genetic control of oxidative phosphorylation and experimental

Human Reproduction, Vol. 15, (Suppl. 2), pp. 18-27, 2000

Genetic control of oxidative phosphorylation andexperimental models of defects

Ian Trounce1

Mutation Research Centre, 7th Floor, Daly Wing St Vincent's Hospital,Fitzroy, Melbourne, Victoria 3065, Australia

'To whom correspondence should be addressed at: Mutation Research Centre,7th Floor, Daly Wing, St Vincent's Hospital, Fitzroy, Melbourne, Victoria 3065,

Australia. E-mail: [email protected]

Energy in the form of ATP is continuallyproduced by all cells for normal growth andfunction. Anaerobic glycolysis can provideenough ATP for some cells, but energeticcells such as cardiomyocytes and neuronsrequire a more efficient ATP supply, whichcan only be provided by mitochondrialoxidative phosphorylation. Invented by bac-teria that became symbiotically associatedwith other bacteria to form eukaryotic cellsbillions of years ago, oxidative phosphoryla-tion carries with it a genetic legacy that isunique. The mitochondrial oxidativephosphorylation complexes are assembledfrom protein subunits encoded by both themitochondrial genome (mtDNA) and thenuclear genome (nDNA, located in the chro-mosomes). The mtDNA is a remnantgenome of the bacterial progenitor of mito-chondria, and (unlike the biparental dip-loidy that characterizes the nucleargenome) is present in thousands of copiesper cell, is replicated through life, and isinherited (cytoplasmically) only from thefemale parent. Oxidative phosphorylationcomprises five multimeric enzyme com-plexes that act as a redox pathway, passingelectrons from oxidizable intermediatesproduced by the metabolism of food tomolecular oxygen in the mitochondrial mat-

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rix, while producing an electrochemical gra-dient by pumping protons into theintermembranal space. The proton (hydro-gen ion) gradient across the inner mitochon-drial membrane is used by the H + -transporting ATP synthase to produce ATPfrom ADP and inorganic phosphate, withthe protons released into the mitochondrialmatrix then combining with electronatedoxygen to form water. Many of the detailsregarding the control of the synthesis ofoxidative phosphorylation enzyme com-plexes remain to be elucidated. Transmit-ochondrial cell culture systems have beendeveloped so that defective oxidativephosphorylation can be studied in a con-trolled nuclear background. Such systemsmay soon enable the development ofmtDNA 'knockout' mice in order to bettermodel mtDNA transmission and mitochon-drial disease.Key words: electron transport chain/mitochon-drial DNA/oxidative phosphorylation/respirat-ory chain/transmitochondrial cells

Introduction

A glance at any biochemistry textbook willreveal that mitochondria house a large number

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Oxidative phosphorylation and defect models

ND6

NAD+

NADH

Figure 1. Genetic map of human mtDNA (upper) and scheme showing how the gene products contribute to the complexes ofoxidative phosphorylation (lower). The circular mtDNA genome is represented in the upper figure as a linear map, with tRNAgenes indicated by vertical lines, often separating reading frames. Heavy (H-) strand genes are above the line and aretranscribed from right to left; light (L-) strand genes are below the line and are read from left to right. As electrons pass fromcomplex I down the chain, oxygen is reduced to water and protons are pumped into the intermembranal space (lower right).cyt b encodes apocytochrome b of complex III; ND genes encode NADH dehydrogenase subunits 1 to 6 of complex I;CO genes encode cytochrome oxidase subunits I—III of complex IV; ATP6/8 encode ATP synthase subunits of complex V.

of metabolic pathways. Some of these, e.g.part of the urea cycle in liver mitochondriaand thermogenesis in brown fat, are uniqueto particular organs; others are common tomitochondria in all cells, e.g. the citric acidcycle and other catabolic pathways that gener-ate reduced (i.e. readily oxidizable) forms ofnicotinamide- and flavin-adenine dinucleotideintermediates (NADH and FADH2).

The respiratory chain (or electron transportchain) together with the H+-transporting ATPsynthase comprise the final common fueloxidation pathway, known as oxidativephosphorylation (or OXPHOS). Overall, oxid-ative phosphorylation involves oxidizableintermediate metabolic substrates, whichinclude NADH and FADH2, generating ATPfrom ADP and inorganic phosphate, and inthe process reducing molecular oxygen towater. The details are explained below. Ener-getic cells require mitochondrially-producedATP, whereas quiescent cells can derive suffi-cient ATP from anaerobic, cytosolic glyco-lysis, so mitochondria are most abundant in

cells with the highest demand for energy. Theoxidative phosphorylation pathway comprisesfive multisubunit enzyme complexes, in turncomposed of >80 different polypeptide units,13 of which are encoded by a separate genometo those contained in the (nuclear) chromo-somes, the mitochondrial genome, composedof mitochondrial DNA (mtDNA). The geneticcontrol of oxidative phosphorylation is there-fore unusual, requiring the co-ordinatedexpression of both nuclear and mitochondrialgenes. However, mitochondrial biogenesis andself-replication, including mtDNA replication,appear to be regulated entirely by nucleargenes.

The mitochondrial genome

It is now accepted that mitochondria andtheir genomes are vestiges of aerobic bacteria(prokaryotes) that long ago became symbiotic-ally incorporated into anaerobic prokaryotesto give rise to eukaryotic cells (Margulis,1981; Gray et al., 1999; Jansen, 2000). Most

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of the endosymbiont's genome was then lostor transferred to the nucleus, leaving a small,circular, histone-free 'chromosome' ofmtDNA to remain within the cytoplasmicorganelle. In humans, mtDNA forms a 16.5 kbcircle, which (as in all multicellular animalsknown) encodes 13 subunits of the OXPHOSenzyme complexes, a complete set of 22transfer RNAs (for 20 amino acids, two ofwhich have two distinct tRNAs), and tworRNA components of the mitochondrial ribo-somes (Figure 1). Replication and transcriptionof mtDNA are controlled by nuclear-encodedgene products and are discussed elsewhere isthis volume (Clayton, 2000).

The proteins encoded by mtDNA includeseven subunits of the 42-subunit NADH-ubiquinone oxidoreductase complex (complexI), the apocytochrome b of the 11-subunitubiquinol-cytochrome c oxidoreductase com-plex (complex III), three core proteins ofthe 13 subunits that comprise cytochrome coxidase (complex IV), and two of the 16subunits of the H+ATPsynthase (complex V)(Anderson et al., 1981; Saraste, 1999;Figure 1).

Cytoplasmic inheritance, suspected sincethe observations of Ephrussi (1950) with yeast,was confirmed when mtDNA in the fungusNeurospora was shown to be inherited fromthe female parent (Reich and Luck, 1966).Maternal inheritance of mtDNA was found tobe the rule for most of the (multicelled) animalkingdom, including humans (Giles et al.,1980). Plants also show uniparental inherit-ance of mtDNA (and plastid, or chloroplast,DNA), although the contributing parent canoften be the male (Levings, 1983; Hoekstra,2000). While tiny in comparison to the nucleargenome, animal mtDNA exists in thousandsof copies per cell; in typical somatic cellsmtDNA comprises - 1 % of DNA mass, andin the mature mammalian oocyte it can accountfor as much as a half or more of total DNAmass (Dawid, 1972). Mitochondria are

dependent on the nuclear genome for most ofthe 80 or so OXPHOS proteins, and all of theregulatory factors for mtDNA replication andtranscription. Enzymes of the many otherimportant metabolic functions housed in themitochondria of different cells are entirelynuclear-encoded.

Plants have relatively large and complexmitochondrial genomes compared withanimals (Levings, 1983; Gray et al., 1999).An important class of mtDNA mutations inplants result in cytoplasmic sterility, wherebyaffected plants suffer an inability to makeviable pollen (Levings, 1983, 1996). A widevariety of human diseases (individually rare,but collectively important) are now also attrib-uted to mtDNA mutations (Lightowlers et al.,1997; DiMauro and Schon, 1998; Wallace,1999; Christodoulou, 2000; Naviaux andMcGowan, 2000) and are gradually becomingbetter understood. Controversy, however, stillengulfs the idea that it is an accumulation ofwider mtDNA mutations that contributes tothe manifestations of age and degenerativedisease (Beal, 1996; Lightowlers et al., 1999;Wallace, 1999); discovery of mitochondrialinvolvement in the signalling for cell death(Newmayer et al., 1994; Kluck et al, 1997;Yang et al., 1997), has heightened interest isthis area (Wallace, 1999), and the possibilitythat altered mitochondrial function consequentto mtDNA mutations could interfere with orpotentiate apoptotic signals warrants investi-gation. ATP is normally exported from themitochondrion by the adenine nucleotidetranslocator, which forms part of the mitochon-drial permeability transition pore, activatedduring apoptosis. Generation of reactiveoxygen species (see below) and release ofcytochrome c through this pore are importantevents in apoptotic cell death (Matsuyamaetal, 1998; Raff, 1998, Martinou, 1999).

Oxidative phosphorylation

Organisms need to produce ATP continuously,to do cellular work. Glycolysis, converting

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glucose to pyruvate, produces two ATP molec-ules per glucose molecule, releasing around238 kJ/mole. The citric acid cycle and oxidat-ive phosphorylation, housed in the mitochon-drion, catalyses the stepwise oxidation ofpyruvate to carbon dioxide and water, yieldingaround 2870 kJ/mole and 38 ATP moleculesfor each initial glucose molecule (Lehninger,1964). Long before eukaryotes appeared, bac-teria evolved many ways of producing ATP.Oxidative phosphorylation was one of thesebacterial inventions and takes advantage ofthe oxidizing power of molecular oxygen,respiration being the controlled combustionof carbohydrates, producing ATP instead ofexcess heat.

Oxidative phosphorylation is achieved byfive multimeric enzyme complexes presentwithin the inner mitochondrial membrane.Complexes I-IV (the respiratory, or electrontransport chain) pass electrons from NADHand FADH2 (reduced intermediates derivedfrom the oxidation of carbohydrates and fattyacids, ultimately to carbon dioxide) to molecu-lar oxygen, so that electronated oxygenproducts produced by the respiratory chainenter the mitochondrial matrix. The energyreleased is partly conserved by the pumpingof protons (hydrogen ions) into the intermem-branal space (three protons for each electronpair traversing the chain), which produces aproton gradient with respect to both the cytosoland the mitochondrial matrix. The concentra-tion of protons is high enough to reverse aninner mitochondrial membrane-locatedenzyme H+-dependent ATPase (complex V;the reversed ATPase is named H+-transportingATP synthase) and thus to produce ATP frominorganic phosphate and ADP (when, by itspresence, the need for ATP-production is sig-nalled). Meanwhile the protons dischargedinto the mitochondrial matrix reduce the res-piratory chain's electronated oxygen products(which, potentially, constitute 'reactive oxygenspecies') further to form water (Figure 1).

A wealth of exciting new structural informa-tion on the respiratory chain enzymes (forreview see Saraste, 1999) has paralleled con-tinuing advances in mitochondrial genetics.Complex I is the largest and least understoodOXPHOS complex, containing multiple iron-sulphur centres that carry electrons fromNADH to ubiquinone. Complex II (succinatedehydrogenase) contains only four subunits,none encoded by mtDNA; this complex alsocontains several iron-sulphur centres, as wellas FAD and a b-type cytochrome, to carryelectrons from succinate to ubiquinone.Reduced ubiquinone (ubiquinol) is oxidizedin a two-step manner by complex III, aselectrons are passed via iron-sulphur centresin the Rieske protein, and via cytochromes band c1? finally to reduce cytochrome c. Thelast step in the respiratory chain sees complexIV, or cytochrome c oxidase, pass electronsthrough two copper atoms and two a-typecytochromes, finally delivering four electronsto molecular oxygen in the mitochondrialmatrix and, by employing four hydrogen ions(protons) there for each molecule of oxygen,producing two water molecules. Complexes I,III and IV also pump a proton across themitochondrial membrane to the intermem-branal space as they pass a pair of electrons,producing the proton gradient and flow utilizedby complex V to condense ATP from ADPand inorganic phosphate.

While it is tempting to view the electrontransport chain (and hence oxidativephophorylation) as the linear passage of elec-trons, the molar ratios of complexes I, II,III, IV and V in the inner membrane isapproximately 1:2:3:6:6 (Capaldi et al, 1988).From inhibitor studies it is clear that complexI and II, feeding electrons from NADH andFADH2 to complex III, exert the greatestcontrol over respiratory chain flux; complexesIII, IV and V, however, exhibit specific activit-ies more than an order of magnitude greater.

Figure 2 shows how oxidative phosphoryla-

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mito (0.5 mg)I \ ADP 125 nmols

ADP 125 nmols

'. DNP

Figure 2. Polarographic (oxygen electrode) estimation ofoxidative phosphorylation in intact mouse cell mitochondriashowing coupling of active respiration (indicated by oxygenconsumption) with ADP stimulation, and the effects ofuncoupling oxidative phosphorylation with dinitrophenol(DNP) (see text). Oxygen saturation (y axis) is measured as afunction of time (x axis), allowing comparison of specificrates of respiration (O2 consumed/min/mg mitochondrialprotein). Mito = addition of mitochindria, 0.5 mg.

tion can be studied in vitro using freshlyisolated mitochondria in a polarograph cham-ber. Isolated from cultured mouse cells withtheir membranes intact, the organelles main-tain a proton gradient so that phosphorylationas well as maximal respiratory flux can bedirectly measured. This (real) polarographtrace illustrates the basic principle of couplingof respiration to ADP availability (Chance andWilliams, 1955); substrates are added to thestirred chamber containing mitochondria in anisosmotic buffer with phosphate present, anda slow (state II) rate of respiration follows;additions of ADP stimulate state III respiration(the 'active' state in which phosphorylationoccurs); then, when added ADP has all beenphosphorylated to ATP, the slower state IVrespiration ensues. Figure 2 shows how theextraordinary kinetics of oxygen utilization bythe respiratory chain ensures that respirationcan proceed maximally even at the lowestoxygen levels measurable with the polaro-

graph, - 1 % saturated. Respiration can beuncoupled, by adding dinitrophenol, whichcauses the leakage of protons from the inter-membranal compartment, dissipating theproton gradient. The even faster respirationobserved with dinitrophenol-uncoupled mito-chondria in Figure 2 is typical, and implies thatcomplex V shares some control of respiratorychain electron flux in the phosphorylating state(state III).

Mitochondria from cultured cells can alsobe used to assay the individual respiratorychain enzyme complexes to gain a full pictureof nuclear or mtDNA mutation effects onoxidative phosphorylation (Trounce et al.,1996).

During normal respiratory chain activitysome incompletely reduced reactive oxygenspecies (ROS) are produced, with complexes Iand III being the sites of most ROS generation(Boveris and Chance, 1973). An estimated 1-4% of oxygen consumed is directly convertedto such free radicals under normal conditions.Inhibition of the respiratory chain greatlyincreases ROS production (Kwong and Sohal,1998). When the electron transport chain isinhibited, electrons accumulate in the proximalportion of the electron transport chain (com-plex I and ubiquinone) (Kwong and Sohal,1998), and can be added to molecular oxygen,forming superoxide anion (O2°~). Superoxideis converted by mitochondrial manganesesuperoxide dismutase (MnSOD) to give hydro-gen peroxide. Hydrogen peroxide, which inthe presence of reduced transition metals canitself be reduced to the highly reactivehydroxyl radical (OH°) by the Fenton reaction,is then converted to water by glutathioneperoxidase. Using a MnSOD knockout mouseas a model of acute oxidative stress, Melovet al. (1999) found the iron-sulphur centres ofcomplexes I, II, and III, as well as of aconitasein the citric acid cycle, were inactivated,severely disrupting mitochondrial energy pro-duction. Chronic exposure to free radicals

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is thought to result in oxidative damage tomitochondrial and cellular proteins, lipids andnucleic acids, increasing the prevalence ofmtDNA mutations (Wallace, 1999).

Transmitochondrial cells

It can be useful to consider oxidatively-derivedATP as an energy supply that is supplementary(to anaerobic glycolysis) for many cells, butessential for cells of highly energetic tissues,such as myocardium, skeletal muscle, pancre-atic islets, the central nervous system, retinalcells, the kidneys and the liver. That humancells can grow in vitro without OXPHOS hasbeen made clear by studies over the pastdecade using chemically-derived rho-zero (p°)cells, i.e. cells that have been completelydepleted of mtDNA. First described for aviancells (Morais et al., 1988), King and Attardi(1989) succeeded in producing a human p°cell line by the same approach, namely treatingcells with the y-polymerase inhibitor, ethidiumbromide; as the cells divide in the presenceof the ethidium bromide the mtDNA copynumber is reduced until clones can be isolatedthat have no mtDNA.

These p° cells, whether primary or trans-formed, can survive and grow with mitochon-dria that are without a functional respiratorychain. The mitochondria show grossly abnor-mal morphology, being greatly enlarged andlacking cristae (Figure 3). The cells producehigh levels of lactate and, unlike their p +

parents, require 'redox therapy' (additionalpyruvate and uridine in the culture medium)for survival. Such uridine-dependence, or aux-otrophy, among p° cells was found (Moraiset al., 1988) to be because dihydroorotatedehydrogenase (an enzyme of the pyrimidinebiosynthesis pathway — thus essential fornuclear DNA synthesis and mitosis - locatedon the outer surface of the mitochondrion'sinner membrane), normally needs to passelectrons released from the oxidation of dihy-

Figure 3. Electron microcrographs showing (A) a cybridmade by fusion of enucleated mouse 3T3 cells with a mousep° cell line, showing normal mitochondrial morphology, and(B) the p° cell line depleted of mtDNA, showing swollen andhypodense mitochondria with few cristae. (A, B)Magnification X15 000. The mouse p° cell line was producedby the author in the laboratory of D.C.Wallace.

droorotate on to ubiquinone and thence tocomplex III and the remainder of the electrontransport chain, all of which is missing in themitochondria of p° cells. The requirement forexogenous pyruvate (King and Attardi, 1989)probably provides oxidized NAD (NAD+)necessary for ATP production from glycolysisin the cytosol by further pushing lactate pro-duction via lactate dehydrogenase.

Omission of the nutritional requirementsfor p° cells allows the selection of p + cybridsmade by fusing enucleated cells (cytoplasts)from patients with mutant mtDNA with p°cells, in turn permitting demonstration of theassociation of different mtDNA diseasemutations with different OXPHOS phenotypesin vitro (Chomyn et al., 1991; King et al.,1992; Trounce et al, 1994).

Towards mtDNA 'knockout' mice

Successful mouse models of MendelianOXPHOS disease have been produced recently

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by knockout of the heart-muscle adenine nuc-leotide translocator isoform (Graham et al,1997) and knockout of mitochondrial tran-scription factor A (mtTFA, or Tfam) (Wanget al, 1999; Rantanen and Larsson, 2000).Knockout mice with different mtDNA defectswill no doubt also be valuable tools. Byenabling direct study of segregation of dele-terious heteroplasmic mutations throughdevelopment they will greatly aid the studyof the pathogenesis of, and possible treatmentmodalities for, mitochondrial diseases, as wellas being useful to researchers investigatingnuclear genes that affect OXPHOS (includingmediators of apoptosis).

The unique features of mitochondrialgenetics present some technical barriers toproducing mtDNA knockout mice. Recomb-ination has not been clearly demonstratedin mtDNA (but the subject continues to becontroversial; Arctander, 1999), meaning thatthe targeted knockout approach used for nuc-lear genes,utilizing homologous recombina-tion of a mutated construct, cannot be usedfor mtDNA. Transfection approaches usingartificial mtDNA constructs have not yet suc-ceeded in transferring the construct into mito-chondria within cells.

Transmitochondrial somatic cell techniquesprovide another approach to producingmtDNA knockout mice, but still requiremtDNA mutant cells as the starting point. Theinbred mouse strains exhibit little variationin mtDNA sequence, although polymorphicvariants between two strains were used toadvantage in a pioneering study byShoubridge's group (Jenuth et al., 1996).These authors constructed heteroplasmicmouse embryos by fusion of a cytoplast fromone strain with a fertilized oocyte from anotherstrain. In other experiments, Meirelles andSmith (1998) have used a similar approach ofeither cytoplast or karyoplast fusion to furthershow that oocyte mitochondria of perinuclear(karyoplast) origin tend to show less stringent

segregation than peripheral (cytoplast origin)mitochondria during preimplantation devel-opment.

A handful of drug-resistant mouse cellmtDNA mutants have been identified in cellculture, the best characterized being chloram-phenicol resistance (CAP1) due to a 16S rRNApoint mutation. CAPr cells are not growth-inhibited by chloramphenicol, providing a con-venient selectable marker, and the cells havedefects in OXPHOS, probably due to disruptedmitochondrial protein synthesis (Howell andNalty, 1988). Wallace et al. have recentlyreported an attempt to make a transmit-ochondrial mouse with the CAPr mutation(Levy et al., 1999). While succeeding intransferring the mutant mtDNA into embry-onic stem (ES) cells, they failed to get germlinetransmission of the clone. However, a recentupdate form the same group claimed to haveachieved germline transmission using a differ-ent XX ES cell line (Wallace et al, 1999).

Creation of homoplasmic transmitochon-drial cells without drug-resistant markers orp° cells is possible with the use of the toxicdye rhodamine 6-G (R6G) (Christodoulou,2000). Pretreatment of cells including ES cells(Levy et al, 1999) with low levels of R6Gfor several days was shown to kill cells, butthe cells could be rescued by fusion withenucleated untreated mitochondrial donorcells. The R6G appears to irreversibly collapsethe mitochondrial membrane potential, andthe utility of the drug in creating transmit-ochondrial mouse cells has been demonstrated(Trounce and Wallace, 1996).

Another approach is suggested by the find-ings of Moraes's group that mtDNA fromclosely related primate species can be replic-ated in human p° cells, but that the 'xenomito-chondrial' cybrids show OXPHOS defects(Kenyon and Moraes, 1997). We have sim-ilarly produced a range a xenomitochondrialcybrids by introducing mitochondria from sev-eral murinae species into a mouse p° cell

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XX ES cellMouse cell withmtDNA mutation

R-6G treatment

transfect mito. or rho-zero cellsrho-zero cellwith homoplasmicmtDNA mutation

blastocyst injectionT

mice

Figure 4. Transmitochondrial cell approaches to producing mtDNA knockout mice (see text).

line, resulting in various OXPHOS defects(McKenzie and I.Trounce, unpublishedresults). Irwin et at. (1999) have also usedmitochondria from another species (Musspretus), directly injecting isolated mitochon-dria into oocytes. We are also attempting toisolate mouse mtDNA mutants using classicaland novel mutagenesis approaches combinedwith efficient mutant scanning using chemicalcleavage of mismatch, which identifies differ-ences between heteroduplexed DNA molec-ules (see Forrest et ai, 1995). DifferentmtDNA mutant cells with OXPHOS or ROSphenotypes similar to human mtDNA diseasemutations may then be used to create mtDNAknockout mice. Figure 4 shows a schematicsummary of these approaches.

Conclusions

Oxidative phosphorylation defects consequentto mtDNA mutations cause an important groupof inherited diseases, and may be involved indegenerative diseases of ageing and cancer. Tobetter understand pathogenesis and inheritanceof heteroplasmic mtDNA mutations, and todevelop new treatment modalities, mousemodels of mtDNA disease are required. In-vitro manipulation of mitochondrial genotypes

in cultured somatic cells is now well estab-lished, as are techniques for karyoplast orcytoplast transfer into fertilized oocytes orearly embryos. To mimic human mtDNAmutations in mouse models we now needtechniques for creating custom mtDNAknockouts and for isolating mtDNA mutantsin cell culture.

AcknowledgementsThe author's research is supported by NHMRC grants970543 and 98002, and grants-in-aid from the RamaciottiFoundation, ANZ Trustees and the Percy Baxter Trust.

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