, 2010; and Winge, 2012), are compatible with a model in which individual OXPHOS complexes are not randomly distributed but organized in supercomplexes (or 'respirasomes'). The finding that CIII and CIV are not essential for the assembly/stability of CI in fungi (Maas et al, 2009) suggests that respirasome formation and/or stability might be species and/or tissue dependent. Although it was previously suggested that CIII interacts with CII (Chen et al, 2008), the current view is that respirasomes consist of CI, CIII and CIV (Boekema and Braun, 2007; Wittig and Schägger, 2009; Dudkina et al, 2010; Althoff et al, 2011; Winge, 2012). In order of decreasing abundance, respirasome composition in bovine heart is predicted to be I-III2-IV1, I-III2, I-III2-IV2 and I-III2-IV3–4 (Schägger and Pfeiffer, 2001; Winge, 2012). In silico evidence highlighted the involvement of lipids in the gluing together of the OXPHOS complexes at the interfaces (Dudkina et al, 2011). Based on biochemical evidence, respiratory strings of CI, CIII and CIV have been proposed meaning that respirasome