Fatty acid biosynthesis in mitochondria of grasses: malonyl-coenzyme A is generated by a mitochondrial-localized acetyl-coenzyme A carboxylase

We present biochemical evidence for the occurrence of a 250-kD multifunctional acetyl-coenzyme A carboxylase in barley (Hordeum vulgare) mitochondria. Organelles from 6-d-old barley seedlings were purified by differential centrifugation and Percoll density gradient centrifugation. Upon analysis by two-dimensional Blue-native (BN)/SDS-PAGE, an abundant 250-kD protein can be visualized, which runs at 500 kD on the native gel dimension. A similar 500-kD complex is present in etioplasts from barley. The mitochondrial 250-kD protein is biotinylated as indicated by specific reaction with an antibody directed against biotin. Peptide sequence analysis by electrospray ionization tandem mass spectrometry of the 250-kD proteins from both organellar fractions revealed amino acid sequences that are 100% identical to plastidic acetyl-coenzyme A carboxylase from wheat (Triticum aestivum). The 500-kD complex was also detected in wheat mitochondria, but is absent in mitochondrial fractions from Arabidopsis. Specific acetyl-coenzyme A carboxylation activity in barley mitochondria is higher than in etioplasts, suggesting an important role of mitochondria in fatty acid biosynthesis. Functional implications are discussed.     

Functional definition of outer membrane proteins involved in preprotein import into mitochondria

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of beta-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.     

Cloning and sequence analysis of the gene encoding 19-kD subunit of Complex I from Dunaliella salina

NADH:ubiquinone oxidoreductase (complex I ) of the mitochondrial respiratory chain catalyzes the transfer of electrons from NADH to ubiquinone coupled to proton translocation across the membrane. The cDNA sequence of Dunaliella salina mitochondrial NADH: ubiquinone oxidoreductase 19-kD subunit contains a 682-bp ORF encoding a protein with an apparent molecular mass of 19 kD. The sequence has been submitted to the GenBank database under Accession No. EF566890 (cDNA sequences) and EF566891 (genomic sequence). The deduced amino-acid sequence is 74% identical to Chlamydomonas reinhardtii mitochondrial NADH:ubiquinone oxidoreductase 18-kD subunit. The 19-kD subunit mRNA expression was observed in oxygen deficiency, salt treatment, and rotenone treatment with lower levels. It demonstrate that the 19-kD subunit of Complex I from Dunaliella salina is regulated by these stresses .     

The NADH-binding subunit of respiratory chain complex I is nuclear-encoded in plants and identified only in mitochondria

In higher plants, genes for subunits of respiratory chain complex I (NADH:ubiquinone oxidoreductase) have so far been identified solely in organellar genomes. At least nine subunits are encoded by the mitochondrial DNA and 11 homologues by the plastid DNA. One of the 'key' components of complex I is the subunit binding the substrate NADH. The corresponding gene for the mitochondrial subunit has now been cloned and identified in the nuclear genome from potato ( Solanum tuberosum ). The mature protein consists of 457 amino acids and is preceded by a mitochondrial targeting sequence of 30 amino acids. The protein is evolutionarily related to the NADH-binding subunits of complex I from other eukaryotes and is well conserved in the structural domains predicted for binding the substrate NADH, the FMN and one iron-sulphur cluster. Expression examined in different potato tissues by Northern blot analysis shows the highest steady-state mRNA levels in flowers.
Precursor proteins translated in vitro from the cDNA are imported into isolated potato mitochondria in a ΔΨ-dependent manner. The processed translation product has an apparent molecular mass of 55 kDa, identical to the mature protein present in the purified plant mitochondrial complex I. However, the in-vitro translated protein is not imported into isolated chloroplasts. To further investigate whether the complex I-like enzyme in chloroplasts contains an analogous subunit for binding of NAD(P)H, different plastid protein fractions were tested with a polyclonal antiserum directed against the bovine 51 kDa NADH-binding subunit. In none of the different thylakoid or stroma protein fractions analysed were specific crossreactive polypeptides detected. These results are discussed particularly with respect to the structure of a potential complex I in chloroplasts and the nature of its acceptor site.     

Biogenesis of the yeast cytochrome bc1 complex

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.     

MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.     

Immunological detection of potential signal-transduction proteins expressed during wheat somatic tissue culture.

An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (G-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to G-protein (alpha subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-G alpha antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multiplication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrstin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed.     

Inactivation of Genes Encoding Subunits of the Peripheral and Membrane Arms of Neurospora Mitochondrial Complex I and Effects on Enzyme Assembly

We have isolated and characterized the nuclear genes encoding the 12.3-kD subunit of the membrane arm and the 29.9-kD subunit of the peripheral arm of complex I from Neurospora crassa. The former gene was known to be located in linkage group I and the latter is now assigned to linkage group IV of the fungal genome. The genes were separately transformed into different N. crassa strains and transformants with duplicated DNA sequences were isolated. Selected transformants were then mated with other strains to generate repeat-induced point mutations in both copies of the genes present in the nucleus of the parental transformant. From the progeny of the crosses, we were then able to recover two individual mutants lacking the 12.3- and 29.9-kD proteins in their mitochondria, mutants nuo12.3 and nuo29.9, respectively. Several other subunits of complex I are present in the mutant organelles, although with altered stoichiometries as compared with those in the wild-type strain. Based on the analysis of Triton-solubilized mitochondrial complexes in sucrose gradients, neither mutant is able to fully assemble complex I. Our results indicate that mutant nuo12.3 separately assembles the peripheral arm and most of the membrane arm of the enzyme. Mutant nuo29.9 seems to accumulate the membrane arm of complex I and being devoid of the peripheral part. This implicates the 29.9-kD protein in an early step of complex I assembly.     

Localization of Proteins Immunologically Related to Subunits of Stress 310-kD Protein in Winter Wheat Mitochondria

We studied the localization of polypeptides immunochemically related to subunits of cold-shock 310-kD protein from winter rye (Secale cerealeL.) in mitochondria and submitochondrial structures of winter wheat (Triticum aestivumL.) seedlings. Polypeptides were separated by SDS-PAGE and probed with the antibody against 310-kD protein from rye seedlings. Wheat mitochondria contained the following polypeptides cross-reacting with this antibody: 66, 60, 55, and 23 kD in the inner membrane; 60 and 58 kD in the outer membrane; and 66 and 55 kD in the matrix.     

Proteinaceous complexes from mitochondrial contact sites

A Triton X-100 extract from rat brain mitochondria was obtained using low detergent/protein ratio. From this extract a proteinaceous complex was purified; its molecular weight was as high as 880 kD. The complex contained both hexokinase and creatine kinase activity. When incorporated into phospholipid bilayer membranes, the complex formed a channel whose activity was different than the channel activity of purified porin isolated either by adsorption chromatography or by dissociation from protein complexes. A ligand of the mitochondrial benzodiazepine receptor (Ro5-4864) in submicromolar concentrations had an apparent influence on the kinetic behavior of enzymatic coupling of hexokinase and creatine kinase. It is suggested that the 880-kD complex is formed by mitochondrial contact sites. The role of the isolated protein complex in the formation of nonspecific permeability in mitochondria is discussed.     

Mitochondrial Low-Molecular-Weight Heat-Shock Proteins and the Tolerance of Cereal Mitochondria to Hyperthermia

Relationships between the appearance of low-molecular-weight heat-shock proteins (LMW HSPs) in maize, winter wheat, and winter rye mitochondria and the tolerance of the mitochondria to hyperthermia (42°C, 3 h) were studied using one-dimensional SDS-PAGE, immunochemical methods, and polarography. Heat shock inhibited respiration to a greater extent in the wheat and rye than in the maize mitochondria. A single 20-kD LMW HSP was found both inside and on the surface of mitochondria isolated from heat-treated wheat and rye seedlings. After heating maize seedlings, two LMW HSPs (28 and 23 kD) appeared inside the mitochondria, and three proteins (22, 20, and 19 kD) appeared on their surface. We suppose that the latter three proteins play an essential role in the protection of mitochondria from hyperthermic damage. It seems likely that the diversity of the hyperthermia-induced LMW HSPs in plant mitochondria affects their thermal stability.     

Purification,Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase

An NADH dehydrogenase activity from red beet (Beta vulgaris L.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays L. B73) mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other less prevalent proteins. Polyclonal antibodies prepared against the 58-kD protein of red beet roots were found to immunoprecipitate the NAD(P)H dehydrogenase activity. The antibodies cross-reacted to similar proteins in mitochondria from a number of plant species but not to rat liver mitochondrial proteins. The polyclonal antibodies were used in conjunction with maize mitochondrial fractionation to show that the 58-kD protein was likely part of a protein complex loosely associated with the membrane fraction. A membrane-impermeable protein cross-linking agent was used to further show that the majority of the 58-kD protein was located on the outer surface of the inner mitochondrial membrane or in the intermembrane space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD protein doublet. The NAD(P)H dehydrogenase activity was not affected by ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or CaCl2, was stimulated somewhat (21%) by flavin mononucleotide, was inhibited by p-chloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containing platanetin (61%).     

Two overlapping genes in bovine mitochondrial DNA encode membrane components of ATP synthase.

Two hydrophobic proteins have been purified to homogeneity from a mixture of about 13 proteins that are extracted from bovine mitochondria with a chloroform:methanol mixture. Sequence analysis shows that the smaller is a protein of 66 amino acids and is the product of a mitochondrial gene, A6L. The larger, a protein of 226 amino acids, is ATPase-6, a membrane component of ATP synthase, also encoded in mitochondrial DNA. The protein sequences determined establish that the genes for the two proteins overlap by 40 bases and indicate that translation of the second gene, ATPase-6, is initiated within the coding region of A6L. The A6L and the ATPase-6 proteins have also been isolated from the ATP synthase complex and so appear to be bona fide components of the enzyme. The function of A6L is unknown. However, weak structural homology suggests a functional similarity to the yeast mitochondrial protein, aapI, which is required for assembly of the fungal ATP synthase complex. Homologies between ATPase-6 and subunit a of the Escherichia coli ATP synthase complex indicate that the ATPase-6 protein has a similar role in the mitochondrial complex to its bacterial counterpart, being essential for the formation of an active proton channel.     

14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants

Transit sequences of chloroplast-destined precursor proteins are phosphorylated on a serine or threonine residue. The amino acid motif around the phosphorylation site is related to the phosphopeptide binding motif for 14-3-3 proteins. Plant 14-3-3 proteins interact specifically with wheat germ lysate-synthesized chloroplast precursor proteins and require an intact phosphorylation motif within the transit sequence. Chloroplast precursor proteins do not interact with 14-3-3 when synthesized in the heterologous reticulocyte lysate. In contrast, a precursor protein destined for plant mitochondria was found to be associated with 14-3-3 proteins present in the reticulocyte lysate but not with 14-3-3 from wheat germ lysate. This indicates an unrecognized selectivity of 14-3-3 proteins for precursors from mitochondria and plastids in plants in comparison to fungi and animals. The heterooligomeric complex has an apparent size of 200 kD. In addition to the precursor protein, it contains 14-3-3 (probably as a dimer) and a heat shock protein Hsp70 isoform. Dissociation of the precursor complex requires ATP. Protein import experiments of precursor from the oligomeric complex into intact pea chloroplasts reveal three- to fourfold higher translocation rates compared with the free precursor, which is not complexed. We conclude that the 14-3-3-Hsp70-precursor protein complex is a bona fide intermediate in the in vivo protein import pathway in plants.     

Purification and Characterization of the Voltage-Dependent Anion-Selective Channel Protein from Wheat Mitochondrial Membranes

An approximately 29-kD protein was purified from the membrane fraction of wheat (Triticum aestivum cv Dganit) mitochondria by the utilization of standard liquid chromatography techniques. The protein, designated MmP29 for mitochondrial membrane protein having a molecular mass of approximately 29 kD, exhibited cationic properties in a buffering solution, adjusted to pH 7.5. This positive charge enabled its passage through a diethylaminoethyl column, without interaction with the positively charged matrix. Subsequently, this protein was separated from the remaining polypeptides by a preferential elution from a hydroxylapatite/celite mixed column. Reconstituted liposomes containing this protein were characterized as being permeable to 8-amino-naphthalene 1,3,6-trisulfonic acid disodium salt (Mr 445) but non-permeable to dextran fluorescein (Mr 40,000). Additionally, MmP29 was inserted into planar phospholipid membranes, and anion-selective, voltage-dependent channels were demonstrated. All of the MmP29 properties mentioned highly resemble voltagedependent, anion-selective channel (VDAC) proteins, suggesting that MmP29 is the mitochondrial outer membrane VDAC protein of wheat.     

Association of dehydrins with wheat mitochondria during low-temperature adaptation

In mitochondria from the crowns of field-grown winter wheat plants or their seedlings hardened in the laboratory, thermostable proteins immunologically related to dehydrins were detected. It was found that two dehydrins with mol wts of 63 and 52 kD bound with the outer mitochondrial membrane during autumnal hardening or during adaptation to low temperature in the laboratory. Dehydrins of similar mol wts were detected among proteins in the total membrane fraction from low-temperature-adapted wheat plants. In addition, dehydrins with mol wts of 209 and 196 kD were present in this fraction as well. Dehydrins of similar mol wts were bound with mitochondria from seedlings adapted to low temperature and those from the crowns of plants after autumnal hardening. In spring, the amount of dehydrins associated with mitochondria from the crowns declined to the level characteristic of early autumn. Dehydrin association with mitochondria is evidently an important defense mechanism of frost-resistant plants.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 221–226.Original Russian Text Copyright © 2005 by Borovskii, Stupnikova, Antipina, Anuchina, Voinikov.This revised version was published online in April 2005 with a corrected cover date.     

Subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria is involved in electron transport at center o and faces the matrix side of the membrane

The functional role and topographical orientation in the inner membrane of subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria has been investigated. The apparent molecular weight of this subunit on sodium dodecyl sulfate-urea gels was calculated to be 15,500, while its amino acid composition was similar to that of the Q-binding proteins present in the cytochrome b-c1 complexes isolated from both beef heart and yeast mitochondria. The specific antibody obtained against subunit VII inhibited 30-47% of the ubiquinol-cytochrome c reductase activity in the isolated cytochrome b-c1 complex and in submitochondrial particles but had no effect on cytochrome c reductase activity in mitoplasts, mitochondria from which the outer membrane has been removed. Furthermore, the antibody against subunit VII strongly inhibited (74%) the reduction of cytochrome b by succinate in the presence of antimycin, an inhibitor of center i, but had no effect on cytochrome b reduction in the presence of myxothiazol, an inhibitor of center o. These results suggest that subunit VII, the Q-binding protein, is involved in electron transport at center o of the cytochrome b-c1 complex of the respiratory chain and that subunit VII is localized facing the matrix side of the inner mitochondrial membrane.     

Intramitochondrial sorting of the precursor to yeast cytochrome c oxidase subunit Va

We have continued our studies on the import pathway of the precursor to yeast cytochrome c oxidase subunit Va (pVa), a mitochondrial inner membrane protein. Previous work on this precursor demonstrated that import of pVa is unusually efficient, and that inner membrane localization is directed by a membrane-spanning domain in the COOH- terminal third of the protein. Here we report the results of studies aimed at analyzing the intramitochondrial sorting of pVa, as well as the role played by ancillary factors in import and localization of the precursor. We found that pVa was efficiently imported and correctly sorted in mitochondria prepared from yeast strains defective in the function of either mitochondrial heat shock protein (hsp)60 or hsp70. Under identical conditions the import and sorting of another mitochondrial protein, the precursor to the beta subunit of the F1 ATPase, was completely defective. Consistent with previous results demonstrating that the subunit Va precursor is loosely folded, we found that pVa could be efficiently imported into mitochondria after translation in wheat germ extracts. This results suggests that normal levels of extramitochondrial hsp70 are also not required for import of the protein. The results of this study enhance our understanding of the mechanism by which pVa is routed to the mitochondrial inner membrane. They suggest that while the NH2 terminus of pVa is exposed to the matrix and processed by the matrix metalloprotease, the protein remains anchored to the inner membrane before being assembled into a functional holoenzyme complex.