A homologue of a nuclear-coded iron-sulfur protein subunit of bovine mitochondrial complex I is encoded in chloroplast genomes

The chloroplast genomes of Marchantia polymorpha, Nicotiana tabacum, and Oryza sativa contain open reading frames (ORFs or potential genes) encoding homologues of some of the subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I). Seven of these subunits (ND1-ND4, ND4L, ND5, and ND6) are products of the mitochondrial genome, and two others (the 49- and 30-kDa components of the iron-sulfur protein fraction) are nuclear gene products. These findings have been taken to indicate the presence in chloroplasts of an enzyme related to complex I, possibly an NAD(P)H:plastoquinone oxidoreductase, participating in chlororespiration. This view is reinforced by the present work in which we have shown that chloroplast genomes encode a homologue of the 23-kDa subunit, another nuclear-encoded component of bovine complex I. The 23-kDa subunit is in the hydrophobic protein fraction of the enzyme, the residuum after removal of the flavoprotein and iron-sulfur protein fractions. The sequence motif CysXXCysXXCysXXXCysPro, which provides ligands for tetranuclear iron-sulfur centers in ferredoxins, occurs twice in its polypeptide chain and is evidence of two associated 4Fe-4S clusters. This is the only iron-sulfur protein identified so far in the hydrophobic protein fraction of complex I, and so it is possible that one of these centers is that known as N-2, the donor of electrons to ubiquinone. The sequence of the 23-kDa subunit is closely related to potential proteins, which also contain the cysteine-rich sequence motifs, encoded in the frxB ORFs in chloroplast genomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine complex I is a complex of 45 different subunits

Mammalian mitochondrial complex I is a multisubunit membrane-bound assembly with a molecular mass approaching 1 MDa. By comprehensive analyses of the bovine complex and its constituent subcomplexes, 45 different subunits have been characterized previously. The presence of a 46th subunit was suspected from the consistent detection of a molecular mass of 10,566 by electrospray ionization mass spectrometry of subunits fractionated by reverse-phase high pressure liquid chromatography. The component was found associated with both the intact complex and subcomplex Ibeta, which represents most of the membrane arm of the complex, and it could not be resolved chromatographically from subunit SGDH (the subunit of bovine complex I with the N-terminal sequence Ser-Gly-Asp-His). It has now been characterized by tandem mass spectrometry of intact protein ions and shown to be a C-terminal fragment of subunit SGDH arising from a specific peptide bond cleavage between Ile-55 and Pro-56 during the electrospray ionization process. Thus, the subunit composition of bovine complex I has been established. It is a complex of 45 different proteins plus non-covalently bound FMN and eight iron-sulfur clusters.     

The first twelve amino acids of a yeast mitochondrial outer membrane protein can direct a nuclear-coded cytochrome oxidase subunit to the mitochondrial inner membrane.

We have used an in vivo complementation assay to test whether a given polypeptide sequence can direct an attached protein to the mitochondrial inner membrane. The host is a previously described yeast deletion mutant that lacks cytochrome oxidase subunit IV (an imported protein) and, thus neither assembles cytochrome oxidase in its mitochondrial inner membrane nor grows on the non-fermentable carbon source, glycerol. Growth on glycerol and cytochrome oxidase assembly are restored to the mutant if it is transformed with the gene encoding authentic subunit IV precursor, a protein carrying a 25-residue transient pre-sequence. No restoration is seen with a plasmid encoding a subunit IV precursor whose pre-sequence has been shortened to seven residues. Partial, but significant restoration is achieved by an artificial subunit IV precursor in which the authentic pre-sequence has been replaced by the first 12 amino acids of a 70-kd protein of the mitochondrial outer membrane. If this dodecapeptide is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein), the resulting fusion protein is imported into the matrix of yeast mitochondria in vitro and in vivo. Import in vitro requires an energized inner membrane. We conclude that the extreme amino terminus of the 70-kd outer membrane protein can direct an attached protein across the mitochondrial inner membrane.     

Neurospora strains harboring mitochondrial disease-associated mutations in iron-sulfur subunits of complex I

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.     

Congenital deficiency of a 20-kDa subunit of mitochondrial complex I in fibroblasts.

The first component of the mitochondrial electron-transport chain is especially complex, consisting of 19 nuclear and seven mitochondrion-encoded subunits. Accordingly, a wide range of clinical manifestations are produced by the various mutations occurring in human populations. In this study, we analyze the subunit structure of complex I in fibroblasts from two patients who have distinct clinical phenotypes associated with complex I deficiency. The first patient died in the second week of life from overwhelming lactic acidosis. Severe complex I deficiency was evident in her fibroblasts, since alanine oxidation was markedly reduced whereas succinate oxidation was normal. Absence of a 20-kDa subunit was demonstrable when newly synthesized proteins were immunoprecipitated from pulse-labeled fibroblasts by anti-complex I antibody. Disordered assembly or decreased stability of the complex was suggested by deficiency of multiple subunits on Western immunoblots. The second patient exhibited a milder clinical phenotype, characterized by moderate lactic acidosis and developmental delay in childhood and by onset of seizures at 8 years of age. Oxidation studies demonstrated expression of the complex I deficiency in fibroblasts, but no subunit abnormalities were detected by immunoprecipitation or Western immunoblotting. This report demonstrates the utility of cultured fibroblasts in studying mutations affecting synthesis and assembly of complex I.     

Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A.

Protein phosphatase 2A (PP2A) is an abundant heterotrimeric serine/threonine phosphatase containing highly conserved structural (A) and catalytic (C) subunits. Its diverse functions in the cell are determined by its association with a highly variable regulatory and targeting B subunit. At least three distinct gene families encoding B subunits are known: B/B55/CDC55, B'/B56/RTS1 and B"/PR72/130. No homology has been identified among the B families, and little is known about how these B subunits interact with the PP2A A and C subunits. In vitro expression of a series of B56alpha fragments identified two distinct domains that bound independently to the A subunit. Sequence alignment of these A subunit binding domains (ASBD) identified conserved residues in B/B55 and PR72 family members. The alignment successfully predicted domains in B55 and PR72 subunits that similarly bound to the PP2A A subunit. These results suggest that these B subunits share a common core structure and mode of interaction with the PP2A holoenzyme.     

Carbonic anhydrase subunits form a matrix-exposed domain attached to the membrane arm of mitochondrial complex I in plants

Complex I of Arabidopsis includes five structurally related subunits representing gamma-type carbonic anhydrases termed CA1, CA2, CA3, CAL1, and CAL2. The position of these subunits within complex I was investigated. Direct analysis of isolated subcomplexes of complex I by liquid chromatography linked to tandem mass spectrometry allowed the assignment of the CA subunits to the membrane arm of complex I. Carbonate extraction experiments revealed that CA2 is an integral membrane protein that is protected upon protease treatment of isolated mitoplasts, indicating a location on the matrix-exposed side of the complex. A structural characterization by single particle electron microscopy of complex I from the green alga Polytomella and a previous analysis from Arabidopsis indicate a plant-specific spherical extra-domain of about 60 A in diameter, which is attached to the central part of the membrane arm of complex I on its matrix face. This spherical domain is proposed to contain a heterotrimer of three CA subunits, which are anchored with their C termini to the hydrophobic arm of complex I. Functional implications of the complex I-integrated CA subunits are discussed.     

Carbonic anhydrase subunits of the mitochondrial NADH dehydrogenase complex (complex I) in plants

The mitochondrial nicotinamide adenine dinucleotide, reduced (NADH) dehydrogenase complex (complex I) of plants has a molecular mass of about 1000 kDa and is composed of more than 40 distinct protein subunits. About three quarter of these subunits are homologous to complex I subunits of heterotrophic eukaryotes, whereas the remaining subunits are unique to plants. Among them are three to five structurally related proteins that resemble an archaebacterial γ-type carbonic anhydrase (γCA). The γCA subunits are attached to the membrane arm of complex I on the matrix-exposed side and form an extra spherical domain. At the same time, they span the inner mitochondrial membrane and are essential for assembly of the protein complex. Expression of the genes encoding γCA subunits is reduced if plants are cultivated in the presence of elevated CO₂ concentration. The functional role of these subunits within plant mitochondria is currently unknown but might be related to photorespiration. We propose that the complex I–integrated γCAs are involved in mitochondrial HCO₃⁻ formation to allow efficient recycling of inorganic carbon for CO₂ fixation in chloroplasts under high light conditions.     

Two subunits of the canine signal peptidase complex are homologous to yeast SEC11 protein

Canine microsomal signal peptidase activity was previously isolated as a complex of five subunits (25, 22/23, 21, 18, and 12 kDa). Two of the signal peptidase complex (SPC) subunits (23/23 and 21 kDa) have been cloned and sequenced. One of these, the 21-kDa subunit, was observed to be a mammalian homolog of SEC11 protein (Sec11p) (Greenburg, G., Shelness, G. S., and Blobel, G. (1989) J. Biol. Chem. 264, 15762-15765) a gene product essential for signal peptide processing and cell growth in yeast (B?hni, P.C., Deshqies, R.J., and Schekman, R.W. (1988) J. Cell Biol. 106, 1035-1042). cDNA clones for the 18-kDa SPC subunit have now been characterized and found to encode a second SEC11p homolog. Both the 18- and 21-kDa canine SPC subunits are integral membrane proteins by virtue of their resistance to alkaline extraction. Upon detergent solubilization, both proteins are found in a complex with the 22/23 kDa SPC subunit, the only SPC subunit containing N-linked oligosaccharide. No steady-state pool of canine Sec11p-like monomers is detected in microsomal membranes. Alkaline extraction of microsomes prior to solubilization or solubilization at alkaline pH causes partial dissociation of the SPC. The Sec11p-like subunits displaced from the complex under these conditions demonstrate no signal peptide processing activity by themselves. The existence of homologous subunits is common to a number of known protein complexes and provides further evidence that the association between SPC proteins observed in vitro may be physiologically relevant to the mechanism of signal peptide processing and perhaps protein translocation.     

The nuclear-coded subunits of yeast cytochrome c oxidase. II. The amino acid sequence of subunit VIII and a model for its disposition in the inner mitochondrial membrane

The amino acid sequence of subunit VIII from yeast cytochrome c oxidase is reported. This 47-residue (Mr = 5364) amphiphilic polypeptide has a polar NH2 terminus, a hydrophobic central section, and a dilysine COOH terminus. An analysis of local hydrophobicity and predicted secondary structure along the peptide chain predicts that the hydrophobic central region is likely to be transmembranous. Subunit VIII from yeast cytochrome c oxidase exhibits 40.4% homology to bovine heart cytochrome c oxidase subunit VIIc , at the level of primary structure. Secondary structures and hydrophobic domains predicted from the sequences of both polypeptides are also highly conserved. From the location of hydrophobic domains and the positions of charged amino acid residues we have formulated a topological model for subunit VIII in the inner mitochondrial membrane.     

Shotgun proteomics for the characterization of subunit composition of mitochondrial complex I

Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.     

Regulation of the 75-kDa subunit of mitochondrial complex I by iron

Iron homeostasis is tightly regulated, as cells work to conserve this essential but potentially toxic metal. The translation of many iron proteins is controlled by the binding of two cytoplasmic proteins, iron regulatory protein 1 and 2 (IRP1 and IRP2) to stem loop structures, known as iron-responsive elements (IREs), found in the untranslated regions of their mRNAs. In short, when iron is depleted, IRP1 or IRP2 bind IREs; this decreases the synthesis of proteins involved in iron storage and mitochondrial metabolism (e.g. ferritin and mitochondrial aconitase) and increases the synthesis of those involved in iron uptake (e.g. transferrin receptor). It is likely that more iron-containing proteins have IREs and that other IRPs may exist. One obvious place to search is in Complex I of the mitochondrial respiratory chain, which contains at least 6 iron-sulfur (Fe-S) subunits. Interestingly, in idiopathic Parkinson's disease, iron homeostasis is altered, and Complex I activity is diminished. These findings led us to investigate whether iron status affects the Fe-S subunits of Complex I. We found that the protein levels of the 75-kDa subunit of Complex I were modulated by levels of iron in the cell, whereas mRNA levels were minimally changed. Isolation of a clone of the 75-kDa Fe-S subunit with a more complete 5'-untranslated region sequence revealed a novel IRE-like stem loop sequence. RNA-protein gel shift assays demonstrated that a specific cytoplasmic protein bound the novel IRE and that the binding of the protein was affected by iron status. Western blot analysis and supershift assays showed that this cytosolic protein is neither IRP1 nor IRP2. In addition, ferritin IRE was able to compete for binding with this putative IRP. These results suggest that the 75-kDa Fe-S subunit of mitochondrial Complex I may be regulated by a novel IRE-IRP system.     

Altered anesthetic sensitivity of mice lacking ndufs4, a subunit of mitochondrial complex I

Anesthetics are in routine use, yet the mechanisms underlying their function are incompletely understood. Studies in vitro demonstrate that both GABA(A) and NMDA receptors are modulated by anesthetics, but whole animal models have not supported the role of these receptors as sole effectors of general anesthesia. Findings in C. elegans and in children reveal that defects in mitochondrial complex I can cause hypersensitivity to volatile anesthetics. Here, we tested a knockout (KO) mouse with reduced complex I function due to inactivation of the Ndufs4 gene, which encodes one of the subunits of complex I. We tested these KO mice with two volatile and two non-volatile anesthetics. KO and wild-type (WT) mice were anesthetized with isoflurane, halothane, propofol or ketamine at post-natal (PN) days 23 to 27, and tested for loss of response to tail clamp (isoflurane and halothane) or loss of righting reflex (propofol and ketamine). KO mice were 2.5 - to 3-fold more sensitive to isoflurane and halothane than WT mice. KO mice were 2-fold more sensitive to propofol but resistant to ketamine. These changes in anesthetic sensitivity are the largest recorded in a mammal.     

Defective mitochondrial translation differently affects the live cell dynamics of complex I subunits

Complex I (CI) of the oxidative phosphorylation system is assembled from 45 subunits encoded by both the mitochondrial and nuclear DNA. Defective mitochondrial translation is a major cause of mitochondrial disorders and proper understanding of its mechanisms and consequences is fundamental to rational treatment design. Here, we used a live cell approach to assess its consequences on CI assembly. The approach consisted of fluorescence recovery after photobleaching (FRAP) imaging of the effect of mitochondrial translation inhibition by chloramphenicol (CAP) on the dynamics of AcGFP1-tagged CI subunits NDUFV1, NDUFS3, NDUFA2 and NDUFB6 and assembly factor NDUFAF4. CAP increased the mobile fraction of the subunits, but not NDUFAF4, and decreased the amount of CI, demonstrating that CI is relatively immobile and does not associate with NDUFAF4. CAP increased the recovery kinetics of NDUFV1-AcGFP1 to the same value as obtained with AcGFP1 alone, indicative of the removal of unbound NDUFV1 from the mitochondrial matrix. Conversely, CAP decreased the mobility of NDUFS3-AcGFP1 and, to a lesser extent, NDUFB6-AcGFP1, suggestive of their enrichment in less mobile subassemblies. Little, if any, change in mobility of NDUFA2-AcGFP1 could be detected, suggesting that the dynamics of this accessory subunit of the matrix arm remains unaltered. Finally, CAP increased the mobility of NDUFAF4-AcGFP1, indicative of interaction with a more mobile membrane-bound subassembly. Our results show that the protein interactions of CI subunits and assembly factors are differently altered when mitochondrial translation is defective.     

The 30-kilodalton subunit of bovine mitochondrial complex I is homologous to a protein coded in chloroplast DNA

In cattle, 7 of the 30 or more subunits of the respiratory enzyme NADH:ubiquinone reductase (complex I) are encoded in mitochondrial DNA, and potential genes (open reading frames, orfs) for related proteins are found in the chloroplast genomes of Marchantia polymorpha and Nicotiana tabacum. Homologues of the nuclear-coded 49- and 23-kDa subunits are also coded in chloroplast DNA, and these orfs are clustered with four of the homologues of the mammalian mitochondrial genes. These findings have been taken to indicate that chloroplasts contain a relative of complex I. The present work provides further support. The 30-kDa subunit of the bovine enzyme is a component of the iron-sulfur protein fraction. Partial protein sequences have been determined, and synthetic oligonucleotide mixtures based on them have been employed as hybridization probes to identify cognate cDNA clones from a bovine library. Their sequences encode the mitochondrial import precursor of the 30-kDa subunit. The mature protein of 228 amino acids contains a segment of 57 amino acids which is closely related to parts of proteins encoded in orfs 169 and 158 in the chloroplast genomes of M. polymorpha and N. tabacum. Moreover, the chloroplast orfs are found near homologues of the mammalian mitochondrial genes for subunit ND3. Therefore, the plant chloroplast genomes have at least two separate clusters of potential genes encoding homologues of subunits of mitochondrial complex I. The bovine 30-kDa subunit has no extensive sequences of hydrophobic amino acids that could be folded into membrane-spanning alpha-helices, and although it contains two cysteine residues, there is no clear evidence in the sequence that it is an iron-sulfur protein.     

The secondary structures of the Xenopus laevis and human mitochondrial small ribosomal subunit RNA are similar

Extensive corrections of the nucleotide sequence of the Xenopus laevis mitochondrial small ribosomal subunit RNA gene [Roe et al. (1985) J. Biol. Chem. 260, 9759-9774] are reported. We found an additional fragment of 142 nucleotides and describe 25 nucleotide differences scattered in the gene. The nucleotide sequence the same gene of bovine mitochondrion. We propose a new secondary structure for the product of the X. laevis gene. Contrary to the finding of Roe et al., we observed the same general organization of stems and loops as for the human mitochondrial 12 S rRNA gene product. On the other hand, the structural homology observed between the mitochondrial and cytoplasmic small subunit rRNAs of X. laevis appears much lower. These results strongly suggest that animal vertebrate mitochondrial DNAs have followed the same evolutionary pathway.     

The biogenesis of rat mitochondrial ATPase. Two subunits are synthesized inside the mitochondria

Isolated mitochondria from regenerating rat liver synthesize at least five different polypeptides with molecular weights ranging from 19 000 to 43 000. Among these, two polypeptides with molecular weights of 22 000 and 25 ooo could be identified as ATPase subunits. It has previously been shown that these subunits, designated 6 and 7, are lacking in the ATPase complex that is formed in vivo when mitochondrial protein synthesis is blocked by thiamphenicol treatment. The 22 000 Mr protein is enriched in the fraction containing the fully assembled ATPase complex, whereas the 25 000 Mr protein is not.     

The C8ORF38 homologue Sicily is a cytosolic chaperone for a mitochondrial complex I subunit

Mitochondrial complex I (CI) is an essential component in energy production through oxidative phosphorylation. Most CI subunits are encoded by nuclear genes, translated in the cytoplasm, and imported into mitochondria. Upon entry, they are embedded into the mitochondrial inner membrane. How these membrane-associated proteins cope with the hydrophilic cytoplasmic environment before import is unknown. In a forward genetic screen to identify genes that cause neurodegeneration, we identified sicily, the Drosophila melanogaster homologue of human C8ORF38, the loss of which causes Leigh syndrome. We show that in the cytoplasm, Sicily preprotein interacts with cytosolic Hsp90 to chaperone the CI subunit, ND42, before mitochondrial import. Loss of Sicily leads to loss of CI proteins and preproteins in both mitochondria and cytoplasm, respectively, and causes a CI deficiency and neurodegeneration. Our data indicate that cytosolic chaperones are required for the subcellular transport of ND42.     

Leber hereditary optic neuropathy mutations in the ND6 subunit of mitochondrial complex I affect ubiquinone reduction kinetics in a bacterial model of the enzyme

LHON (Leber hereditary optic neuropathy) is a maternally inherited disease that leads to sudden loss of central vision at a young age. There are three common primary LHON mutations, occurring at positions 3460, 11778 and 14484 in the human mtDNA (mitochondrial DNA), leading to amino acid substitutions in mitochondrial complex I subunits ND1, ND4 and ND6 respectively. We have now examined the effects of ND6 mutations on the function of complex I using the homologous NuoJ subunit of Escherichia coli NDH-1 (NADH:quinone oxidoreductase) as a model system. The assembly level of the NDH-1 mutants was assessed using electron transfer from deamino-NADH to the 'shortcut' electron acceptor HAR (hexammine ruthenium), whereas ubiquinone reductase activity was determined using DB (decylubiquinone) as a substrate. Mutant growth in minimal medium with malate as the main carbon source was used for initial screening of the efficiency of energy conservation by NDH-1. The results indicated that NuoJ-M64V, the equivalent of the common LHON mutation in ND6, had a mild effect on E. coli NDH-1 activity, while nearby mutations, particularly NuoJ-Y59F, NuoJ-V65G and NuoJ-M72V, severely impaired the DB reduction rate and cell growth on malate. NuoJ-Met64 and NuoJ-Met72 position mutants lowered the affinity of NDH-1 for DB and explicit C-type inhibitors, whereas NuoJ-Y59C displayed substrate inhibition by oxidized DB. The results are compatible with the notion that the ND6 subunit delineates the binding cavity of ubiquinone substrate, but does not directly take part in the catalytic reaction. How these changes in the enzyme's catalytic properties contribute to LHON pathogenesis is discussed.