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. 2005 Sep;171(1):91–99. doi: 10.1534/genetics.105.041517

Neurospora Strains Harboring Mitochondrial Disease-Associated Mutations in Iron-Sulfur Subunits of Complex I

Margarida Duarte *, Ulrich Schulte †, Alexandra V Ushakova *, Arnaldo Videira *,‡,1
PMCID: PMC1456533  PMID: 15956670

Abstract

We subjected the genes encoding the 19.3-, 21.3c-, and 51-kDa iron-sulfur subunits of respiratory chain complex I from Neurospora crassa to site-directed mutagenesis to mimic mutations in human complex I subunits associated with mitochondrial diseases. The V135M substitution was introduced into the 19.3-kDa cDNA, the P88L and R111H substitutions were separately introduced into the 21.3c-kDa cDNA, and the A353V and T435M alterations were separately introduced into the 51-kDa cDNA. The altered cDNAs were expressed in the corresponding null-mutants under the control of a heterologous promoter. With the exception of the A353V polypeptide, all mutated subunits were able to promote assembly of a functional complex I, rescuing the phenotypes of the respective null-mutants. Complex I from these strains displays spectroscopic and enzymatic properties similar to those observed in the wild-type strain. A decrease in total complex I amounts may be the major impact of the mutations, although expression levels of mutant genes from the heterologous promoter were sometimes lower and may also account for complex I levels. We discuss these findings in relation to the involvement of complex I deficiencies in mitochondrial disease.

THE proton-pumping NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain, or complex I (EC.1.6.5.3), is located in the inner membrane of the eukaryotic organelle. It contains up to 46 polypeptide subunits of both nuclear and mitochondrial origin, as well as flavin mononucleotide (FMN) and several iron-sulfur clusters (Walker 1992; Carroll et al. 2003). The enzyme is composed of two distinct domains undergoing independent assembly, the peripheral and membrane arms, which are arranged perpendicularly to each other in an L-shaped structure (Tuschen et al. 1990; Hofhaus et al. 1991). Complex I is also found in many prokaryotes (NDH1) with fewer protein constituents but similar constitution of prosthetic groups (Yagi et al. 1998). The prokaryotic enzymes contain 14 proteins considered as the minimal assembly required for coupling electron transfer with proton translocation. All subunits are homologous to subunits of the eukaryotic complex I: 7 proteins are homologous to nuclear-encoded polypeptides and the other 7 are homologous to mitochondrial-encoded polypeptides.

Complex I is important for several cellular processes in different organisms (Videira 1998). Deficiencies in complex I activity and/or its mitochondrial or cytoplasmic-synthesized subunits have been implicated in the development of several pathogenic human conditions. In fact, disturbances of mitochondrial energy metabolism occurring with an estimated incidence of 1 in 10,000 live births are often caused by isolated complex I deficiency (Wallace 1992; Smeitink et al. 2001). The relationship between gene alterations and clinical disease symptoms or, e.g., tissue-specific phenotypes, remains rather obscure and there is no effective therapy for these diseases. As direct studies of human material are subject to strong restrictions and, in particular, many complex I patients die young, the development of nonhuman models of the diseases is very desirable. Research on mitochondrial complex I deficiency has been conducted in cell cultures (Hofhaus and Attardi 1993; Hofhaus et al. 1996), the yeast Yarrowia lipolytica (Ahlers et al. 2000), and Caenorhabditis elegans (Grad and Lemire 2004). Models of mutations in mitochondrial genes have been generated especially in prokaryotes (Lunardi et al. 1998; Zickermann et al. 1998) due to the specific difficulties associated with gene replacement in mitochondria.

A very useful model for investigating the effects of mutations in complex I is Neurospora crassa. Complex I from N. crassa has been extensively characterized [it is highly similar to that of mammals (Videira 1998)] and the genetics and physiology of the fungus are well known (Davis and Perkins 2002). In this article, we describe the development of Neurospora strains harboring mutations in single subunits relevant to human diseases.

During the past years, several specific point mutations in nuclear genes have been discovered, specifically in those coding for the human homologs of the 75-kDa (Benit et al. 2001), 51-kDa (Schuelke et al. 1999), 49-kDa (Loeffen et al. 2001), 30-kDa (Benit et al. 2004), 24-kDa (Benit et al. 2003), TYKY (Loeffen et al. 1998), PSST (Triepels et al. 1999), AQDQ (van den Heuvel et al. 1998), and IP13 (Kirby et al. 2004) subunits of complex I. Mimicked in our study are two compound heterozygous mutations in TYKY (P79L and R102H) and a homozygous mutation in PSST (V122M) in patients suffering from Leigh syndrome (Loeffen et al. 1998; Triepels et al. 1999), as well as mutations in the 51-kDa polypeptide involved in the development of leukodystrophy and myoclonic epilepsy (associated with a homozygous A341V mutation and a compound heterozygous T423M and R59X mutation) (Schuelke et al. 1999). These three proteins have homologs in the fungus N. crassa, namely the 21.3c-kDa (Duarte et al. 1996), 19.3-kDa (Sousa et al. 1999), and 51-kDa (Preis et al. 1991) subunits, respectively, and are highly conserved from bacteria to mammals. They belong to the peripheral domain of complex I in N. crassa (Videira 1998) or to the IλS subcomplex in bovine (Finel et al. 1994). The amino acid sequences of the 21.3c- and 19.3-kDa proteins display consensus motives for binding of the two [4Fe-4S] clusters N6a and N6b (Rasmussen et al. 2001) and of the [4Fe-4S] cluster N2 (Duarte et al. 2002), respectively. The 51-kDa subunit forms the NADH- and FMN-binding sites of complex I and harbors iron-sulfur cluster N3 (Fecke et al. 1994). All these polypeptide subunits have an essential function in electron transfer.

MATERIALS AND METHODS

N. crassa manipulations:

We used the N. crassa strain 74-OR23-1A (wild type), the mutant strains nuo19.3 and nuo21.3c obtained by repeat-induced point mutations of the nuo-19.3 (Duarte et al. 2002) and nuo-21.3c genes (Duarte and Videira 2000), respectively, and mutant nuo51 obtained after disruption of the nuo-51 gene by homologous recombination (Fecke et al. 1994). These mutant strains lack the 19.3-, 21.3c-, and 51-kDa subunits of complex I, respectively. The former two mutants still display transcription from the mutated genes (a smaller transcript in the case of nuo21.3c, not shown). Anyway, they can be considered as null-mutants because the relevant proteins are not detected. In addition, there is no detectable complex I assembly (by different criteria) and no detectable complex I activity (different activities measured). The failure to undergo homozygous genetic crosses of the nuo19.3 (H. Pópulo, unpublished results) and nuo21.3c mutants (Duarte and Videira 2000) also supports this conclusion. Growth and handling of N. crassa were carried out according to standard procedures (Davis and de Serres 1970). Conidia from 7-day-old cultures were used to prepare spheroplasts (Duarte et al. 1995), which were then transformed with recombinant pMYX2 vectors and selected on plates containing benomyl. For expression of cDNAs under the control of the quinic acid promoter of pMYX2 (Campbell et al. 1994), 10 mm quinic acid was added to the medium.

Site-directed mutagenesis:

The cDNAs coding for the 21.3c- and 19.3-kDa subunits cloned in pGEM4 (Duarte et al. 1996; Sousa et al. 1999) were cleaved with EcoRI, treated with Klenow to create blunt ends, and then cloned in the SmaI site of the expression vector pMYX2, downstream of the qa-2 inducible promoter. A N. crassa cDNA mycelial library M-1 cloned in UniZAP XR (obtained from the Fungal Genetics Stock Center) was screened by hybridization with an incomplete cDNA coding for the 51-kDa subunit of complex I to obtain the complete reading frame. The pBluescript plasmid was excised from a positive phage and a 1.8-kb SmaI/PvuII fragment containing the entire cDNA was also cloned in the SmaI site of pMYX2. Site-directed mutagenesis was achieved using the Quik Change site-directed mutagenesis kit (Stratagene, La Jolla, CA). Briefly, the pMYX2-recombinant vectors and pairs of synthetic complementary oligonucleotide primers containing the desired mutation were used in PCR reactions to create mutated plasmids. The mutagenic oligonucleotide primers used were: P88L, 5′-TTCAGGCCGCTCTACACAATCTATTACC-3′; R111H, 5′-CACGCCCTTCACCGTTACCCGTCG-3′; V135M, 5′-GTCATGATTATGGCGGGCACC-3′; A353V, 5′-GACTTCGATGTCCTCAAGGACAGC-3′; T435M, 5′-GAAGGTCACATGATTTGCGCTCTC-3′, and their complementary strands. The underlined nucleotides represent substitutions that change codons within the cDNAs. They result in the replacement of proline 88 by leucine (P88L) and of arginine 111 by histidine (R111H) in the 21.3c-kDa protein, of valine 135 by methionine (V135M) in the 19.3-kDa subunit, and of alanine 353 by valine (A353V) and threonine 435 by methionine (T435M) in the 51 kDa protein. The mutagenesis was confirmed by complete sequencing of the PCR products. The mutated plasmids were transformed into the respective null-mutant strains. Transformants expressing higher amounts of each mutated protein were selected for further analysis.

Northern blot analysis:

Total RNA was purified from mycelial tissue (Sokolovsky et al. 1990) and treated with RNase free-DNase I (Boehringer Mannheim, Indianapolis) for 20 min at 37° in 20 mm Tris/HCl, 5 mm MgSO4, pH 7.6. Then it was extracted once with phenol/chloroform, precipitated with ethanol, and stored at −20°. The RNA was resolved by electrophoresis in formaldehyde/formamide denaturing gels before blotting (Sambrook and Russell 2001). Hybridization was conducted with a 51-kDa cDNA probe labeled with Gene Images labeling kit (Amersham Biosciences).

Characterization of isolated mutant complexes:

Mitochondria were isolated from hyphae grown in the presence of 10 mm quinic acid. Ten milliliters of water-suspended organelles (0.6 g protein) were solubilized with 5 ml of 20% Triton X-100. After centrifugation for 20 min at 20,000 × g, the supernatant was applied to three sucrose gradients of 30 ml each (10–25% sucrose in 50 mm Tris/HCl pH 7.5, 50 mm NaCl, 0.1% Triton). Mitochondrial proteins were separated at 100,000 × g for 22 hr. The gradients were fractionated and analyzed for NADH:ferricyanide reductase activity. Fractions corresponding to the lower quarter of the gradients showing high activity were pooled and applied to an 8-ml source Q anion exchange column (Pharmacia, Piscataway, NJ). Complex I was eluted by means of a 50-ml salt gradient: 50–400 mm NaCl in 50 mm Tris/HCl pH 7.5, 0.05% Triton. Fractions with high NADH:ferricyanide reductase activity were pooled. The NADH:decylquinone reductase activity was determined after reconstitution of the enzyme in phospholipids. To obtain electron paramagnetic resonance (EPR) spectra, the enzyme preparation was concentrated fivefold by ultrafiltration on a Diaflo XM 300 filter and treated as described (Wang et al. 1991). Spectra were taken with a Bruker EMX 6/1 EPR spectrometer.

Other protein and enzymatic analysis:

The oxidation of dNADH was measured photometrically (ε340 = 6.22 mm−1 cm−1) in a standard assay mixture (50 mm Tris/HCl, 0.25 m sucrose, 0.2 mm EDTA, pH 8.0). When dNADH:Q1 reductase was assayed, 2 mm KCN, 120 μm dNADH, and 80 μm Q1 were added to the reaction mixture. NADH:hexaammineruthenium III (HAR) reductase activity was measured photometrically by the oxidation of NADH (ε380 = 1.25 mm−1 cm−1) in the presence of 2 mm KCN, 120 μm NADH, and 2 mm HAR. NADH:ferricyanide reductase activity was measured photometrically (ε420 = 1.05 mm−1 cm−1) in the standard assay mixture containing 2 mm KCN, 120 μm NADH, and 0.5 mm ferricyanide.

The techniques used for the preparation of N. crassa mitochondria (Werner 1977) and mitochondrial membranes (Melo et al. 2001), the development of rabbit antisera directed to subunits of complex I (Videira and Werner 1989), protein determination (Bradford 1976), SDS-polyacrylamide gel electrophoresis (Zauner et al. 1985), blotting and incubation of blots with antisera, detection of alkaline phosphatase second antibodies, and sucrose-gradient centrifugation analysis of detergent-solubilized mitochondrial proteins (Alves and Videira 1994) have been described.

RESULTS

Mutants of the 21.3c-, 19.3-, and 51-kDa proteins in N. crassa:

The null-mutants nuo21.3c and nuo19.3, which respectively lack the mammalian TYKY-homologous 21.3c-kDa subunit and the PSST-homologous 19.3-kDa subunit of fungal complex I, were generated by repeat-induced point mutations in the correspondent genes (Duarte and Videira 2000; Duarte et al. 2002). Inactivation of the gene encoding the 51-kDa subunit of complex I was obtained by homologous recombination (Fecke et al. 1994). The cDNAs encoding the 21.3c-, 19.3-, and 51-kDa polypeptides were cloned in the pMYX2 vector, allowing its transformation and controlled expression in the fungus. Missense mutations were separately introduced into the cDNAs by site-directed mutagenesis. The mutations P88L or R111H, equivalent to those found in a patient with Leigh syndrome, were thus applied to the 21.3c-kDa subunit. Likewise, the V135M mutation was generated in the 19.3-kDa cDNA. Finally, mutations A353V and T435M relevant to the development of leukodystrophy and myoclonic epilepsy were applied to the 51-kDa subunit. All pMYX2-recombinant plasmids were separately transformed into the corresponding null-mutants.

Assembly of complex I in strains harboring site-specific mutations:

In the null-mutants nuo21.3c and nuo19.3, assembly of the peripheral arm of complex I is prevented and only the membrane arm is formed (Duarte and Videira 2000; Duarte et al. 2002). Lack of the 51-kDa subunit led to the formation of an inactive enzyme comprising all but the missing subunit (Fecke et al. 1994). To find out whether complex I assembly occurs in strains harboring the point mutations separately introduced into the three subunits, we performed sucrose-gradient centrifugation analysis of their mitochondrial proteins. Mitochondria from the wild-type strain, null-mutants rescued by wild-type cDNAs, and the different mutants were solubilized with Triton X-100 and centrifuged in linear sucrose gradients. The NADH:ferricyanide reductase activity as well as the distribution of several complex I subunits throughout the gradients were followed.

Figure 1 depicts an experiment with the site-directed 21.3c mutants, where we immunostained the 12.3-kDa (Videira et al. 1993) and 20.8-kDa polypeptides (Videira et al. 1990a) as markers for the membrane arm and the 29.9-kDa (van der Pas et al. 1991) and 30.4-kDa polypeptides (Videira et al. 1990b) as markers for the peripheral arm of complex I. In the wild-type strain, the reductase activity is found in fractions 9–11 of the gradient, with a peak in fraction 10 (Figure 1A), in agreement with the sedimentation profile of the complex I proteins (Figure 1B). This represents the typical behavior of complex I, migrating about two-thirds of the gradients. As negative control in this experiment, we included the null-mutant nuo21.3c, which is unable to assemble the peripheral arm of complex I, as can be deduced from the lack of activity in the gradients (Figure 1A) and the fact that the 29.9- and 30.4-kDa protein markers of the peripheral arm of complex I are found in the low-molecular-weight region of the gradients (fractions 2–4, Figure 1C). Results similar to wild type were obtained with mitochondrial proteins from mutants P88L (Figure 1, A and D) and R111H (Figure 1, A and E). Similar results were obtained with the V135M mutant as well (results not shown). We conclude that, in contrast to the respective null-mutants, expression of the mutated 21.3c-kDa proteins (carrying either P88L or R111H) as well as expression of the mutated 19.3-kDa protein (carrying V135M) support the assembly of an active complex I. The observation of decreased activity (Figure 1A) and intensity of the protein bands (Figure 1, B, D, and E) in the gradients of the site-directed mutants reflects mainly that lower amounts of complex I are present in these strains in comparison with wild type (see also below).

Figure 1.

Figure 1.

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Analysis of complex I assembly in 21.3c-kDa mutant strains. Mitochondria (2.5 mg protein) were isolated, solubilized with 5% Triton X-100, and centrifuged through 12-ml sucrose gradients (7.5–25%). Fractions of the gradients (labeled 1–12) were collected and assayed for NADH:ferricyanide reductase activity. (A) Solid circles, wild type; open circles, mutant nuo21.3c; open squares, strain P88L; solid squares, strain R111H. Aliquots of the fractions obtained with material from the wild-type strain (B) and the mutants nuo21.3c (C), P88L (D), and R111H (E) were also analyzed by Western blotting with antisera against the 30.4-, 29.9-, 20.8-, and 12.3-kDa subunits of complex I, as indicated at the left in B–E.

Assembly of complex I in strains harboring mutations in the 51-kDa subunit is shown in Figure 2. The NADH:ferricyanide reductase activity as well as the distribution of the 30.4-kDa (Videira et al. 1990b), 51-kDa, and 78-kDa subunits (Preis et al. 1991) of the peripheral arm of complex I were followed in this experiment. In the null-mutant strain rescued by wild-type cDNA, the reductase activity and the complex I proteins elute in fractions 8 and 9 of the gradient, with a peak in fraction 9 (Figure 2, A and B), representing the entire complex I. As a negative control, we included in these experiments the mutant strain nuo51, which assembles an inactive complex I (Figure 2, A and C). The A353V mutant behaves like the knockout nuo51 mutant. The complex I subunits elute in fractions 8 and 9, but the 51-kDa polypeptide is not detected and no reductase activity is observed (Figure 2, A and D). The results obtained with mitochondrial proteins from mutant T435M (Figure 2, A and E) are essentially similar to those of the cDNA-rescued strain. We conclude that, in contrast to the null-mutant nuo51, expression of a 51-kDa protein carrying the point-mutation T435M supports the assembly of an active complex I, quite similar to the wild-type enzyme. In fact, analysis of the catalytic properties of the T435M complex I indicate that all activities of the mutant enzyme are present in roughly twofold lower amounts than in wild type. Moreover, there are no significant differences between mutant and wild-type enzymes in terms of rotenone sensitivity or affinity to the substrate (Table 1).

Figure 2.

Figure 2.

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Analysis of complex I assembly in 51-kDa mutant strains. The experiment was performed as described in the legend of Figure 1. NADH:ferricyanide reductase activities are shown in A (solid circles, wild-type cDNA-rescued strain; open circles, mutant nuo51; open squares, A353V strain; solid squares, T435M strain). Western blots are from the cDNA-rescued strain (B) and from the nuo51 (C), A353V (D), and T435M (E) mutants.

TABLE 1.

Catalytic properties of mitochondrial membranes from different N. crassa mutant strains

N. crassa strain
Wild type P88L R111H V135M T435M
dNADH:Q1 reductase activity (μmol dNADH/min/mg) 0.436 ± 0.115 0.236 ± 0.038 0.148 ± 0.053 0.189 ± 0.045 0.242 ± 0.044
% rotenone sensitivity 95 82 82 89 89
Inline graphic (μm) 6 7 8 5 9
Inline graphic (μm) 24 26 16 22 ND
NADH:HAR reductase activity (μmol NADH/min/mg) 1.543 ± 0.285 0.629 ± 0.218 0.365 ± 0.156 0.559 ± 0.042 1.112 ± 0.215
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Gene expression from mutant cDNAs:

We were surprised by the fact that assembly of the 51-kDa protein carrying the A353V substitution into complex I was not detected. Therefore, we tested additional isolates from the transformation with the A353V cDNA. The mutated 51-kDa polypeptide could not be detected among 24 benomyl-resistant transformants analyzed. This was in contrast to the T435M-mutated protein, which was found in most of the strains transformed with T435M cDNA. We subjected 10 of the strains transformed with the A353V cDNA to Southern blot analysis and found the presence of several copies of the altered cDNA in all of them (not shown). To further clarify this issue, we checked the mRNA expression in different strains. Total RNA was prepared from mycelial tissue of the wild-type, cDNA-rescued, nuo51, A353V, and T435M strains and analyzed by Northern blotting with the 51-kDa cDNA as probe (Figure 3). With the exception of nuo51 (obtained by homologous recombination), the transcript encoding the 51-kDa polypeptide could be identified in all other strains. The A353V mutant expresses the cDNA to the same extent as the cDNA-rescued strain, where the 51-kDa subunit of complex I is clearly detected. These results indicate that the point-mutated 51-kDa polypeptide is likely synthesized in the A353V strain but is then degraded during its route to mitochondria and/or assembly into complex I.

Figure 3.

Figure 3.

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Analysis of gene expression in 51-kDa mutant strains. Total RNA was prepared from wild type grown for 12–16 hr (E, early exponential phase) or 20–24 hr (L, late exponential phase) and from the cDNA-rescued, nuo51, A353V, and T435M strains grown to late exponential phase. The transcript encoding the 51-kDa protein (1.7 kb) was identified by Northern blotting using the correspondent labeled-cDNA as probe. (Bottom) 28S rRNA, a direct photograph of the ethidium-bromide-stained gel.

We also examined the expression of other cDNAs introduced into the N. crassa strains (not shown). Expression ranged from about half (nuo21.3c rescued with wild-type cDNA and the P88L strain) to similar (R111H strain) or slightly higher (nuo19.3 rescued with wild-type cDNA and the V135M strain) than the expression of the wild-type endogenous gene. Thus, considerable gene expression was observed in all cases, suggesting that it may not be a limiting factor in complex I assembly. In the case of P88L, the lower expression may account for the lower levels of complex I observed in the correspondent strain.

Spectroscopic and enzymatic characteristics of complex I from strains P88L, R111H, and V135M:

We studied complex I activities with different electron donors/acceptors in mitochondrial membrane preparations from wild type and the mutants. The NADH:HAR reductase activity, which can roughly estimate complex I amounts (Sled and Vinogradov 1993), is about half of the wild-type activity in P88L and V135M and ∼25% of the wild-type value in R111H (Table 1). These results, in agreement with the data from the sucrose gradients and the dehydrogenase activities depicted in Table 2, further indicate that the amount of complex I in the mutants is lower than that in wild type. To determine the more physiological NADH:ubiquinone reductase activity of complex I in the mutants, we specifically used dNADH as the electron donor because it is less efficiently used by the alternative NADH dehydrogenases present in fungal mitochondria (Friedrich et al. 1994). The dNADH:Q1 reductase activity in membranes from all three mutants is lower than that in wild type, again reflecting the lower complex I content in these strains. The kinetic parameters of dNADH:Q1 reductase activity, such as the KM for the substrates and the rotenone sensitivity, differ very slightly from the wild-type values.

TABLE 2.

NADH dehydrogenase-specific activities (units/mg) of N. crassa strains

Mitochondria: Isolated enzyme
N. crassa strains NADH:ferricyanide NADH:ferricyanide NADH:DecQ (% rotenone sensitivity)
Wild type 0.9 ± 0.1 120 ± 10 0.4 ± 0.1 (70)
P88L 0.4 ± 0.1 102 ± 20 0.3 ± 0.1 (66)
R111H 0.2 ± 0.1 95 ± 20 0.4 ± 0.1 (74)
V135M 0.5 ± 0.1 100 ± 20 0.3 ± 0.1 (70)
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To further characterize complex I formed in the mutants, we isolated the enzyme from mitochondria of the strains by a two-step procedure involving sucrose-gradient centrifugation and anion exchange chromatography. Complex I thus isolated was ∼85% pure. The yield in the mutants was two- to fourfold lower compared to that of complex I isolated from wild-type mitochondria. The polypeptide subunit composition visible on SDS-PAGE gels revealed no significant differences from the wild-type enzyme (data not shown).

Table 2 depicts NADH dehydrogenase activities of mitochondria and isolated complex I from the mutants and the wild-type strain. In agreement with the results obtained with the sucrose gradients, the NADH:ferricyanide reductase activities of mitochondria from mutants P88L, R111H, and V135M are only about one-half, one-quarter, and one-half, respectively, of the activities found in wild type. This most likely reflects the fact that lower than wild-type amounts of complex I are being formed in the mutants. In fact, when the same activity is determined in isolated enzyme preparations, the values obtained with the mutants and the wild-type strain are not significantly different. Nevertheless, a slightly decreased NADH:ferricyanide reductase activity was noted in the mutant strains. We did not find significant differences between the mutants and wild-type complex I concerning NADH:quinone reductase activities or rotenone sensitivity.

EPR spectra of the isolated enzymes from mutants P88L and R111H show signals for all detectable iron-sulfur clusters, namely N1, N2, N3, and N4 (Figure 4). The ratios of the signals show slight variations between both enzymes and compared to the wild-type enzyme. Especially the cluster N2 is somewhat diminished in mutant R111H. Other variations are in a range usually observed between different complex I preparations and do not indicate significant alterations in the spectroscopic properties of the EPR-detectable iron-sulfur clusters of the mutant enzymes. We obtained similar results with the V135M mutant, namely an EPR spectrum not significantly different from the wild-type spectrum (data not shown).

Figure 4.

Figure 4.

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EPR spectra of complex I from 21.3c-kDa mutant strains reduced by NADH. Spectra from P88L (a), R111H (b), and wild type (c) were recorded at 13 K and 2 mW microwave power. Microwave frequency was 9.46 GHz, modulation amplitude was 6 mT, time constant was 0.032 sec, and scan rate was 17.9 mT/min.

DISCUSSION

We have developed a eukaryotic model to study mutations in respiratory chain complex I subunits associated with mitochondrial disease. In particular, we describe N. crassa strains with mutations in the nuclear-coded TYKY and PSST polypeptides, which are associated with Leigh syndrome in humans, and mutations in the 51-kDa polypeptide, equivalent to mutations associated with leukodystrophy and myoclonic epilepsy. In human patients, homozygous as well as heterozygous mutations are associated with disease. The V122M substitution in the PSST subunit (Triepels et al. 1999) and also the A341V mutation in the 51-kDa subunit (Schuelke et al. 1999) were found to be homozygous in the human patients. Two human patients were compound heterozygous. One carried two compound heterozygous missense mutations in the TYKY protein, amino acid substitutions P79L and R102H, respectively, arising from the two heterozygous and apparently healthy parents (Loeffen et al. 1998). The other patient is a compound heterozygous expressing both a 51-kDa subunit with a T435M substitution and a truncated form of the polypeptide (Schuelke et al. 1999). The fact that N. crassa is a haploid organism allowed us to test separately the effects of single mutations in complex I assembly and function.

The null-mutants nuo21.3c (lacking the fungal TYKY homolog) and nuo19.3 (lacking the PSST homolog) are unable to assemble the peripheral arm of complex I (Duarte and Videira 2000; Duarte et al. 2002). The null mutant nuo51 assembles an inactive complex I lacking only the 51-kDa subunit (Fecke et al. 1994). When the null-mutants are complemented with mutant versions of the disrupted genes, four of the five single mutations analyzed in this study support assembly of the fungal enzyme. These are the P88L and the R111H mutations in the 21.3c-kDa subunit, the V135M mutation in the 19.3-kDa subunit, and the T435M mutation in the 51-kDa subunit. None of these mutations seem to affect significantly the specific activity of isolated complex I. Also the sensitivity to inhibitors is not significantly affected by the mutations. EPR spectra of the 21.3c- and 19.3-kDa site-directed mutants show that all detectable iron-sulfur clusters are present in the mutant enzymes.

Considerable differences between mutants and wild type are apparent, however, regarding the specific complex I activities of mitochondria. This is paralleled with decreased complex I proteins in mitochondria of the N. crassa mutants. Clearly, our data indicate that the reduction in complex I activity in mutant mitochondria is mainly due to a decreased accumulation of active complex I in the membrane and not to a diminished activity of the mutated enzyme. The reduced assembly/stability of the mutant enzymes is also apparent in the low yield during their isolation. Mutation R111H in the 21.3c-kDa polypeptide has a stronger effect on both the complex I content in mitochondria and the stability of the isolated enzyme.

A drastic effect in complex I activity was found in the human situation, where a marked reduction in NADH:quinone reductase activity was detected in several tissues of the patient affected in TYKY (Loeffen et al. 1998). Reconstruction of the human pathogenic mutations in the TYKY and PSST subunits of complex I in the aerobic yeast Y. lipolytica led to complex I catalytic defects. The yeast mutants showed a 50% decrease in the Vmax of the mutated complex I, elevated Km values, and/or elevated I50 values for quinone-analogous inhibitors (Ahlers et al. 2000) and are presumably not affected in complex I assembly. We have obtained different results in N. crassa, suggesting that neither the catalytic activity nor the affinity of complex I to substrates is greatly affected in the correspondent mutant strains. We suggest that the diminished formation and the stability of complex I most likely are the major factors in the development of disease in these cases. In fact, analysis by blue-native-PAGE of mitochondrial proteins from patients carrying the TYKY and PSST mutations revealed that decreased levels of fully assembled complex I correlate with the low enzyme activities, suggesting an assembly/stability defect as the primary pathogenic mechanism (Ugalde et al. 2004).

Only one mutation presented in this study prevents assembly of active complex I. We were unable to detect any assembly of the A353V-mutated 51-kDa polypeptide into complex I, although expression of the protein was clearly visible in Northern blots. We assume that the mutant subunit is degraded at some stage before integration in complex I. Recently, C. elegans transgenic strains composing the two mutations in the 51-kDa subunit were generated as disease models (Grad and Lemire 2004). The worm mutants demonstrated hallmark features of complex I dysfunction such as lactic acidosis and decreased NADH-dependent mitochondrial respiration, although the authors did not study the assembly of the mutated enzymes. Furthermore, the C. elegans mutants displayed a decrease in the assembly and/or activity of complex IV and hypersensitivity to oxidative stress (Grad and Lemire 2004). In agreement with the results observed in this system, we found that the A353V mutation has a more drastic effect on N. crassa complex I than the T435M mutation does, which results in an approximately twofold reduction in complex I amounts. It is not clear if a similar situation occurs and is primarily responsible for the phenotype of the human patient. It should be noted that the patient is a compound heterozygous expressing a truncated 51-kDa protein in addition to the T435M polypeptide (Schuelke et al. 1999).

It is conceivable that complex I proteins evolved an optimum structure to interact with other proteins [some amino acid residues are even responsible for species-specific phenotypes (Yadava et al. 2002)] and alterations interfere negatively with the efficiency of synthesis, mitochondrial import, or enzyme assembly, limiting the amounts of complex I. This seems to be particularly evident in the case of the 51-kDa protein containing a A353V substitution, which seems to be expressed but does not reach complex I. Our interpretation is supported from the findings that mutations in the mitochondrial signal sequence of the precursor of the 24-kDa subunit of complex I, which may influence the levels of the protein in mitochondria, result in increased susceptibility to Parkinson's disease (Hattori et al. 1998). Other mutations that lower the mitochondrial amounts of the 24-kDa polypeptide (Benit et al. 2003) and of other complex I subunits (Ugalde et al. 2004) were found as well.

It has been shown that complex I is essential for different cellular processes in several unrelated organisms (Videira 1998). Since mammals do not possess alternative NADH dehydrogenases as, for instance, fungi and plants do, they are expected to rely completely on complex I activity. Human diseases have been associated with more or less pronounced deficiencies but not with a complete impairment of complex I function, indicating that this is incompatible with mammalian life and/or development, as recently shown in mice by homologous deletion of the GRIM-19 subunit of complex I (Huang et al. 2004). In fact, point mutations of cysteine ligands of iron-sulfur clusters have more drastic effects in preventing complex I assembly and function (Chevallet et al. 1997; Almeida et al. 1999; Duarte and Videira 2000) and this type of mutation has not been found in human disorders (Smeitink and van den Heuvel 1999; Ugalde et al. 2004). Nevertheless, further work is required to better understand the development of mitochondrial disease associated with complex I and why different mutations lead to similar phenotypes. The more obvious explanation is that disease arises from an energetic deficiency as a result of either altered catalytic or assembly/stability properties of the mutant enzymes. Although our analysis of a few cases suggests that mutations have a higher impact on complex I formation than on enzyme catalytic activity, we cannot exclude that subtle changes in catalytic activity represent an important issue in the mammalian enzyme. Extensive studies performed directly on human material demonstrated that both catalytic properties and complex I amounts are associated with disease (Smeitink et al. 2004; Ugalde et al. 2004). Complex I disease was also claimed to arise from other causes, like deficiency of other respiratory complexes or an increased oxidative stress (Budde et al. 2000; van der Westhuizen et al. 2003; Smeitink et al. 2004). Disease development likely arises from a combination of different events. Comparison of the outcome from the study of different systems will certainly contribute to our understanding of the processes involved in disease and our ability to control them.

Acknowledgments

We thank Thorsten Friedrich for EPR spectroscopy. This work was supported by Fundação para a Ciência e a Tecnologia from Portugal and the Programa Operacional “Ciência, Tecnologia, Inovação” program of Quadro Comunitário de Apoio III (coparticipated by Fundo Europeu de Desenvolvimento Regional) through research grants to A.V. and grants from the Deutsche Forschungsgemeinschaft to U.S.

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