The iron–sulphur protein Ind1 is required for effective complex I assembly
2008; Springer Nature; Volume: 27; Issue: 12 Linguagem: Inglês
10.1038/emboj.2008.98
ISSN1460-2075
AutoresKatrine Bych, Stefan Kerscher, Daili J. A. Netz, Antonio J. Pierik, Klaus Zwicker, Martijn A. Huynen, Roland Lill, Ulrich Brandt, Janneke Balk,
Tópico(s)Enzyme Structure and Function
ResumoArticle22 May 2008free access The iron–sulphur protein Ind1 is required for effective complex I assembly Katrine Bych Katrine Bych Department of Plant Sciences, University of Cambridge, Cambridge, UK Search for more papers by this author Stefan Kerscher Stefan Kerscher Fachbereich Medizin, Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Centre of Excellence Frankfurt 'Macromolecular Complexes', Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany Search for more papers by this author Daili J A Netz Daili J A Netz Institut für Zytobiologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Antonio J Pierik Antonio J Pierik Institut für Zytobiologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Klaus Zwicker Klaus Zwicker Fachbereich Medizin, Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Centre of Excellence Frankfurt 'Macromolecular Complexes', Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany Search for more papers by this author Martijn A Huynen Martijn A Huynen Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Roland Lill Roland Lill Institut für Zytobiologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Ulrich Brandt Ulrich Brandt Fachbereich Medizin, Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Centre of Excellence Frankfurt 'Macromolecular Complexes', Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany Search for more papers by this author Janneke Balk Corresponding Author Janneke Balk Department of Plant Sciences, University of Cambridge, Cambridge, UK Search for more papers by this author Katrine Bych Katrine Bych Department of Plant Sciences, University of Cambridge, Cambridge, UK Search for more papers by this author Stefan Kerscher Stefan Kerscher Fachbereich Medizin, Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Centre of Excellence Frankfurt 'Macromolecular Complexes', Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany Search for more papers by this author Daili J A Netz Daili J A Netz Institut für Zytobiologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Antonio J Pierik Antonio J Pierik Institut für Zytobiologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Klaus Zwicker Klaus Zwicker Fachbereich Medizin, Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Centre of Excellence Frankfurt 'Macromolecular Complexes', Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany Search for more papers by this author Martijn A Huynen Martijn A Huynen Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Roland Lill Roland Lill Institut für Zytobiologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Ulrich Brandt Ulrich Brandt Fachbereich Medizin, Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Centre of Excellence Frankfurt 'Macromolecular Complexes', Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany Search for more papers by this author Janneke Balk Corresponding Author Janneke Balk Department of Plant Sciences, University of Cambridge, Cambridge, UK Search for more papers by this author Author Information Katrine Bych1,‡, Stefan Kerscher2,‡, Daili J A Netz3,‡, Antonio J Pierik3, Klaus Zwicker2, Martijn A Huynen4, Roland Lill3, Ulrich Brandt2 and Janneke Balk 1 1Department of Plant Sciences, University of Cambridge, Cambridge, UK 2Fachbereich Medizin, Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Centre of Excellence Frankfurt 'Macromolecular Complexes', Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany 3Institut für Zytobiologie, Philipps-Universität Marburg, Marburg, Germany 4Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands ‡These authors contributed equally to this work *Corresponding author. Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK. Tel.: +44 1223 330225; Fax: +44 1223 333953; E-mail: [email protected] The EMBO Journal (2008)27:1736-1746https://doi.org/10.1038/emboj.2008.98 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron–sulphur (Fe–S) clusters. Little is known about the assembly of complex I and its Fe–S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe–S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe–S clusters showed that only a minor fraction (∼20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe–4S] cluster that was readily transferred to an acceptor Fe–S protein. Our data suggest that Ind1 facilitates the assembly of Fe–S cofactors and subunits of complex I. Introduction Iron–sulphur (Fe–S) clusters are ubiquitous and versatile cofactors of proteins mediating electron transfer, enzymatic catalysis and regulation of gene expression (Beinert, 2000). The most commonly found clusters are the rhombic [2Fe–2S] and the cubane [4Fe–4S] clusters. Despite the simple structure of Fe–S cofactors and spontaneous assembly of Fe–S proteins by chemical means, in living cells Fe–S protein maturation is an enzymatic process. Many of the genes involved have been identified over the past decade, and are markedly conserved from bacteria to higher eukaryotes (for reviews, see Balk and Lobréaux, 2005; Johnson et al, 2005; Lill and Mühlenhoff, 2008). At least three Fe–S cluster assembly systems can be distinguished. The NIF system is specialized for the maturation of nitrogenase in nitrogen-fixing bacteria. A second system called ISC (for iron–sulphur cluster assembly) is present in most bacteria, and is conserved in mitochondria. A third system termed SUF mediates Fe–S cluster assembly under iron-limiting and oxidative stress conditions. Each system, encoded by operons or gene clusters in bacteria but dispersed genes in eukaryotic genomes, consists of a cysteine desulphurase for the generation of sulphane sulphur (Nfs1 in Baker's yeast, Saccharomyces cerevisiae); a scaffold protein to which the sulphur is transferred and assembled with iron (Isu1 and Isu2 in S. cerevisiae mitochondria); and auxiliary proteins, the functions of which are less well understood, including frataxin that is thought to facilitate iron delivery. The mitochondrial ISC machinery is also required for the assembly of Fe–S proteins in the cytosol and nucleus (Kispal et al, 1999; Lill and Mühlenhoff, 2008). An as yet unknown compound is exported through the ATP-binding cassette transporter of the mitochondria, Atm1, to assist iron–sulphur protein assembly in the cytosol and nucleus. The first known cytosolic Fe–S cluster assembly protein was the essential P-loop NTPase Cfd1, which was identified in a mutant screen in Baker's yeast (Roy et al, 2003). Two highly conserved cysteine residues in a CxxC motif (Cys201 and Cys204) were found to be important for its function. Cfd1 forms a stable complex with the related Nbp35 protein (Hausmann et al, 2005), and together they bind up to three [4Fe–4S] clusters. Rapid transfer of the labile Fe–S clusters to target Fe–S proteins in vitro and the requirement of Cfd1–Nbp35 for Fe–S cluster assembly in vivo demonstrated that the Cfd1–Nbp35 complex can function as a novel Fe–S scaffold in the yeast cytosol (Netz et al, 2007). Cfd1 and Nbp35 are classified as P-loop GTPases based on a unique set of sequence and structural signatures (Leipe et al, 2002). The small subfamily of Mrp/NBP35-like proteins can be further divided into five groups based on sequence similarities and conserved domains. Nbp35 has a highly conserved N terminus that binds an Fe–S cluster (Hausmann et al, 2005), whereas Cfd1 lacks this domain (Figure 1A and Supplementary Figure S1). A third group of Mrp/NBP35-like proteins was identified in the model plant Arabidopsis in screens for photosynthesis mutants displaying high chlorophyll fluorescence. hcf101 mutant alleles failed to accumulate photosystem I, which has three [4Fe–4S] clusters in its reaction centre (Lezhneva et al, 2004; Stöckel and Oelmüller, 2004). The mutants also had decreased levels of the [4Fe–4S] protein ferredoxin–thioredoxin reductase, suggesting a specific role of HCF101 in the assembly of Fe–S proteins (Lezhneva et al, 2004). The HCF101 group has a conserved N-terminal domain of unknown function, designated DUF59. The fourth group of Mrp/NBP35-like proteins has an N-terminal sequence that is predicted to target the protein to mitochondria. Although this protein is not found in Baker's yeast, it is present in most eukaryotes, including plants, mammals and fungi. The fifth group of Mrp/NBP35-like sequences is found in bacteria. Whereas the Escherichia coli Mrp (MetG-related protein) lends its name to the subfamily, only the ApbC orthologue in Salmonella enterica has been studied experimentally. Initially identified as a step in the alternative pyrimidine biosynthetic pathway (Petersen and Downs, 1996), the ApbC protein was later implicated in Fe–S cluster assembly. Deletion of apbC affected aconitase and succinate dehydrogenase activities by 30–35%, and this decrease was additive with mutations in the alternative Fe–S scaffold protein iscA, suggesting that the gene products function in the same biochemical pathway (Skovran and Downs, 2003). Figure 1.Ind1 is targeted to mitochondria. (A) Cartoon of Mrp/NBP35-like proteins found in Yarrowia lipolytica. Cfd1, YALI0E19074g; Nbp35, YALI0E02354g; Ind1, YALI0B18590g. The proteins are 40% similar in amino-acid sequence but differ in their N termini. MTS, mitochondrial targeting sequence. Conserved cysteine motifs are indicated in black. Cys279 in Ind1 is drawn in grey, as it is not conserved in the mitochondrial group. (B) Polyclonal antibodies raised against recombinant Ind1–strep recognized a 30–31 kDa protein in cell extracts (20 μg protein per lane) of Y. lipolytica expressing full-length IND1 or IND1–strep (IND1s) under the control of its own promoter from plasmid pUB4. (C) Immunoblot showing the mitochondrial localization of Ind1. Y. lipolytica cells expressing IND1–strep were treated with zymolyase, broken in a Dounce homogenizer (Tot=total lysate) and fractionated in mitochondria (Mit) and post-mitochondrial supernatant (PMS). Protein (20 μg) was separated by SDS–PAGE, blotted and labelled with antibodies against Ind1, the mitochondrial proteins aconitase (Aco1) or cysteine desulphurase (Nfs1) or cytosolic actin. (D) Immunodetection of Ind1 in the mitochondrial matrix, associated with membranes. Mitochondria (50 μg protein) of Y. lipolytica expressing IND1–strep were incubated in hypotonic buffer to swell the organelles and break the outer membrane, followed by 10 min incubation and centrifugation in the presence of 150 mM KCl to separate soluble and membrane-bound proteins of the intermembrane space (Ims) from mitoplasts (Mp). The pellet (Mp) was resuspended in hypotonic buffer and subjected to three rounds of freeze–thawing, followed by centrifugation to separate soluble matrix proteins (Mtx) and membranes (Mem). The volume of each mitochondrial fraction was adjusted to 50 μl in 1 × gel loading buffer, and 20 μl of each fraction was analysed by SDS–PAGE and immunoblotting to visualize Ind1, Nfs1, the Fe–S scaffold protein Isu1 (soluble matrix protein), Rieske Fe–S protein (integral membrane protein of complex III) and cytochrome c (Cytc; protein of the intermembrane space). Note that the separation of cytochrome c from the mitoplasts was incomplete in this experiment. Similar results were obtained for cells expressing Ind1 without Strep tag (not shown). Download figure Download PowerPoint Although S. cerevisiae has proved to be an excellent model organism for unravelling the biochemical pathways of Fe–S protein maturation in the mitochondria and cytosol, it is lacking respiratory complex I (proton pumping NADH:ubiquinone oxidoreductase), a multi-subunit Fe–S-containing protein complex. Complex I contains 8 Fe–S clusters in mitochondria and 8–9 Fe–S clusters in bacteria. From X-ray crystallographic studies of the peripheral arm of complex I of the bacterium Thermus thermophilus, it was deduced that seven clusters form an 'electrical wire' transferring electrons from the substrate NADH, through FMN, to ubiquinone (Hinchliffe and Sazanov, 2005). Little is known about the assembly of complex I or the insertion of its Fe–S clusters. Only five assembly factors of complex I have been identified to date, of which three, CIA30, CIA84 and B12.7L, are conserved from fungi to man (Küffner et al, 1998; Ogilvie et al, 2005; Vogel et al, 2005, 2007; Dunning et al, 2007; Saada et al, 2008). These proteins have been classified as molecular chaperones, as they assist in the protein folding and/or assembly of intermediates but are not part of the fully assembled complex. Furthermore, it was shown that a functional sulphurtransferase associated with complex I in the yeast Yarrowia lipolytica was not required for Fe–S cluster assembly on complex I (Abdrakhmanova et al, 2006). It is therefore likely that the cysteine desulphurase Nfs1 and other ISC assembly components are involved in Fe–S insertion into complex I. In support of this, two recent reports described patients with strongly decreased levels of the mitochondrial scaffold protein ISCU who displayed a general deficiency in mitochondrial Fe–S proteins. The biochemical symptoms included a decrease in the activity and subunit abundance of complex I, although this effect was minor compared with the strongly affected succinate dehydrogenase and aconitase enzymes (Mochel et al, 2008; Olsson et al, 2008 and references therein). Also, frataxin may be involved in providing iron to complex I, as deletion of the bacterial frataxin homologue cyaY resulted in 30% less of the protein complex (Pohl et al, 2007). The presence of a putative, mitochondrial Mrp/NBP35-like protein in eukaryotes with the exception of Baker's yeast led us to investigate the function of this protein in the yeast Y. lipolytica, with particular attention to complex I. Here, we describe a deletion mutant of the gene called IND1 that lacked approximately 80% of complex I activity due to a similar decrease in the abundance of complex I. Survival of the mutant was dependent on expression of an alternative NADH dehydrogenase transgene. Moreover, aconitase and succinate dehydrogenase activities were not affected, indicating that Ind1 is specifically involved in the assembly of complex I. Ind1 has a CxxC motif that is essential for its function. Similar to the cytosolic scaffold proteins Cfd1–Nbp35, Ind1 bound an Fe–S cluster that could be transferred to an Fe–S apo-protein, suggesting that Ind1 performs a specific role in assembling Fe–S clusters in complex I. Results Y. lipolytica expresses an Mrp/NBP35-like protein targeted to mitochondria The genome of the obligate respiratory yeast Y. lipolytica encodes three P-loop NTPases classified as Mrp/NBP35-like proteins (Leipe et al, 2002). Two of these, YALI0E02354 and YALI0E19074, bear high sequence similarity and domain organization to the S. cerevisiae proteins Nbp35 and Cfd1, respectively (Supplementary Figure S2). The third P-loop NTPase, YALI0B18590, carries the Mrp family signature and the conserved cysteine motif (CxxC) found in Nbp35 and Cfd1 (Figure 1A and Supplementary Figure S2). In addition, YALI0B18590, which we have named IND1 for iron–sulphur protein required for NADH-dehydrogenase, encodes a protein with an N terminus comprising a putative mitochondrial targeting signal. To investigate expression of IND1, antibodies were raised against the recombinant Ind1 protein and applied to immunoblots of total extracts of Y. lipolytica cells expressing full-length IND1, with or without a C-terminal Strep tag, under the control of its own promoter. A protein of 30–31 kDa was detected in cells expressing IND1(–strep) (Figure 1B, lanes 5 and 6), which is smaller than expected from the full-length protein (34.365 kDa including Strep tag). Affinity purification of Ind1–strep from Y. lipolytica cell extract and N-terminal sequencing gave the sequence SxE(N), corresponding with cleavage after Arg34, thus giving a calculated molecular mass of 30.550 kDa including Strep tag. These results are consistent with the cleavage site predicted by TargetP (Emanuelsson et al, 2000), and with the observed molecular mass. To determine the subcellular localization of Ind1, cells were fractionated in mitochondria and post-mitochondrial supernatant. Equal amounts of protein were separated by SDS–PAGE and subjected to immunoblotting. Ind1 was detected in the mitochondrial fraction but not in the post-mitochondrial supernatant of cells expressing Ind1–strep (Figure 1C, lanes 3–5). The purity of the mitochondrial fraction was confirmed by probing the same nitrocellulose membrane for the mitochondrial proteins aconitase (Aco1) and cysteine desulphurase (Nfs1), and for cytosolic actin. To establish in which mitochondrial subcompartment Ind1 is localized, mitochondria were further fractionated into mitoplasts, intermembrane space, matrix and membranes. Comparison with marker proteins showed that Ind1 is located in the matrix and is mostly membrane bound (Figure 1D). Taken together, these data show that the predicted N-terminal targeting sequence of Ind1 is cleaved and that the mature protein is localized to the mitochondrial matrix with a large fraction bound to the inner membrane. IND1 is implicated with complex I What is the function of Ind1? Because of sequence similarity to Nbp35, Cfd1 and the bacterial Mrp/ApbC, Ind1 may have a role in Fe–S protein assembly. Moreover, the absence of Ind1 in S. cerevisiae suggested a specific mitochondrial target Fe–S protein that is not present in S. cerevisiae: complex I (NADH:ubiquinone oxidoreductase). We therefore examined the presence of IND1 and complex I genes among 77 published eukaryotic genomes, using PSI-Blast searches to find homologues and phylogenetic analyses to determine orthology relations (Gabaldón et al, 2005). There is a strong genomic link between IND1 and complex I genes, as they occur together in 53 genomes, and are both absent from 18 genomes. Furthermore, assuming a standard eukaryotic phylogeny, complex I genes and IND1 have clearly co-evolved, as they have been lost concertedly five times in evolution, whereas in Schizosaccharomyces pombe IND1 has been lost with a subset of complex I genes (Figure 2A). This indicates a functional link between IND1 and complex I. Nevertheless, there are a few exceptions to the pattern: two ciliates appear to have replaced IND1 with an IND1 paralogue that has gained a mitochondrial targeting signal (Figure 2A and Supplementary Figures S1 and S4). Furthermore, the nematodes have a complete set of complex I genes without IND1, and Trichomonas vaginalis has an IND1 with the same subset of complex I genes as S. pombe. The reverse, a genome that has IND1 without having any complex I genes was not found, consistent with the hypothesis that Ind1 is required for the formation of complex I. Figure 2.IND1 is phylogenetically and functionally linked to complex I. (A) Coevolution of IND1 and genes for Fe–S protein subunits of complex I. A cross indicates the loss of IND1 in that lineage, and +MTS indicates the gain of a mitochondrial targeting signal in an IND1 paralogue. White and black squares represent the absence or presence, respectively, of the gene indicated above each column. Grey indicates the presence of an IND1 paralogue with a mitochondrial targeting signal. Abbreviated species names: Saccharomyces s.l. Saccharomyces sensu lato; S. (Schizosaccharomyces) pombe; E. (Encephalitozoon) cuniculi; S. (Strongylocentrotus) purpuratus; D. (Dictyostelium) discoideum; E. (Entamoeba) histolytica; G. (Giardia) lamblia; T. (Trichomonas) vaginalis. (B) Growth of Y. lipolytica strain ind1Δ in which the IND1 gene was deleted (centre, transformed with an empty plasmid) and the ind1Δ strain expressing the wild-type IND1 gene (left), or IND1 fused to the Strep tag coding sequence (right), expressed under the control of the IND1 promoter from the pUB4 plasmid. (C) Activities of Fe–S enzymes in purified mitochondria from ind1Δ cells (black bars) or the IND1-complemented wild type (cWT, white bars). Complementation with IND1 or IND1–strep gave similar results (not shown). Complex I was measured as NADH:HAR oxidoreductase activity in alamethicin-permeabilized mitochondria. Complex II plus III activity was measured following the electron transfer from succinate to cytochrome c in intact mitochondria. Aconitase activity was assayed following cis-aconitate consumption. Citrate synthase activity served as a non-Fe–S enzyme control. Error bars represent the standard deviation, n=3. Download figure Download PowerPoint We then investigated whether Ind1 is functionally required for complex I activity. A deletion mutant was obtained in Y. lipolytica by replacing the entire coding sequence of IND1 with a URA3 marker using homologous recombination (Supplementary Figure S3). The haploid strain used contains the Y. lipolytica NDH2 transgene fused to a mitochondrial targeting sequence (NDH2i), resulting in the expression of a version of the alternative NADH dehydrogenase on the matrix side of the inner mitochondrial membrane, bypassing the absolute requirement for respiratory complex I (Kerscher et al, 2001). The absence of IND1 and its expression in the deletion mutant was confirmed by PCR (Supplementary Figure S3) and immunoblotting (Figure 1B). The resulting ind1Δ (NDH2i) strain (in the following abbreviated as ind1Δ) was viable, but grew slowly. Growth of the ind1Δ strain was restored following complementation with a plasmid expressing the full-length IND1 gene, with or without Strep tag, under the control of its own promoter (Figure 2B). Backcrossing the ind1Δ strain with a strain lacking NDH2i resulted in spores that had either NDH2i, or IND1, or both genes (Supplementary Figure S5). These data indicated that IND1 is essential for viability and is required for NADH oxidation in the mitochondrial matrix. Next, we assayed the activities of several key Fe–S enzymes in mitochondria purified from ind1Δ cells. No significant differences were found between ind1Δ and the complemented wild-type strain (cWT) for the enzyme activities of aconitase, succinate dehydrogenase (complex II) and cytochrome bc1 complex (complex III), or the non-Fe–S enzyme citrate synthase (Figure 2C). In contrast, electron transfer from NADH to the artificial electron acceptor hexaammineruthenium(III) chloride (HAR), known to accept electrons from the 51-kDa NUBM subunit of complex I, was significantly decreased in the ind1Δ mutant. These results indicate that Ind1 is required for normal function specifically of complex I, as suggested by the co-evolution of Ind1 and complex I genes in eukaryotes. Cys242 and Cys245 are essential for the function of Ind1 To analyse NADH oxidation by complex I in more detail, we isolated unsealed mitochondrial membranes and measured the electron transfer rate from NADH to HAR, as well as electron transfer from deamino-NADH (dNADH) to n-decylubiquinone (DBQ) that is sensitive to the complex-I-specific inhibitor 2-decyl-4-quinazolinyl amine (DQA). Whereas the NADH:HAR activity involves only the primary electron acceptor FMN bound to the 51-kDa subunit, the DQA-sensitive dNADH:DBQ oxidoreductase activity involves all cofactors of the peripheral arm, therefore reflecting the physiological NADH:ubiquinone oxidoreductase activity of complex I. Comparison of the two activities has been useful in the past to identify impaired electron transport due to mutations in the ubiquinone-binding pocket (Tocilescu et al, 2007). We found that both the NADH:HAR and the DQA-sensitive dNADH:DBQ activity of complex I were decreased by approximately 70% in mitochondrial membranes isolated from cells lacking Ind1 (Table I). Table 1. Complex I activities in mitochondrial membranes from Y. lipolytica IND1 mutants Strain NADH:HAR activity DQA-sensitive dNADH:DBQ activity U mg−1 % of wild type U mg−1 % of wild type ind1Δ+pUB4 0.43±0.01 28 0.23±0.01 28 ind1Δ+pUB4-IND1 1.56±0.05 100 0.83±0.02 100 ind1Δ+pUB4-IND1–strep 1.47±0.08 94 0.82±0.01 99 C242A 0.39±0.02 25 0.19±0.01 23 C242E 0.32±0.02 21 0.17±0.01 20 C242S 0.47±0.03 30 0.21±0.01 25 C245A 0.45±0.02 29 0.23±0.01 28 C245E 0.31±0.02 20 0.15±0.01 18 C245S 0.32±0.02 21 0.17±0.01 20 C279A 1.25±0.06 80 0.67±0.02 81 C279E 0.25±0.01 16 0.12±0.01 14 C279S 1.50±0.07 96 0.83±0.02 100 Activities are given as mean±standard deviation (n=5). We then investigated the importance of the CxxC motif in Ind1 (Figure 1A) using site-directed mutagenesis of Cys242 and Cys245 to alanine, glutamate or serine. All single amino-acid changes resulted in a decrease in NADH:HAR and DQA-dependent dNADH:DBQ activity (Table I). The magnitude of the decrease was similar to that observed in the deletion mutant, demonstrating that the CxxC motif is essential for the function of Ind1. In contrast, mutations of Cys279, which is not evolutionary conserved (Supplementary Figure S4), to Ala or Ser had no effect on complex I activity. Changing Cys279 to Glu did abolish most of complex I activity, possibly because this change affected protein folding. Immunoblot analysis showed that all mutant Ind1 proteins were stably expressed (Figure 3A, lower panels). Figure 3.Deletion of IND1 leads to a major decrease in fully assembled complex I. (A) In-gel staining of complex I in mitochondrial membranes (upper panels) and immunostaining of Ind1 protein (lower panels). Mitochondrial membrane proteins were separated by BN–PAGE and incubated with NADH and NBT to visualize NADH dehydrogenase activity. Monomeric complex I (with a molecular mass of 947 kDa) is marked by an arrow. Activity-stained bands of higher molecular mass represent supercomplexes containing complex I. cWT (ind1Δ+pUB4-IND1); nubmΔ, a strain lacking the gene encoding the 51-kDa NUBM subunit of complex I; ind1Δ carrying the pUB4 plasmid; C242A–C279S, point mutations that exchange cysteines at positions 242, 245 or 279 in pUB4-IND1. (B) Two-dimensional (BN/SDS) PAGE analysis of mitochondrial membranes from complemented wild-type (cWT) and ind1Δ cells, stained with silver. The following multiprotein assemblies are indicated by dashed grey lines: VD and VM, dimeric and monomeric forms of complex V; I, complex I; S, an incompletely characterized supercomplex that contains complex III, IIID, dimeric form of complex III. The 75-kDa NUAM subunit of complex I is circled. Download figure Download PowerPoint We concluded from these enzyme assays that the function of Ind1 is required for full activity of complex I and that this function strictly depends on the cysteine residues of the conserved CxxC motif. ind1 mutants have minor amounts of fully assembled complex I The parallel decrease in NADH:HAR and dNADH:DBQ activities in the ind1Δ strain is indicative of a decrease in the amount, rather than a dysfunction, of complex I. To investigate this further, we analysed the amount and integrity of the 40-subunit protein complex using blue-native polyacrylamide gel electrophoresis (BN–PAGE) in combination with SDS–PAGE. First, mitochondrial membranes were subjected to BN–PAGE to separate the intact respiratory complexes, and stained using NADH and nitroblue tetrazolium (NBT) as a complex I-specific stain. The intensity of the major activity associated with monomeric, fully assembled complex I (Figure 3A, arrow) was strongly decreased in membranes from the ind1Δ strain and the C242/C245 cysteine mutants compared with the complemented wild type (cWT). However, a fraction of intact, active complex I was still present in the ind1 mutants, in contrast to the mutant lacking the 51-kDa subunit (nubmΔ), which did not have any complex I staining at all. In the IND1 mutants, no novel activities of smaller molecular mass were detected, indicating that subcomplexes containing a functional NBT reduction site (within the 51-kDa subunit) were absent. Second, the assembly and subunit composition of complex I and other respiratory complexes were investigated by separating the individual subunits in the second dimension using SDS–PAGE and silver staining. This revealed that the abundance of all complex I subunits was strongly diminished in the ind1Δ mutant (Figure 3B). None of the other respiratory comple
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