Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH:ubiquinone reductase ofNeurospora mitochondria

Mv, da Silva; Alves, Paulo C.; Duarte, M. M. L.; Mota, N. O.; Lobo‐da‐Cunha, Alexandre; Ta, Harkness; Fe, Nargang; Videira, Arnaldo

doi:10.1007/bf02173218

Cited by 26 publications

(6 citation statements)

References 41 publications

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“…The accessory subunits of hydrophilic Iλ matrix arm sub-complex (Figure 3, λ) show a tendency towards the top, while those of the Iα sub-complex that are not part of Iλ (3a, α-λ) tend towards the membrane arm subunits at the bottom. The association of this latter group of subunits with the proximal matrix and membrane arm is strongly supported by experimental data (S5 [42,43], A9 [20,44-46], A3 [1,2,47], A6 [48], A8 [48,49], A11 [50], A1 [21]). All assembly factors are located close to the matrix arm subunits.…”

Section: Resultssupporting

confidence: 56%

A three-dimensional topology of complex I inferred from evolutionary correlations

Kensche

Duarte

Huynen

2012

BMC Structural Biology

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BackgroundThe quaternary structure of eukaryotic NADH:ubiquinone oxidoreductase (complex I), the largest complex of the oxidative phosphorylation, is still mostly unresolved. Furthermore, it is unknown where transiently bound assembly factors interact with complex I. We therefore asked whether the evolution of complex I contains information about its 3D topology and the binding positions of its assembly factors. We approached these questions by correlating the evolutionary rates of eukaryotic complex I subunits using the mirror-tree method and mapping the results into a 3D representation by multidimensional scaling.ResultsMore than 60% of the evolutionary correlation among the conserved seven subunits of the complex I matrix arm can be explained by the physical distance between the subunits. The three-dimensional evolutionary model of the eukaryotic conserved matrix arm has a striking similarity to the matrix arm quaternary structure in the bacterium Thermus thermophilus (rmsd=19 Å) and supports the previous finding that in eukaryotes the N-module is turned relative to the Q-module when compared to bacteria. By contrast, the evolutionary rates contained little information about the structure of the membrane arm. A large evolutionary model of 45 subunits and assembly factors allows to predict subunit positions and interactions (rmsd = 52.6 Å). The model supports an interaction of NDUFAF3, C8orf38 and C2orf56 during the assembly of the proximal matrix arm and the membrane arm. The model further suggests a tight relationship between the assembly factor NUBPL and NDUFA2, which both have been linked to iron-sulfur cluster assembly, as well as between NDUFA12 and its paralog, the assembly factor NDUFAF2.ConclusionsThe physical distance between subunits of complex I is a major correlate of the rate of protein evolution in the complex I matrix arm and is sufficient to infer parts of the complex’s structure with high accuracy. The resulting evolutionary model predicts the positions of a number of subunits and assembly factors.

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Section: Resultssupporting

confidence: 56%

A three-dimensional topology of complex I inferred from evolutionary correlations

Kensche

Duarte

Huynen

2012

BMC Structural Biology

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“…Because we initially thought that NDE1 was acting on the inner side of the inner mitochondrial membrane, oxidizing matrix NADH as an alternative to complex I, we crossed the nde-1 mutant with several mutants in subunits of complex I. Surprisingly, we obtained double mutants in crosses between nde-1 and the complex I mutants nuo51 (39), nuo24 (40), nuo21.3a (26), nuo21.3c (41), nuo20.8 (42), and nuo12.3 (43), respectively, which display various phenotypes in terms of complex I assembly and function. Fig.…”

Section: Resultsmentioning

confidence: 99%

The External Calcium-dependent NADPH Dehydrogenase from Neurospora crassa Mitochondria

Melo

Duarte

Møller

et al. 2001

Journal of Biological Chemistry

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We have inactivated the nuclear gene coding for a putative NAD(P)H dehydrogenase from the inner membrane of Neurospora crassa mitochondria by repeat-induced point mutations. The respiratory rates of mitochondria from the resulting mutant (nde-1) were measured, using NADH or NADPH as substrates under different assay conditions. The results showed that the mutant lacks an external calcium-dependent NADPH dehydrogenase. The observation of NADH and NADPH oxidation by intact mitochondria from the nde-1 mutant suggests the existence of a second external NAD(P)H dehydrogenase. The topology of the NDE1 protein was further studied by protease accessibility, in vitro import experiments, and in silico analysis of the amino acid sequence. Taken together, it appears that most of the NDE1 protein extends into the intermembrane space in a tightly folded conformation and that it remains anchored to the inner mitochondrial membrane by an Nterminal transmembrane domain.In nonphotosynthetic eukaryotes, the mitochondrion is the cellular organelle responsible for producing most of the energy required for cellular metabolism. The process of oxidative phosphorylation takes place in the inner mitochondrial membrane, whereby the electrons produced by the oxidation of substrates like NAD(P)H are transported through the electron transport chain to oxygen, coupled to the generation of a transmembrane proton gradient that eventually leads to ATP synthesis (1). In contrast to mammals, the electron transport chains of plants and fungi possess several nonproton-pumping NAD(P)H dehydrogenases for transferring electrons to ubiquinone (2). In the case of potato tubers, four rotenone-insensitive NAD(P)H dehydrogenases have been identified in the inner mitochondrial membrane, two with the catalytic site facing the matrix (3, 4) and two facing the intermembrane space (5). In mitochondria from Saccharomyces cerevisiae, where the proton-pumping complex I is not present, the oxidation of NADH and NADPH is performed exclusively by three nonproton-pumping enzymes, one facing the matrix and two facing the intermembrane space (6 -8). In addition, the genome analysis of Synechocystis revealed three open reading frames that may code for such type II NAD(P)H dehydrogenases (9). On the other hand, only one external type II NADH dehydrogenase was reported for the fungus Yarrowia lipolytica (10). Although NAD(P)H dehydrogenases have been studied for a long time, our understanding of protein function at the molecular level is still very incomplete. The cloning of genes encoding several of these rotenoneinsensitive NAD(P)H dehydrogenases from mitochondria of different organisms (7, 10 -13) provides important tools for further research in this field. These enzymes might constitute a wasteful system acting to prevent the overreduction of the electron transport components and the production of reactive oxygen species, but their exact roles remain unclear.Both proton-pumping and nonproton-pumping NAD(P)H dehydrogenases have been described in Neurospora crassa mitochon...

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“…The fourth peripheral arm mutant used in this study, nuo30.4, cannot assemble a peripheral arm and accumulates membrane arm intermediates (22). The other four mutants investigated (nuo9.8, nuo11.5, nuo14, and nuo20.8) each lack a membrane arm subunit of complex I and are able to assemble the peripheral arm and fragments of the membrane arm (17,54,55).…”

Section: Vol 6 2007 Inactive Respiratory Supercomplexes In a Nuo51 mentioning

confidence: 99%

Supramolecular Organization of the Respiratory Chain in Neurospora crassa Mitochondria

et al. 2007

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The existence of specific respiratory supercomplexes in mitochondria of most organisms has gained much momentum. However, its functional significance is still poorly understood. The availability of many deletion mutants in complex I (NADH:ubiquinone oxidoreductase) of Neurospora crassa, distinctly affected in the assembly process, offers unique opportunities to analyze the biogenesis of respiratory supercomplexes. Herein, we describe the role of complex I in assembly of respiratory complexes and supercomplexes as suggested by blue and colorless native polyacrylamide gel electrophoresis and mass spectrometry analyses of mildly solubilized mitochondria from the wild type and eight deletion mutants.As an important refinement of the fungal respirasome model, we found that the standard respiratory chain of N. crassa comprises putative complex I dimers in addition to I-III-IV and III-IV supercomplexes. Three Neurospora mutants able to assemble a complete complex I, lacking only the disrupted subunit, have respiratory supercomplexes, in particular I-III-IV supercomplexes and complex I dimers, like the wild-type strain. Furthermore, we were able to detect the I-III-IV supercomplexes in the nuo51 mutant with no overall enzymatic activity, representing the first example of inactive respirasomes. In addition, III-IV supercomplexes were also present in strains lacking an assembled complex I, namely, in four membrane arm subunit mutants as well as in the peripheral arm nuo30.4 mutant. In membrane arm mutants, high-molecular-mass species of the 30.4-kDa peripheral arm subunit comigrating with III-IV supercomplexes and/or the prohibitin complex were detected. The data presented herein suggest that the biogenesis of complex I is linked with its assembly into supercomplexes.

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Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH:ubiquinone reductase ofNeurospora mitochondria

Cited by 26 publications

References 41 publications

A three-dimensional topology of complex I inferred from evolutionary correlations

A three-dimensional topology of complex I inferred from evolutionary correlations

The External Calcium-dependent NADPH Dehydrogenase from Neurospora crassa Mitochondria

Supramolecular Organization of the Respiratory Chain in Neurospora crassa Mitochondria

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