Stability of Complex I in Fungi

doi:10.1016/j.jmb.2008.12.025

J. Mol. Biol. (2009) 387, 259–269

Available online at www.sciencedirect.com

Respiratory Complexes III and IV Are Not Essential for the Assembly/Stability of Complex I in Fungi Marc F.P.M. Maas 1 , Frank Krause 2 ⁎, Norbert A. Dencher 2 and Annie Sainsard-Chanet 1,3 1

Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France 2

Department of Chemistry, Technische Universität Darmstadt, Petersenstrasse 22, D-64287 Darmstadt, Germany 3

Université Paris-Sud, 91405 Orsay, France Received 7 October 2008; received in revised form 8 December 2008; accepted 10 December 2008 Available online 24 December 2008

The functional relevance of respiratory supercomplexes in various eukaryotes including mammals, plants, and fungi is hitherto poorly elucidated. However, substantial evidence indicates as a major role the assembly and/or stabilization of mammalian complex I by supercomplex formation with complexes III and IV. Here, we demonstrate by using native electrophoresis that the long-lived Podospora anserina mutant Cyc1-1, respiring exclusively via the alternative oxidase (AOX), lacks an assembled complex III and possesses complex I partially assembled with complex IV into a supercomplex. This resembles the situation in complex-IV-deficient mutants displaying a corresponding phenotype but possessing I–III supercomplexes instead, suggesting that either complex III or complex IV is in a redundant manner necessary for assembly/stabilization of complex I as previously shown in mammals. To corroborate this notion, we constructed the double mutant Cyc1-1,Cox5::ble. Surprisingly, this mutant lacking both complexes III and IV is viable and essentially a phenocopy of mutant Cyc1-1 including the reversal of the phenotype towards wild-type-like characteristics by the several-fold overexpression of the AOX in mutant Cyc1-1,Cox5::ble (Gpd-Aox). Fungal specific features (not found in mammals) that must be responsible for assembly/stabilization of fungal complex I when complexes III and IV are absent, such as the presence of the AOX and complex I dimerization, are addressed and discussed. These intriguing results unequivocally prove that complexes III and IV are dispensable for assembly/ stability of complex I in fungi contrary to the situation in mammals, thus highlighting the imperative to unravel the biogenesis of complex I as well as the true supramolecular organization of the respiratory chain and its functional significance in a variety of model eukaryotes. In summary, we present the first obligatorily aerobic eukaryote with an artificial, simultaneous lack of the respiratory complexes III and IV. © 2008 Elsevier Ltd. All rights reserved.

Edited by J. Karn

Keywords: aging; alternative oxidase; complex I biogenesis; mitochondria; respiratory supercomplexes

Introduction *Corresponding author. E-mail address: [email protected]. Present address: M. F. P. M. Maas, Department of Chronobiology, Faculty of Mathematics and Natural Sciences, Center for Life Sciences, University of Groningen, PO Box 14, 9750 AA, Haren, The Netherlands. Abbreviations used: AOX, alternative oxidase; OXPHOS, oxidative phosphorylation; BN, blue-native; NADH-DH, NADH dehydrogenase; CN, colorless-native; COX, cytochrome c oxidase; DBQ, decylubiquinone.

The mitochondrial oxidative phosphorylation (OXPHOS) system is the main generator of cellular ATP in most eukaryotes, whose major components are the four respiratory chain complexes and the FOF1-ATP synthase located in the inner mitochondrial membrane.1,2 Complexes I (NADH:ubiquinone oxidoreductase), III (ubiquinol:cytochrome c oxidoreductase), and IV (cytochrome c oxidase, COX) transduce the energy of nutritional compounds into an electrochemical proton gradient across the inner membrane, used by the FOF1-ATP synthase (complex

0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.

260 V) to generate ATP. Strong evidence has been accumulating that in most eukaryotes,3–11 including filamentous ascomycetes,12,13 complexes I, III, and IV are organized as stoichiometric supercomplexes and the ATP synthase is organized as homodimers/ homooligomers (reviewed in Refs. [14–18]). Of particular importance is the recent determination of single-particle structures of respiratory supercomplexes.19–22 The functional significance of respiratory supercomplexes is still poorly understood, but there is substantial evidence that one of their major roles is the assembly and/or stabilization of complex I at least in mammals.5,23–26 Unlike mammals, many fungi, plants, algae, and protists possess alternative respiratory enzymes branching the standard respiratory chain without energy-conserving proton pumping, particularly the alternative NADH:ubiquinone oxidoreductases27–31 and the so-called alternative oxidase (AOX).29,32 The latter bypasses complexes III and IV, is insensitive against cyanide and other COX inhibitors, and was even confirmed to occur in some lower animals.33 Filamentous fungi such as the pezizomycete (formerly euascomycete) Neurospora crassa are invaluable model organisms necessary to explore essential molecular features of eukaryotes,34 for example, in mitochondria research such as investigation of the protein import machinery (e.g., Refs. [35,36]) and the biogenesis of complex I.30,31,37 In contrast to most fungi, capable of infinite vegetative growth, Podospora anserina, a close relative of N. crassa, is prone to a senescence process. P. anserina has been used as a model eukaryote to investigate molecular aspects of aging for decades, more so as a mitochondrial etiology, particularly the age-dependent systematic reorganization of mtDNA in all wild-type isolates is well established.38–40 Of particular interest is the apparently causative link of respiration and longevity: The exclusive use of the AOX respiration due to the specific impairment of either complex IV,41–43 for example, mutant Cox5::ble42 lacking a gene for an essential COX subunit, or complex III (mutant Cyc1-144) with a loss-of-function mutation in the gene encoding cytochrome c1 of complex III leads to mtDNA stabilization and virtual immortality. However, the observation that the constitutive several-fold overexpression of the AOX in those mutants, Cox5::ble (Gpd-Aox)45 and Cyc1-1(Gpd-Aox),44 restores senescence and other wild-type characteristics to a large extent is puzzling. Here, we show that long-lived P. anserina mutants lacking either complex III or complex IV have I–IVor I–III supercomplexes, respectively, suggesting that either complex is involved in a redundant way in assembly/stability of complex I as previously shown in mammals. However, by constructing and analyzing the double mutant Cyc1-1,Cox5::ble, with it being a phenocopy of the single mutants and devoid of complexes III and IV, we prove that both complexes are not essential for assembly/stability of fungal complex I in contrast to the situation in mammals.

A Fungus Lacking Respiratory Complexes III and IV

Results and Discussion The long-lived complex-III-deficient mutant Cyc1-1 has a I–IV supercomplex To gain insight into the molecular basis of lifespan extension in cytochrome-deficient mutants, we analyzed the steady-state OXPHOS system of Cox5::ble, Cyc1-1, the rescue mutants Cox5::ble(GpdAox) and Cyc1-1(Gpd-Aox), and juvenile wild-type mitochondria as a control. This was done by bluenative (BN) PAGE of mitochondria solubilized with the particularly gentle detergent digitonin able to preserve OXPHOS supercomplexes (e.g., Refs. [3–22,24–26,46,47]). For direct comparison of the respiratory chain in these strains, the BN gels were probed for in-gel activity of NADH dehydrogenase (NADH-DH; complex I) and COX (complex IV) (Fig. 1a). Second-dimension SDS gels gave the characteristic subunit patterns of OXPHOS complexes and supercomplexes (Fig. 1b and c). In line with previous results from Podospora12 and Neurospora13 wild-type mitochondria, large amounts of supercomplexes comprising complexes I, III, and IV (I1IV1, I1III2IV1, and IxIIIyIVz) as well as the smaller ones (III2IV1 and III2IV2) along with ATP synthase monomers and dimers (V1 and V2) were found in our wild-type culture (Fig. 1a and b). As expected, the complex-IV-deficient mutants Cox5::ble and Cox5::ble(Gpd-Aox) displayed no bands with COX activity but a pattern of high-molecular-weight species with NADH-DH activity (Fig. 1a). Seconddimension SDS-PAGE (not shown) confirmed those bands to be monomeric and dimeric complex I (I1 and I2) as well as the I–III supercomplexes I1III2 and I2III2, previously found in very similar amounts in the mutants ex1 and grisea12 likewise lacking complex IV. Importantly, since the mutants Cox5::ble, ex1, and grisea carry different mutations either in nuclear or in mitochondrial genes, these results suggest that any specific loss of complex IV leads to a characteristic AOX-dependent respiratory chain comprising monomeric and dimeric complex I as well as I–III supercomplexes.12 In contrast, in the mutants Cyc1-1 and Cyc1-1(Gpd-Aox) devoid of complex III activity,44 the in-gel activity stainings immediately verified the presence of complex IV and monomeric complex I as the predominant complex I species (such as in the COX-deficient mutants) as well as a distinct band with both NADH-DH and COX activity (Fig. 1a). 2D BN/SDS-PAGE demonstrated that this high-molecular-mass species (∼ 1250 kDa) is a supercomplex of each a monomer of complexes I and IV (I1IV1) (Fig. 1c), also found in the wild type (Fig. 1b), N. crassa,13 and bovine heart3,6 by the same approach. Significantly, this direct complex I–complex IV interaction was corroborated in 2D and 3D single-particle structures of the bovine heart supercomplex I1III2IV1.21,22 While complex I dimers could not be detected after digitonin solubilization of Cyc1-1 (Fig. 1a and c) and Cyc1-1(Gpd-Aox) mitochondria (Fig. 1a), very

A Fungus Lacking Respiratory Complexes III and IV

261

Fig. 1. Analysis of the respiratory chain of P. anserina wild type and the cytochrome-deficient mutants Cox5::ble, Cox5::ble(Gpd-Aox), Cyc1-1, and Cyc1-1(Gpd-Aox). Digitonin-solubilized mitochondria were analyzed by (a–c) BN-PAGE or (d) CN-PAGE in the first dimension, which were subsequently used for (a) Coomassie-staining (CBB) or in-gel activity assays of respiratory complexes I (NADH-DH, purple bands) and IV (COX, brown-yellowish bands) and (b–d) seconddimension SDS-PAGE to resolve the subunits of all OXPHOS complexes and their supercomplexes in vertical rows. (a–d) Besides ATP synthase monomers and dimers (V1 and V2), the individual respiratory complexes I to IV as well as the respiratory supercomplexes are indicated. The supercomplex I1IV1 found in wild type, Cyc1-1, and Cyc1-1(Gpd-Aox) is highlighted in red letters. (a) The bands of Cox5::ble, Cox5::ble(Gpd-Aox) displaying NADH-DH activity (I2, I1III2, I2III2) as well as ATP synthase dimers (V2) with bound Coomassie dye from prior BN-PAGE are highlighted in green letters. Note that supercomplexes I1IV1 and ATP synthase dimers (V2) have very similar apparent masses with partially overlapping bands, which is also true for complexes I1 and III2IV1 as well as for the complexes V1, III2, and IV2. (b) Additionally, the discernible subunits of individual complex III (III2) of wild-type mitochondria are marked by arrowheads and numbers. Note that supercomplexes I1IV1 and III2IV2 as well as ATP synthase dimers (V2) have very similar apparent masses. (c) Three missing subunits of the smaller form of individual complex IV (IV1b) are indicated by arrowheads. (a–d) Whereas wild type possesses large amounts of I–III–IV and III–IV supercomplexes as previously described, the mutants Cox5::ble and Cox5::ble(Gpd-Aox) are characterized by the isolated absence of assembled complex IV and the mutants Cyc1-1 and Cyc1-1(Gpd-Aox) are characterized by the specific loss of assembled complex III.

low amounts of complex I dimers were verified after extraction with Triton X-100 at an intermediate detergent/protein ratio of 1.5 g/g (not shown) such as in N. crassa.13 Moreover, individual complex IV in Cyc1-1 and Cyc1-1(Gpd-Aox) had a slightly higher mobility than in wild type (Fig. 1a–c) probably due to more delipidation. In each one culture among several independent ones of Cyc1-1 and Cyc1-1(Gpd-Aox), low amounts of an additional individual complex IV species that missed several subunits (IV1b, Fig. 1a and c) were found, but not under milder colorlessnative (CN) PAGE conditions that also increase the

proportion of preserved supercomplexes from wildtype mitochondria (Fig. 1d). This rather points at a differential effect of the experimental conditions as discussed elsewhere16,17,46 and, in any case, does not distract from the finding that complex I is at least partially assembled with complex IV. Noteworthy, no significant differences between the cytochrome-deficient mutants Cox5::ble and Cyc1-1 on one hand and their respective AOXoverexpressing rescue mutants on the other were evidenced by in-gel activity stainings (Fig. 1a) and 2D BN/SDS gels (not shown), in particular not in the

262

A Fungus Lacking Respiratory Complexes III and IV

abundance of OXPHOS complexes and the ratio of digitonin-stable supercomplexes. This suggests that the supramolecular organization of the respiratory chain is essentially identical, only distinguished by a different content of AOX. The respiratory chain organization of strains lacking either complex III or complex IV resembles each other Taken together, the supramolecular architecture of the AOX-dependent respiratory chains in mutants with the specific absence of either complex III or complex IV mutually resembles each other. Besides dimerization, complex I is at least partially assembled with either complex III or complex IV into hetero-supercomplexes, although neither complex III nor complex IV has a direct respiratory function. Since the formation of protein complexes and supercomplexes is a sophisticated and energyconsuming process, these results suggest that, in a redundant manner, either one of complex III or IV is necessary for assembly/stability of complex I by forming supercomplexes. In fact, such a role of complex III and/or complex IV was deduced in mammals from the study of human patients and cell lines with mutations in cytochrome b5,23 of complex III or an essential subunit25 or assembly factor24 of complex IV leading to combined deficiencies of complexes III and I5,23 or complexes IV and I24,25 and also indicated in bacteria. 47 Accordingly, the reduced amounts of assembled complex I in Barth syndrome patients were ascribed to be a consequence of destabilized I–III–IV supercomplexes due

to cardiolipin deficiency.26 Supporting this interpretation, it appears that fungal complex I is obligatorily incorporated into I–III–IV supercomplexes and/or assembled into homodimers. This was suggested due to the respiratory chain organization in three N. crassa mutants each lacking a peripheral complex I subunit but assembling a “complete” complex I: These have I–III–IV supercomplexes and complex I dimers like the wild type even in the case of the nuo51 mutant whose complex I without the NADH-binding 51-kDa subunit is inactive.13 The double mutant Cyc1-1,Cox5::ble lacks complexes III and IV, proving that both complexes are dispensable for assembly/ stability of complex I To prove the abovementioned conclusion, we constructed a double mutant line lacking both complex III and complex IV. Since the Gpd-Aox transgene restores not only senescence but also, partially, fertility in Cyc1-144 and Cox5::ble,45 we crossed Cox5::ble(GpdAox) with Cyc1-1 and crossed Cox5::ble with Cyc1-1 (Gpd-Aox). All genotypes resulting from these crosses were viable, such that we eventually obtained the double mutant Cyc1-1,Cox5::ble and the double mutant carrying the Gpd-Aox transgene, Cyc1-1, Cox5::ble(Gpd-Aox). The ascospores of Cyc1-1 and the double mutant Cyc1-1,Cox5::ble had low (∼20%) germination rates compared to those of Cox5::ble (∼80%) and wild type (90–100%). Also, Cyc1-1 and Cyc1-1,Cox5::ble ascospores were typically white or green, whereas Cox5::ble and wild-type ascospores were invariably black (Table 1). Hence, with regard to

Table 1. Phenotypes associated with the absence of complex III (Cyc1-1), IV (Cox5::ble), and III + IV (Cyc1-1,Cox5::ble) in P. anserina

Genotype Cyc1+,Cox5+ (wild type) Cyc1+,Cox5+(Gpd-Aox) Cyc1+,Cox5+,Aox::hygc Loss of complex III Cyc1-1,Cox5+ Cyc1-1,Cox5+(Gpd-Aox) Cyc1-1,Cox5+,Aox::hygc Loss of complex IV Cyc1+,Cox5::ble Cyc1+,Cox5::ble(Gpd-Aox) Cyc1+,Cox5::ble,Aox:: hygc Loss of complexes III and IV Cyc1-1,Cox5::ble Cyc1-1,Cox5::ble(Gpd-Aox) Cyc1-1,Cox5::ble,Aox::hygc

Replicative life spana

Growth rate (mm/day), mean ± SE (n)

(mm), mean ± SE (n)

6.6 ± 0.2 (8) 6.6 ± 0.2 (8) 6.6 ± 0.2 (8)

106 ± 3 (8) 123 ± 14 (8) 104 ± 6 (8)

2.9 ± 0.1 (22) 4.5 ± 0.1 (22) (Lethal)

(days), mean ± SE (n)

Germination rate (%)

Spore color

Female fertilityb

16 ± 1 (8) 19 ± 2 (8) 16 ± 1 (8)

90–100 90–100 90–100

Black Black Black

+++ +++ +++

N500 (22) 356 ± 70 (22)

N200 (22) 79 ± 16 (22)

∼20 90–100

White/Green Black

− +

2.4 ± 0.1 (22) 4.8 ± 0.1 (22) (Lethal)

N500 (22) 389 ± 76 (22)

N200 (22) 81 ± 15 (22)

∼80 90–100

Black Black

− +

3.0 ± 0.1 (22) 4.5 ± 0.1 (22) (Lethal)

N500 (22) 774 ± 152 (22)

N200 (22) 172 ± 34 (22)

∼20 90–100

White/Green Black

− +

The complex III, complex IV, or double mutant strains all have similar reductions in growth rate and fertility and a huge increase in replicative life span. The Gpd-Aox transgene partially restores this. With regard to ascospore germination rate and spore color phenotype, Cyc1 is epistatic over Cox5. a Average values reported for Cyc1-1,Cox5+(GpdAox), Cyc1+,Cox5::ble(Gpd-Aox), and Cyc1-1,Cox5::ble(Gpd-Aox) represent estimates based on a doubly truncated distribution, because not every culture died within the time frame of the experiment. None of the Cyc1-1, Cox5::ble or double mutant cultures died within the time frame of the experiment. b +++, normal fertility; +, reduced fertility; −, infertile. c As a control, the AOX was deleted in each genetic background.

A Fungus Lacking Respiratory Complexes III and IV

263

Fig. 2. Phenotypes associated with loss of complex III (Cyc1-1), IV (Cox5::ble), and III + IV (Cyc1-1,Cox5::ble). All deficiencies cause a similar reduction in mycelial growth rate and pigmentation. When an ectopic copy of the Aox gene is introduced, under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene Gpd, the wild-type phenotype is partially restored.

germination rate and spore color, the Cyc1-1 allele was epistatic over Cox5::ble, but concerning crucial phenotypic parameters such as growth rate and life span, the single mutants and the double mutant were identical (Fig. 2; Table 1).

2D BN/SDS-PAGE of digitonin-solubilized mitochondria from the double mutant Cyc1-1,Cox5::ble demonstrated the presence of complex I as well as ATP synthase monomers and dimers but neither assembled complex III or IV (Fig. 3a). Similar to

Fig. 3. The simultaneous loss of complexes III and IV has no detrimental effect on fungal complex I assembly/stability as demonstrated in mutants Cyc1-1,Cox5::ble and Cyc1-1,Cox5::ble(Gpd-Aox). Digitonin-solubilized mitochondria were analyzed by BN-PAGE and subsequent (a and b) in-gel activity assays of NADH-DH or COX or (a) 2D SDS-PAGE. (a) The complex-III/IV-deficient double mutant Cyc1-1,Cox5::ble contains complex I monomers and dimers (I1 and I2) along with ATP synthase monomers and dimers (V1 and V2). The subunits of complex I monomers and dimers are marked by boxes. Note that the subunits of dimeric complex I (I2), displaying in-gel NADH-DH activity, are only faintly discernible due to their low amounts. Several complex I subunits are indicated by arrowheads and numbers according to the MS identification of corresponding bands in 2D BN/SDS gels of N. crassa mitochondria:13 band 1, 78-kDa subunit; band 2, 49kDa subunit; band 3, 40-kDa subunit; band 4, 21.3a-kDa and 21.3b-kDa subunits; band 5, 20.9-kDa subunit. (b) The in-gel NADH-DH stainings of both mutants, in particular the presence of complex I monomers and dimers (I1 and I2), are essentially indistinguishable from each other, whereas there is no COX activity as expected. As a control, wild-type mitochondria were loaded as shown in Fig. 1a.

264 COX-deficient strains12 (Fig. 1a), low amounts of digitonin-stable complex I dimers could be separated (Fig. 3a), which were also found in similarly low yield after solubilization with 1.5 g Triton X100/g protein (not shown) such as in N. crassa,13 along with ATP synthase monomers and dimers. Significantly, the double mutant carrying the GpdAox transgene Cyc1-1,Cox5::ble(Gpd-Aox) resembles the AOX-overexpressing rescue mutants of the single mutants Cox5::ble(Gpd-Aox) and Cyc1-1(GpdAox): Its phenotype is reversed towards wild-typelike characteristics (Fig. 2; Table 1), and the native gel experiments revealed no detectable difference of the respiratory chain in comparison with Cyc1-1, Cox5::ble (Fig. 3b). The specific activities of complexes I, III, and IV in the mutants were distinct but similar to the wild type as determined by photometric absorbance measurements of isolated mitochondria, whereas the absence of complexes III and IV in Cyc1-1,Cox5::ble was confirmed (Fig. 4). Together with the native gel analyses, these data demonstrate that the amount of assembled complex I and its enzymatic activity are not impaired by the simultaneous absence of complexes III and IV. In conclusion, it seems that, normally, the biogenesis/integrity of fungal complex I is assisted by complexes III and IV via supercomplex formation with III–IV supercomplexes or each of only complex III or IV, whenever the conditions for the assembly of either complex are in place. Indeed, this is discussed as a major role of respiratory supercomplexes besides enzymatic advantages such as substrate channeling and diminishing generation of reactive oxygen species (e.g., Refs. [5,12–15,17–26,47]). How-

A Fungus Lacking Respiratory Complexes III and IV

ever, our results unequivocally demonstrate that neither complex III nor complex IV is essential for assembly/stability of fungal complex I, in apparent contrast to the situation in mammals5,23–26 and bacteria.47 What is the molecular basis of complex I assembly/stability in fungi when complexes III and IV are absent? Complex I is the most complicated and least understood OXPHOS complex composed of nuclear and mitochondrially encoded proteins,30,31,37,48 at least 39 subunits in fungi.49 It seems clear that many proteins support its biogenesis, but only a few are known or just emerging.31,37,50–52 How is fungal complex I assembled/stabilized when complexes III and IV are absent? Fungal specific features that account for it can be readily proposed. There must be factors that either are obligatorily involved and sufficient for assembly/stability of fungal complex I or fulfill this task as a backup mechanism when both complex III and complex IV are absent. First, peculiarities of complex I composition and its assembly process may be important; for example, at least two fungal specific subunits49 that might have a direct role in biogenesis/stability or that might be significant for other assembly factors are known. Indeed, it is of special note that although the available data of complex I biogenesis in various organisms allow the proposal of a general assembly model,50 they indicate some distinct traits in mammals, fungi, plants, and algae. For example, whereas both membrane and peripheral arm are separately assembled and joined together in N. crassa,30,31,37,50,51,53,54 it

Fig. 4. Specific activities of respiratory complexes I, III, and IV in submitochondrial particles of wild type, Cyc1-1, Cox5::ble, and the double mutant Cyc1-1,Cox5::ble. The activities of the respiratory complexes were measured photospectrometrically by following changes in absorbance due to changes in the redox states of the substrates NADH (complex I) and cytochrome c (complexes III and IV). Data represent the mean values ± SD of at least three independent mitochondrial preparations (n ≥ 3) expressed in nmol/min × mg mitochondrial protein.

265

A Fungus Lacking Respiratory Complexes III and IV

appears that, in mammals, early assembly intermediates of the membrane and peripheral arm combine before the remaining subunits are incorporated.50,51 Second, fungal specific mitochondrial proteins not present in mammals may ensure or contribute to the assembly/stability of complex I either as chaperones50,51 or by forming stable interactions akin to the supercomplexes with complex III and/or complex IV, that is, conceivable peculiarities of the supramolecular organization of the fungal respiratory chain. Apparent candidates are the alternative respiratory enzymes, notably the AOX exclusively conducting the obligate respiration together with complex I as the only coupling site to provide ATP in cytochrome-deficient mutants missing complex III and/or complex IV. As another peculiar trait, the existence of complex I dimers is so far clearly shown only in filamentous fungi12,13 (this study), although it is cogitable that mammals and others might also contain complex I dimers.17 Consequently, the dimerization or even oligomerization of complex I might ensure the assembly/ stability of complex I. The AOX is a candidate protein contributing to assembly/stability of fungal complex I As demonstrated for complexes III and IV in the present study, it is already known that the AOX per se is not essential for complex I assembly/stability, since the wild type is unaffected by an isolated loss of the AOX45 (Cyc1+,Cox5+,Aox::hyg, Table 1). Unfortunately, since the loss of the AOX is lethal per se in all the complex-III/IV-deficient mutants due to its exclusive use as the only available respiratory pathway, the assumed role of the AOX for complex I assembly/stabilization cannot be proven by generating a nonviable AOX-disrupted mutant, that is, the triple mutant Cyc1-1,Cox5::ble,Aox::hyg (Table 1). Engineering a reduction in the expression level or activity of AOX may give important clues if a corresponding decrease in assembled complex I would be observed. Vice versa, the several-fold AOX overexpression by the Gpd promoter44,45 (this study) results in an unaltered supramolecular organization of the respiratory chain as well as unchanged levels of complex I compared with the respective complex-III/IV-deficient mutants (Figs. 1a and 3b) and likewise does not allow a conclusion whether the AOX is involved in complex I assembly/stability. Despite these experimental constraints, a stable interaction of complex I with the AOX would strongly argue in favor of such a role as mentioned above. Hence, we tested whether the AOX is physically associated with respiratory complexes or supercomplexes by anti-AOX immunoblots of 2D BN/ SDS and 2D CN/SDS gels. In fact, the blots of the six cytochrome-deficient strains revealed only specific signals near the running front, indicating individual AOX (not shown) as in the ex1 mutant.12 Nonetheless, it is possible that a complex I–AOX interaction is not stable under the experimental conditions of detergent treatment and native electrophoresis as

discussed elsewhere.16,17 To our knowledge, there is only one report suggesting a physical interaction of the AOX with the respiratory chain in any organism; namely, tomato and amoeba AOX was found to comigrate with complex III in BN-PAGE,55 but this must be corroborated by further evidence. Importantly, an intriguing fact that has hitherto escaped attention when discussing the assembly/ stabilization of complex I as a major role of I–III–IV supercomplexes5,12–15,17–26 sheds more light on this unsolved issue. The occurrence of isolated losses of either complex III or complex IV was reported in various AOX-possessing eukaryotes besides P. anserina. In detail, such mutants with specific deletions of cytochrome b are characterized in the green alga Chlamydomonas reinhardtii56–59 as well as the facultatively aerobic yeast Debaryomyces occidentalis.60,61 Furthermore, knock-downs of cytochrome c1 and the Rieske protein in the procyclic form of the protist Trypanosoma brucei leading to virtual absence of complex III are viable.62 Correspondingly, specific deficiencies of complex IV are also described in C. reinhardtii58,59 and T. brucei.62 Both active complex I and induced AOX respiration are confirmed in these mutants56–62 such as in cytochrome-deficient P. anserina strains lacking complex III and/or complex IV. In particular, the cytochrome b deletions reported in C. reinhardtii and D. occidentalis56–61 correspond to the genetic defects in patients with mutations in cytochrome b leading to severe reduction of assembled complex III and as consequence thereof to a combined complex I + III deficiency.5,23,63–65 Taken together, these correlations trigger the assumption that various (if not most) AOX-expressing eukaryotes may simultaneously omit complexes III and IV for assembly/stability of complex I and that exactly the presence of the AOX is responsible for it. The P. anserina mutants Cyc1-1, Cox5::ble and Cyc1-1,Cox5::ble(Gpd-Aox) are the first known obligatorily aerobic eukaryote strains expressing complex I but devoid of both complexes III and IV. Hence, the generation (if genetic tools are available) and analysis of corresponding viable or nonviable mutants of AOX-expressing species from algae, plants, and other eukaryotic phyla will significantly help to verify and elucidate the proposed role of the AOX in complex I assembly/ stability and its phylogenetical conservation. At least in the case of the alga C. reinhardtii, this ambitious goal is achievable. Accordingly, it would be of particular interest to examine whether the heterologous AOX expression66,67 in patient cell cultures with combined I + III defects due to mutations in cytochrome b5,23,63–65 can restore both complex I levels and the respiratory activity.

Materials and Methods Strains and cultivation All genotypes were studied in the background of P. anserina (Ces.) Rehm standard laboratory strain s as

266 described elsewhere,42,44,45 which is also used as wildtype strain in this study. Cyc1-1 is a loss-of-function mutation in the Cyc1 gene, caused by a single nucleotide substitution within the branch site of the second intron of this gene.44 Cox5::ble and Aox::hyg are targeted knockouts of Cox5 and Aox, respectively.42,45 Gpd-Aox is an ectopic insertion of Aox under the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene Gpd.45 The Cyc1-1 mutation is an A-to-C transversion in the branch site of the second intron of Cyc1. This mutation destroys a BsrI restriction site. We conveniently used a cleaved amplified polymorphic sequence marker or polymerase chain reaction–restriction fragment length polymorphism analysis to distinguish the mutant from the wild-type allele without having to resort to sequencing. DNA was extracted using a standard phenol/ chloroform extraction procedure, and a 301-bp fragment was amplified in a PCR using the oligonucleotide primers 5′-AAG GGT GAG TGG TTG ATC GG-3′ and 5′-GAG ACT GTG TGC GAG CAA GA-3′. The fragment was digested using BsrI, yielding two products of 139 and 162 bp in the wild type and a 301-bp product in the mutant. The Cox5 and Aox knock-outs and the Gpd-Aox transgene were conveniently marked by antibiotic resistance cassettes.42,45 Construction of the double mutant Cyc1-1,Cox5::ble First, we tried to cross Cyc1-1 with Cox5::ble cultures of the opposite mating type by confronting the mycelia. At the interface of the cultures, heterokaryons formed with a fully wild-type phenotype as might be expected. However, these heterokaryons did not form any perithecia or fruiting bodies but were female sterile even though they were hetero-allelic for both genes. Complementation thus occurred at the vegetative level, but unexpectedly not at the sexual level, requiring a circumstantial approach. Therefore, we repeated these crosses using the Gpd-Aox transgene (see above), since Gpd-Aox restores not only senescence in Cyc1-144 and Cox5::ble45 but also, partially, fertility. Cox5::ble(Gpd-Aox) was crossed with a Cyc1-1(Gpd-Aox) culture of the opposite mating type, indeed yielding plentiful asci that were analyzed by a combination of phenotypic and molecular markers: Cox5::ble and Gpd-Aox were marked using phleomycin and hygromycin resistance cassettes, respectively. Cyc1-1 could be distinguished from the wild-type Cyc1 allele using a cleaved amplified polymorphic sequence marker or polymerase chain reaction–restriction fragment length polymorphism analysis. Out of 40 asci that were analyzed, 7 tetratypes, 15 parental ditypes, and 18 nonparental ditypes were recovered, in line with known segregation patterns of Cyc1 (100% first-division segregation) and Cox5 (∼ 75% first-division segregation). All possible genotypes were viable, allowing to recover Cyc1-1,Cox5::ble(Gpd-Aox) cultures. These were identical in phenotype to Cyc1-1 (Gpd-Aox) and Cox5::ble(Gpd-Aox) cultures (i.e., quasiwild type). To get double mutant cultures without the Gpd-Aox transgene, we crossed Cox5::ble(Gpd-Aox) with a Cyc1-1 culture of the opposite mating type and we crossed Cox5::ble with Cyc1-1(Gpd-Aox). This also yielded plentiful asci, of which 36 asci were analyzed. Again, the types of asci were in line with the known segregation patterns of the genes involved. All genotypes were viable, such that we eventually obtained the double mutant Cyc1-1,Cox5::ble.

A Fungus Lacking Respiratory Complexes III and IV

Life-span analysis Growth rates and life spans were routinely measured using approximately 30-cm-long glass “race tubes” at 27 °C and at an approximate humidity of 70%. Crosses and subsequent analyses of the progeny were made as previously described.42,44,45 Isolation of mitochondria Young cultures were derived from monokaryotic ascospores that had been incubated for 1 day or for 2 days in cornmeal (complete) medium supplemented with ammonium acetate to improve germination.44,45 Explants of these were transferred to regular cornmeal medium (MR). For the isolation of mitochondria, they were transferred to solid medium with cellophane disks. After 2–3 days, the mycelium was harvested, fragmented, and transferred to 5-L erlenmeyers with liquid cornmeal (complete) medium. Cultures were incubated (shaken) at 27 °C until they were in mid-logarithmic phase. Next, the mycelium was harvested by filtering the cultures over several layers of cheesecloth, and mitochondria were isolated immediately as described previously.12,68 Briefly, wet mycelium (typically between 10 and 20 g) was disrupted by homogenization using glass beads and suspended in prechilled 0.4 M sucrose, 50 mM Tris–HCl, and 2 mM ethylenediaminetetraacetic acid, pH 7.4, supplemented with 0.1% (v/v) β-mercaptoethanol. This suspension was centrifuged twice for 10 min at low speed (1450g) to pellet nuclei and large cellular debris. The supernatant was filtered and centrifuged for 25 min at high speed (27,000g) to obtain a crude mitochondrial fraction. The crude fraction was resuspended in 0.4 M sucrose buffer, pH7.4, and purified by sucrose gradient centrifugation or immediately flash-frozen in liquid nitrogen and stored at −80 °C. For BN-PAGE and CNPAGE experiments, the crude mitochondria were used. For the in vitro activity assays, the sucrose gradientpurified mitochondria were used. All steps were performed at 4 °C. Electrophoresis and in-gel activity assays Solubilization and electrophoresis were performed as described previously.13,46 In detail, mitochondria were thawed on ice and centrifuged at 20,800g for 8 min. The pellet was suspended in the solubilization buffer containing 50 mM NaCl, 50 mM imidazole/HCl (pH 7.0), 10% glycerol and 5 mM 6-aminocaproic acid (final concentration). Mitochondria were solubilized with digitonin (AppliChem, A1905) using a detergent/protein ratio of 4 g/g or with Triton X-100 (Roche Diagnostics) using a detergent/protein ratio of 1.5 g/g at a final detergent concentration of 1% by adding a freshly prepared 10% detergent solution. The samples were incubated for 30 min at 4 °C with slight agitation followed by centrifugation at 20,800g for 10 min. The extracts were directly loaded onto native gels. For BN-PAGE, linear 3–13% gradient gels overlaid with a 3% stacking gel were used in a Hoefer SE 600 system (18 × 16 × 0.15 cm3) with electrophoresis conditions as described previously.13,46 CN-PAGE was performed identically, except for the omission of Coomassie Blue dye in the cathode buffer.13,46 The apparent molecular masses of the OXPHOS complexes and their supercomplexes were calibrated by digitonin-solubilized bovine heart mitochondria3,6 applied to the same first-

267

A Fungus Lacking Respiratory Complexes III and IV dimension BN gel as earlier described.12,13 Lanes from the first-dimension BN-PAGE or CN-PAGE were then excised and used for in-gel activity staining (see below) or a second-dimension 13% SDS-PAGE8,13,46 (silver staining) or 10% SDS-PAGE46 (immunoblot). The supercomplexes were assigned according to their characteristic subunit compositions revealed in 2D SDS-PAGE and apparent molecular masses. Additionally, many of the subunits of the fungal OXPHOS complexes had already been identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.12,13 NADH-DH activity was probed by the in-gel precipitation of formazan, and COX activity was probed by the ingel precipitation of 3,3′-diaminobenzidine oxides and indamine polymers as described previously.12,13 Immunoblotting and immunodetection For the immunodetection of AOXp, second-dimension 10% SDS gels were electroblotted onto polyvinylidene difluoride membranes in 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol (pH∼ 8.3) using the Bio-Rad Tankblot system. The efficient transfer of proteins was controlled with the SYPRO Ruby blot stain (Bio-Rad Laboratories). The antibody decoration was carried out as described previously,42,44,45 whereas immunodetection was done using the chemiluminescent SuperSignal West Dura Substrate (Pierce Laboratories) in a Fujifilm LAS3000 Imager. Enzymatic analyses The enzymatic activities of the respiratory complexes were quantified using absorbance photospectrometry as described previously.44 Mitochondria were sucrose gradient purified and subjected to osmotic shock and sonication to generate submitochondrial particles. Complex I activity was measured by following the rotenonesensitive rate of β-NADH oxidation at 340 nm (ɛ = 6.22 mM− 1 cm− 1). Decylubiquinone (DBQ, Sigma) was used as an artificial electron acceptor. Assays were made in 50 mM Na2HPO4/NaH2PO4, 50 μM β-NADH, and 50 μM DBQ, pH 7.4, at 27 °C. Complex III activity was measured by following the antimycin-sensitive rate of cytochrome c reduction at 550 nm (ɛ = 19 mM− 1 cm− 1). Reduced DBQ was used as an artificial electron donor. Assays were made in 50 mM Na2HPO4/NaH2PO4, 180 μM reduced DBQ, and 80 μM cytochrome c, pH 7.4, at 27 °C. Complex IV activity was measured by following the cyanide-sensitive rate of cytochrome c oxidation at 550 nm (ɛ = 19 mM− 1 cm− 1). Assays were made in 50 mM Na2HPO4/NaH2PO4 and 80 μM reduced cytochrome c, pH 7.4, at 27 °C.

Acknowledgements This work was supported by the European consortium “MiMage—Role of Mitochondria in Conserved Mechanisms of Ageing”, EC FP6 Contract No. LSHM-CT-2004-512020 to N.A.D. and A.S.-C. as well as by Deutsche Forschungsgemeinschaft grant SFB 472 to N.A.D and Holger Seelert and by grants from the Centre National de la

Recherche Scientifique and the Association Française contre les Myopathies to A.S.-C. This publication reflects only the authors' view. The EC is not liable for any use that may be made from the information herein.

Author contributions M.F.P.M.M., F.K. and A.S.-C. conceived and designed the experiments. M.F.P.M.M. performed the experiments. All authors analyzed the data. M.F. P.M.M. and F.K. wrote the paper. All authors commented on and discussed the paper.

References 1. Saraste, M. (1999). Oxidative phosphorylation at the fin de siècle. Science, 283, 1488–1493. 2. Tielens, A. G., Rotte, C., van Hellemond, J. J. & Martin, W. (2002). Mitochondria as we don't know them. Trends Biochem. Sci. 27, 564–572. 3. Schägger, H. & Pfeiffer, K. (2000). Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J. 19, 1777–1783. 4. Cruciat, C. M., Brunner, S., Baumann, F., Neupert, W. & Stuart, R. A. (2000). The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria. J. Biol. Chem. 275, 18093–18098. 5. Schägger, H., de Coo, R., Bauer, M. F., Hofmann, S., Godinot, C. & Brandt, U. (2004). Significance of respirasomes for the assembly/stability of human respiratory chain complex I. J. Biol. Chem. 279, 36349–36353. 6. Krause, F., Reifschneider, N. H., Goto, S. & Dencher, N. A. (2005). Active oligomeric ATP synthases in mammalian mitochondria. Biochem. Biophys. Res. Commun. 329, 583–590. 7. Wittig, I. & Schägger, H. (2005). Advantages and limitations of clear-native PAGE. Proteomics, 5, 4338–4346. 8. Reifschneider, N. H., Goto, S., Nakamoto, H., Takahashi, R., Sugawa, M., Dencher, N. A. & Krause, F. (2006). Defining the mitochondrial proteomes from five rat organs in a physiologically significant context using 2D blue-native/SDS-PAGE. J. Proteome Res. 5, 1217–1232. 9. Eubel, H., Jänsch, L. & Braun, H. P. (2003). New insights into the respiratory chain of plant mitochondria. Supercomplexes and a unique composition of complex II. Plant Physiol. 133, 274–286. 10. Eubel, H., Heinemeyer, J. & Braun, H. P. (2004). Identification and characterization of respirasomes in potato mitochondria. Plant Physiol. 134, 1450–1459. 11. Krause, F., Reifschneider, N. H., Vocke, D., Seelert, H., Rexroth, S. & Dencher, N. A. (2004). “Respirasome”like supercomplexes in green leaf mitochondria of spinach. J. Biol. Chem. 279, 48369–48375. 12. Krause, F., Scheckhuber, C. Q., Werner, A., Rexroth, S., Reifschneider, N. H., Dencher, N. A. & Osiewacz, H. D. (2004). Supramolecular organization of cytochrome c oxidase- and alternative oxidase-dependent respiratory chains in the filamentous fungus Podospora anserina. J. Biol. Chem. 279, 26453–26461. 13. Marques, I., Dencher, N. A., Videira, A. & Krause, F. (2007). Supramolecular organization of the respiratory chain in Neurospora crassa mitochondria. Eukaryot. Cell, 6, 2391–2405.

268 14. Schägger, H. (2002). Respiratory chain supercomplexes of mitochondria and bacteria. Biochim. Biophys. Acta, 1555, 154–159. 15. Wittig, I., Carrozzo, R., Santorelli, F. M. & Schägger, H. (2006). Supercomplexes and subcomplexes of mitochondrial oxidative phosphorylation. Biochim. Biophys. Acta, 1757, 1066–1072. 16. Krause, F. (2006). Detection and analysis of protein– protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (membrane) protein complexes and supercomplexes. Electrophoresis, 27, 2759–2781. 17. Krause, F. (2007). Oxidative phosphorylation supercomplexes in various eukaryotes: A paradigm change gains critical momentum. In Complex I and Alternative NADH Dehydrogenases (González-Siso, M. I., ed), pp. 179–213, Transworld Research Network, Kerala, India. 18. Genova, M. L., Baracca, A., Biondi, A., Casalena, G., Faccioli, M., Falasca, A. I. et al. (2008). Is supercomplex organization of the respiratory chain required for optimal electron transfer activity? Biochim. Biophys. Acta, 1777, 740–746. 19. Dudkina, N. V., Eubel, H., Keegstra, W., Boekema, E. J. & Braun, H. P. (2005). Structure of a mitochondrial supercomplex formed by respiratory chain complexes I and III. Proc. Natl Acad. Sci. USA, 102, 3225–3229. 20. Heinemeyer, J., Braun, H. P., Boekema, E. B. & Kouřil, R. (2007). A structural model of the cytochrome c reductase/oxidase supercomplex from yeast mitochondria. J. Biol. Chem. 282, 12240–12248. 21. Schäfer, E., Seelert, H., Reifschneider, N. H., Krause, F., Dencher, N. A. & Vonck, J. (2006). Architecture of active mammalian respiratory chain supercomplexes. J. Biol. Chem. 281, 15370–15375. 22. Schäfer, E., Dencher, N. A., Vonck, J. & Parcej, D. (2007). Three-dimensional structure of the respiratory chain supercomplex I1III2IV1 from bovine heart mitochondria. Biochemistry, 46, 12579–12585. 23. Acín-Peréz, R., Bayona-Bafaluy, M. P., FernándezSilva, P., Moreno-Loshuertos, R., Pérez-Martos, A., Bruno, C. et al. (2004). Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol. Cell, 13, 805–815. 24. Diaz, F., Fukui, H., Garcia, S. & Moraes, C. T. (2006). Cytochrome c oxidase is required for the assembly/ stability of respiratory complex I in mouse fibroblasts. Mol. Cell. Biol. 26, 4872–4881. 25. Li, Y., D'Aurelio, M., Deng, J. H., Park, J. S., Manfredi, G., Hu, P. et al. (2007). An assembled complex IV maintains the stability and activity of complex I in mammalian mitochondria. J. Biol. Chem. 282, 17557–17562. 26. McKenzie, M., Lazarou, M., Thorburn, D. R. & Ryan, M. T. (2006). Mitochondrial respiratory chain supercomplexes are destabilized in Barth Syndrome patients. J. Mol. Biol. 361, 462–469. 27. Møller, I. M. (2001). Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 561–591. 28. Melo, A. M., Bandeiras, T. M. & Teixeira, M. (2004). New insights into type II NAD(P)H:quinone oxidoreductases. Microbiol. Mol. Biol. Rev. 68, 603–616. 29. Joseph-Horne, T., Hollomon, D. W. & Wood, P. M. (2001). Fungal respiration: a fusion of standard and alternative components. Biochim. Biophys. Acta, 1504, 179–195. 30. Videira, A. & Duarte, M. (2002). From NADH to ubiquinone in Neurospora mitochondria. Biochim. Biophys. Acta, 1555, 187–191.

A Fungus Lacking Respiratory Complexes III and IV

31. Duarte, M. & Videira, A. (2007). Mitochondrial NAD(P)H dehydrogenases in filamentous fungi. In Complex I and Alternative NADH Dehydrogenases (González-Siso, M. I., ed), pp. 55–68, Transworld Research Network, Kerala, India. 32. Moore, A. L., Albury, M. S., Crichton, P. G. & Affourtit, C. (2002). Function of the alternative oxidase: is it still a scavenger? Trends Plant Sci. 7, 478–481. 33. McDonald, A. & Vanlerberghe, G. (2004). Branched mitochondrial electron transport in the Animalia: presence of alternative oxidase in several animal phyla. IUBMB Life, 56, 333–341. 34. Davis, R. H. & Perkins, D. D. (2002). Timeline: Neurospora: a model of model microbes. Nat. Rev. Genet. 3, 397–403. 35. Kiebler, M., Pfaller, R., Söllner, T., Griffiths, G., Horstmann, H., Pfanner, N. & Neupert, W. (1990). Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature, 348, 610–616. 36. Neupert, W. & Herrmann, J. M. (2007). Translocation of proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749. 37. Schulte, U. (2001). Biogenesis of respiratory complex I. J. Bioenerg. Biomembr. 33, 205–212. 38. Espagne, E., Lespinet, O., Malagnac, F., Da Silva, C., Jaillon, O., Porcel, B. M. et al. (2008). The genome sequence of the model ascomycete fungus Podospora anserina. Genome Biol. 9, R77. 39. Osiewacz, H. D. (2002). Genes, mitochondria and aging in filamentous fungi. Ageing Res. Rev. 1, 425–442. 40. Lorin, S., Dufour, E. & Sainsard-Chanet, A. (2006). Mitochondrial metabolism and aging in the filamentous fungus Podospora anserina. Biochim. Biophys. Acta, 1757, 604–610. 41. Schulte, E., Kück, U. & Esser, K. (1988). Extrachromosomal mutants from Podospora anserina: permanent vegetative growth in spite of multiple recombination events in the mitochondrial genome. Mol. Gen. Genet. 211, 342–349. 42. Dufour, E., Boulay, J., Rincheval, V. & SainsardChanet, A. (2000). A causal link between respiration and senescence in Podospora anserina. Proc. Natl Acad. Sci. USA, 97, 4138–4143. 43. Stumpferl, S. W., Stephan, O. & Osiewacz, H. D. (2004). Impact of a disruption of a pathway delivering copper to mitochondria on Podospora anserina metabolism and life span. Eukaryot. Cell, 3, 200–211. 44. Sellem, C. H., Marsy, S., Boivin, A., Lemaire, C. & Sainsard-Chanet, A. (2007). A mutation in the gene encoding cytochrome c1 leads to a decreased ROS content and to a long-lived phenotype in the filamentous fungus Podospora anserina. Fungal Genet. Biol. 44, 648–658. 45. Lorin, S., Dufour, E., Boulay, J., Begel, O., Marsy, S. & Sainsard-Chanet, A. (2001). Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina. Mol. Microbiol. 42, 1259–1267. 46. Krause, F. & Seelert, H. (2008). Detection and analysis of protein–protein interactions in organellar and prokaryotic proteomes by blue-native and colorlessnative gel electrophoresis. Curr. Protoc. Protein Sci. 51, 14.11.1–14.11.36. republished as 51, 19.18.1–19.18.36. 47. Stroh, A., Anderka, O., Pfeiffer, K., Yagi, T., Finel, M., Ludwig, B. & Schägger, H. (2004). Assembly of respiratory complexes I, III, and IV into NADH oxidase

269

A Fungus Lacking Respiratory Complexes III and IV

48. 49.

50.

51. 52.

53.

54.

55.

56.

57.

58.

supercomplex stabilizes complex I in Paracoccus denitrificans. J. Biol. Chem. 279, 5000–5007. Brandt, U. (2006). Energy converting NADH:quinone oxidoreductase (complex I). Annu. Rev. Biochem. 75, 69–92. Marques, I., Duarte, M., Assunção, J., Ushakova, A. V. & Videira, A. (2005). Composition of complex I from Neurospora crassa and disruption of two “accessory” subunits. Biochim. Biophys. Acta, 1707, 211–220. Vogel, R. O., Smeitink, J. A. & Nijtmans, L. G. (2007). Human mitochondrial complex I assembly: a dynamic and versatile process. Biochim. Biophys. Acta, 1767, 1215–1227. Lazarou, M., Thorburn, D. R., Ryan, M. T. & McKenzie, M. (2009). Assembly of mitochondrial complex I and defects in disease. Biochim. Biophys. Acta, 1793, 78–88. Pagliarini, D. J., Calvo, S. E., Chang, B., Sheth, S. A., Vafai, S. B., Ong, S. E. et al. (2008). A mitochondrial protein compendium elucidates complex I disease biology. Cell, 134, 112–123. Tuschen, G., Sackmann, U., Nehls, U., Haiker, H., Buse, G. & Weiss, H. (1990). Assembly of NADH: ubiquinone reductase (complex I) in Neurospora mitochondria. Independent pathways of nuclearencoded and mitochondrially encoded subunits. J. Mol. Biol. 213, 845–857. Nehls, U., Friedrich, T., Schmiede, A., Ohnishi, T. & Weiss, H. (1992). Characterization of assembly intermediates of NADH:ubiquinone oxidoreductase (complex I) accumulated in Neurospora mitochondria by gene disruption. J. Mol. Biol. 227, 1032–1042. Navet, R., Jarmuszkiewicz, W., Douette, P., SluseGoffart, C. M. & Sluse, F. E. (2004). Mitochondrial respiratory chain complex patterns from Acanthamoeba castellanii and Lycopersicon esculentum: comparative analysis by BN-PAGE and evidence of protein– protein interaction between alternative oxidase and complex III. J. Bioenerg. Biomembr. 36, 471–479. Matagne, R. F., Michel-Wolwertz, M. R., Munaut, C., Duyckaerts, C. & Sluse, F. (1989). Induction and characterization of mitochondrial DNA mutants in Chlamydomonas reinhardtii. J. Cell Biol. 108, 1221–1226. Dorthu, M. P., Remy, S., Michel-Wolwertz, M. R., Colleaux, L., Breyer, D., Beckers, M. C. et al. (1992). Biochemical, genetic and molecular characterization of new respiratory-deficient mutants in Chlamydomonas reinhardtii. Plant Mol. Biol. 18, 759–772. Colin, M., Dorthu, M. P., Duby, F., Remacle, C., Dinant, M., Wolwertz, M. R. et al. (1995). Mutations affecting the mitochondrial genes encoding the

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii. Mol. Gen. Genet. 249, 179–184. Remacle, C., Duby, F., Cardol, P. & Matagne, R. F. (2001). Mutations inactivating mitochondrial genes in Chlamydomonas reinhardtii. Biochem. Soc. Trans. 29, 442–446. Fernet, C. S., Clark-Walker, G. D. & Claisse, M. L. (2002). The mitochondrial genome can be altered or lost without lethal effect in the petite-negative yeast Debaryomyces (Schwanniomyces) occidentalis. Curr. Genet. 42, 94–102. Fernet, C., Claisse, M. & Clark-Walker, G. D. (2003). The mitochondrial genome of Debaryomyces (Schwanniomyces) occidentalis encodes subunits of NADH dehydrogenase complex I. Mitochondrion, 2, 267–275. Horváth, A., Horáková, E., Dunajčíková, P., Verner, Z., Pravdová, E., Slapetová, I. et al. (2005). Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei. Mol. Microbiol. 58, 116–130. Lamantea, E., Carrara, F., Mariotti, C., Morandi, L. & Tiranti, V. (2002). A novel nonsense mutation (Q352X) in the mitochondrial cytochrome b gene associated with a combined deficiency of complexes I and III. Neuromuscular Disord. 12, 49–52. Bruno, C., Santorelli, F. M., Assereto, S., Tonoli, E., Tessa, A., Traverso, M. et al. (2003). Progressive exercise intolerance associated with a new musclerestricted nonsense mutation (G142X) in the mitochondrial cytochrome b gene. Muscle Nerve, 28, 508–511. Blakely, E. L., Mitchell, A. L., Fisher, N., Meunier, B., Nijtmans, L. G., Schaefer, A. M. et al. (2005). A mitochondrial cytochrome b mutation causing severe respiratory chain enzyme deficiency in humans and yeast. FEBS J. 272, 3583–3592. Hakkaart, G. A., Dassa, E. P., Jacobs, H. T. & Rustin, P. (2006). Allotopic expression of a mitochondrial alternative oxidase confers cyanide resistance to human cell respiration. EMBO Rep. 7, 341–345. Perales-Clemente, E., Bayona-Bafaluy, M. P., PérezMartos, A., Barrientos, A., Fernández-Silva, P. & Enriquez, J. A. (2008). Restoration of electron transport without proton pumping in mammalian mitochondria. Proc. Natl Acad. Sci. USA, 105, 18735–18739. Gredilla, R., Grief, J. & Osiewacz, H. D. (2006). Mitochondrial free radical generation and lifespan control in the fungal aging model Podospora anserina. Exp. Gerontol. 41, 439–447.