RESUMO
The essential type of endonuclease that removes 5' leader sequences from transfer RNA precursors is called RNase P. While ribonucleoprotein RNase P enzymes containing a ribozyme are found in all domains of life, another type of RNase P called 'PRORP', for 'PROtein-only RNase P', is composed of protein that occurs only in a wide variety of eukaryotes, in organelles and in the nucleus. Here, to find how PRORP functions integrate with other cell processes, we explored the protein interaction network of PRORP1 in Arabidopsis mitochondria and chloroplasts. Although PRORP proteins function as single subunit enzymes in vitro, we found that PRORP1 occurs in protein complexes and is present in high-molecular-weight fractions that contain mitochondrial ribosomes. The analysis of immunoprecipitated protein complexes identified proteins involved in organellar gene expression processes. In particular, direct interaction was established between PRORP1 and MNU2 a mitochondrial nuclease. A specific domain of MNU2 and a conserved signature of PRORP1 were found to be directly accountable for this protein interaction. Altogether, results revealed the existence of an RNA maturation complex in Arabidopsis mitochondria and suggested that PRORP proteins cooperated with other gene expression factors for RNA maturation in vivo.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Endonucleases/metabolismo , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , Ribonuclease P/metabolismo , Regiões 5' não Traduzidas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Cloroplastos/enzimologia , Endonucleases/genética , Evolução Molecular , Mitocôndrias/enzimologia , Proteínas Mitocondriais , Modelos Moleculares , Complexos Multiproteicos , Domínios Proteicos , Ribonuclease P/genética , Ribossomos/metabolismoRESUMO
The maturation of tRNA precursors involves the 5' cleavage of leader sequences by an essential endonuclease called RNase P. Beyond the ancestral ribonucleoprotein (RNP) RNase P, a second type of RNase P called PRORP (protein-only RNase P) evolved in eukaryotes. The current view on the distribution of RNase P in cells is that multiple RNPs, multiple PRORPs or a combination of both, perform specialised RNase P activities in the different compartments where gene expression occurs. Here, we identify a single gene encoding PRORP in the green alga Chlamydomonas reinhardtii while no RNP is found. We show that its product, CrPRORP, is triple-localised to mitochondria, the chloroplast and the nucleus. Its downregulation results in impaired tRNA biogenesis in both organelles and the nucleus. CrPRORP, as a single-subunit RNase P for an entire organism, makes up the most compact and versatile RNase P machinery described in either prokaryotes or eukaryotes.
Assuntos
Núcleo Celular/metabolismo , Chlamydomonas/metabolismo , Cloroplastos/metabolismo , Mitocôndrias/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Chlamydomonas/genética , RNA de Transferência/genética , Ribonuclease P/genéticaRESUMO
In higher plants, chloroplast and mitochondrial transcripts contain a number of group II introns that need to be precisely spliced before translation into functional proteins. However, the mechanism of splicing and the factors involved in this process are not well understood. By analysing a seed mutant in maize, we report here the identification of Empty pericarp16 (Emp16) that is required for splicing of nad2 intron 4 in mitochondria. Disruption of Emp16 function causes developmental arrest in the embryo and endosperm, giving rise to an empty pericarp phenotype in maize. Differentiation of the basal endosperm transfer layer cells is severely affected. Molecular cloning indicates that Emp16 encodes a P-type pentatricopeptide repeat (PPR) protein with 11 PPR motifs and is localized in the mitochondrion. Transcript analysis revealed that mitochondrial nad2 intron 4 splicing is abolished in the emp16 mutants, leading to severely reduced assembly and activity of complex I. In response, the mutant dramatically increases the accumulation of mitochondrial complex III and the expression of alternative oxidase AOX2. These results imply that EMP16 is specifically required for mitochondrial nad2 intron 4 cis-splicing and is essential for complex I assembly and embryogenesis and development endosperm in maize.
Assuntos
Endosperma/enzimologia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Splicing de RNA , Zea mays/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Endosperma/citologia , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Íntrons/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , NADH Desidrogenase/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Fenótipo , Proteínas de Plantas/genética , Alinhamento de Sequência , Zea mays/citologia , Zea mays/genética , Zea mays/crescimento & desenvolvimentoRESUMO
RNA editing, converting cytidines (C) to uridines (U) at specific sites in the transcripts of mitochondria and plastids, plays a critical role in organelle gene expression in land plants. Recently pentatricopeptide repeat (PPR) proteins were identified as site-specific recognition factors for RNA editing. In this study, we characterized an empty pericarp7 mutant (emp7) in Zea mays (maize), which confers an embryo-lethal phenotype. In emp7 mutants, mitochondrial functions are seriously perturbed, resulting in a strikingly reduced respiration rate. Emp7 encodes an E-subgroup PPR protein that is localized exclusively in the mitochondrion. Null mutation of Emp7 abolishes the C â U editing of ccmF(N) transcript solely at position 1553. CcmF(N) is coding for a subunit of heme lyase complex in the cytochrome c maturation pathway. The resulting Phe â Ser substitution in CcmF(N) leads to the loss of CcmF(N) protein and a strikingly reduced c-type cytochrome. Consequently, the mitochondrial cytochrome-linked respiratory chain is impaired as a result of the disassembly of complex III in the emp7 mutant. These results indicate that the PPR-E subgroup protein EMP7 is required for C â U editing of ccmF(N) -1553 at a position essential for cytochrome c maturation and mitochondrial oxidative phosphorylation, and hence is essential to embryo and endosperm development in maize.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Citocromos c/genética , Regulação da Expressão Gênica de Plantas , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/genética , Sementes/genética , Zea mays/genéticaRESUMO
The plant mitochondrial DNA-binding protein ODB1 was identified from a mitochondrial extract after DNA-affinity purification. ODB1 (organellar DNA-binding protein 1) co-purified with WHY2, a mitochondrial member of the WHIRLY family of plant-specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52-1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo-protein complexes. Both proteins co-immunoprecipitated in a DNA-dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single-stranded than for double-stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co-immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52-like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild-type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology-mediated recombination. These observations identify ODB1 as a further component of homologous recombination-dependent DNA repair in plant mitochondria.
Assuntos
Arabidopsis/genética , Brassica/genética , Reparo do DNA , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassica/metabolismo , Cromatografia de Afinidade , Quebras de DNA de Cadeia Dupla , Dano ao DNA , DNA de Plantas/genética , Proteínas de Ligação a DNA/genética , Flores/genética , Flores/metabolismo , Recombinação Homóloga , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Especificidade de Órgãos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Plântula/genética , Plântula/metabolismoRESUMO
A fast growing number of studies identify pentatricopeptide repeat (PPR) proteins as major players in gene expression processes. Among them, a subset of PPR proteins called PRORP possesses RNase P activity in several eukaryotes, both in nuclei and organelles. RNase P is the endonucleolytic activity that removes 5' leader sequences from tRNA precursors and is thus essential for translation. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins, although some evidence implied that some eukaryotes or cellular compartments did not use RNA for RNase P activity. The characterization of PRORP reveals a two-domain enzyme, with an N-terminal domain containing multiple PPR motifs and assumed to achieve target specificity and a C-terminal domain holding catalytic activity. The nature of PRORP interactions with tRNAs suggests that ribonucleoprotein and protein-only RNase P enzymes share a similar substrate binding process.
Assuntos
Proteínas de Ligação a RNA/metabolismo , Ribonuclease P/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Células Eucarióticas/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ribonuclease P/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) is the largest complex of the mitochondrial respiratory chain. In eukaryotes, it is composed of more than 40 subunits that are encoded by both the nuclear and mitochondrial genomes. Plant Complex I differs from the enzyme described in other eukaryotes, most notably due to the large number of plant-specific subunits in the membrane arm of the complex. The elucidation of the assembly pathway of Complex I has been a long-standing research aim in cellular biochemistry. We report the study of Arabidopsis mutants in Complex I subunits using a combination of Blue-Native PAGE and immunodetection to identify stable subcomplexes containing Complex I components, along with mass spectrometry analysis of Complex I components in membrane fractions and two-dimensional diagonal Tricine SDS-PAGE to study the composition of the largest subcomplex. Four subcomplexes of the membrane arm of Complex I with apparent molecular masses of 200, 400, 450, and 650 kDa were observed. We propose a working model for the assembly of the membrane arm of Complex I in plants and assign putative roles during the assembly process for two of the subunits studied.
Assuntos
Arabidopsis/enzimologia , Membrana Celular/enzimologia , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Anticorpos/imunologia , Arabidopsis/citologia , Arabidopsis/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismoRESUMO
Two Arabidopsis thaliana genes (HCC1 and HCC2), resulting from a duplication that took place before the emergence of flowering plants, encode proteins with homology to the SCO proteins involved in copper insertion during cytochrome c oxidase (COX) assembly in other organisms. Heterozygote HCC1 mutant plants produce 25% abnormal seeds with defective embryos arrested at the heart or torpedo stage. These embryos lack COX activity, suggesting that the requirement of HCC1 during the early stages of plant development is related with its COX assembly function. Homozygote HCC2 mutant plants develop normally and do not show changes in COX2 levels. These plants display increased sensitivity of root growth to increased copper and a higher expression of miR398 and other genes that respond to copper limitation, in spite of the fact that they have a higher copper content than the wild type. HCC2 mutant plants also show increased expression of stress-responsive genes. The results suggest that HCC1 is the protein involved in COX biogenesis and that HCC2, that lacks the cysteines and histidine putatively involved in copper binding, functions in copper sensing and redox homeostasis. In addition, plants that overexpress HCC1 have an altered response of root elongation to changes in copper in the growth medium and increased expression of two low-copper-responsive genes, suggesting that HCC1 may also have a role in copper homeostasis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Homeostase , Proteínas Mitocondriais/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cobre , Complexo IV da Cadeia de Transporte de Elétrons/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas/genética , Homeostase/genética , Isoenzimas/metabolismo , Proteínas Mitocondriais/genética , Mutação/genética , Oxirredução , Fenótipo , Filogenia , Raízes de Plantas/metabolismo , Sementes/enzimologia , Estresse Fisiológico/genética , Superóxido Dismutase/metabolismoRESUMO
Cytochromes c are metalloproteins that function in electron transfer reactions and contain a heme moiety covalently attached via thioether linkages between the co-factor and a CXXCH motif in the protein. Covalent attachment of the heme group occurs on the positive side of all energy-transducing membranes (bacterial periplasm, mitochondrial intermembrane space and thylakoid lumen) and requires minimally: 1) synthesis and translocation of the apocytochromes c and heme across at least one biological membrane, 2) reduction of apocytochromes c and heme and maintenance under a reduced form prior to 3) catalysis of the heme attachment reaction. Surprisingly, the conversion of apoforms of cytochromes c to their respective holoforms occurs through at least three different pathways (systems I, II and III). In this review, we detail the assembly process of soluble cytochrome c and membrane-bound cytochrome c1, the only two mitochondrial c-type cytochromes that function in respiration. Mitochondrial c-type cytochromes are matured in the intermembrane space via the system I or system III pathway, an intriguing finding considering that the biochemical requirements for cytochrome c maturation are believed to be common regardless of the energy-transducing membrane under study.
Assuntos
Citocromos c/química , Citocromos c/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Heme/análise , Heme/metabolismo , Humanos , Modelos Biológicos , OxirreduçãoRESUMO
The mRNAs of the nad6 and ccmC genes of Arabidopsis and cauliflower were found to be processed upstream of the inframe stop codons. This result was confirmed by northern hybridization and by RT-PCR. There is no evidence that an alternative stop codon is created post-transcriptionally, either by RNA editing or by polyadenylation. The non-stop mRNAs are found in the high molecular weight polysomal fractions, suggesting that they are translated. Using antibodies directed against CcmC, the corresponding protein was detected in Arabidopsis mitochondrial extracts. These observations raise the question of how the plant mitochondrial translation system deals with non-stop mRNAs.
Assuntos
Arabidopsis/genética , Brassica/genética , Códon de Terminação/genética , Proteínas Mitocondriais/genética , Proteínas de Plantas/genética , Arabidopsis/metabolismo , Sequência de Bases , Brassica/metabolismo , Linhagem Celular , Regulação da Expressão Gênica de Plantas , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Transcrição GênicaRESUMO
NADPH-dependent thioredoxin reductases (NTR) are homodimeric enzymes that reduce thioredoxins. Two genes encoding NADPH-dependent thioredoxin reductases (AtNTRA and AtNTRB) were found in the genome of Arabidopsis thaliana. These originated from a recent duplication event and the encoded proteins are highly homologous. Previously, AtNTRA was shown to encode a dual targeted cytosol and mitochondrial protein. Here, we show that the AtNTRB gene encodes two mRNAs, presumably by initiating transcription at two different sites. The longer mRNA encodes a precursor polypeptide that is actively imported into mitochondria by a cleavage-associated mechanism, while the shorter mRNA encodes a cytosolic isoform. Isolation of Arabidopsis mutants with knocked-out AtNTRA or AtNTRB genes allowed us to prove that both genes encode cytosolic and mitochondrial isoforms. Interestingly, AtNTRB appeared to express the major mitochondrial NTR, while AtNTRA expresses as the major cytosolic isoform, suggesting that these two recently duplicated genes are evolving towards a specific function.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Mitocôndrias/enzimologia , Tiorredoxina Dissulfeto Redutase/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Mapeamento Cromossômico , Citosol/química , Citosol/enzimologia , Duplicação Gênica , Mitocôndrias/química , Mitocôndrias/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Transporte Proteico , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Tiorredoxina Dissulfeto Redutase/análise , Tiorredoxina Dissulfeto Redutase/metabolismo , Transcrição GênicaRESUMO
In land plant mitochondria, c-type cytochromes are assembled via a mechanism similar to that found in Gram-negative bacteria and different from that used by mitochondria from other eukaryotes. The wheat mitochondrial genome encodes CCM (for cytochrome c maturation) proteins, among them CcmF(C), a protein similar to the C-terminal part of the bacterial CcmF. The gene is transcribed into a 1.7 kb transcript at steady state. However, the 3' termini of the transcript were found to occur upstream of the deduced gene termination codon. This discrepancy was solved by RNA editing that introduces a novel termination codon, thus shortening the reading frame by 27 codons. The processed transcript is translated into a protein integrated in the mitochondrial inner membrane. We also show that the protein is part of a large (700 kDa) protein complex, that possibly represents a cytochrome c assembly complex.
Assuntos
Grupo dos Citocromos c/química , Grupo dos Citocromos c/metabolismo , Proteínas de Membrana/genética , Mitocôndrias/genética , Triticum/genética , Sequência de Aminoácidos , Códon , Códon de Terminação , Sequência Conservada , Grupo dos Citocromos c/genética , Expressão Gênica , Genes de Plantas , Genoma de Planta , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Edição de RNA , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Triticum/metabolismoRESUMO
Mitochondria and chloroplasts are often described as semi-autonomous organelles because they have retained a genome. They thus require fully functional gene expression machineries. Many of the required processes going all the way from transcription to translation have specificities in organelles and arose during eukaryote history. Most factors involved in these RNA maturation steps have remained elusive for a long time. The recent identification of a number of novel protein families including pentatricopeptide repeat proteins, half-a-tetratricopeptide proteins, octotricopeptide repeat proteins and mitochondrial transcription termination factors has helped to settle long-standing questions regarding organelle gene expression. In particular, their functions have been related to replication, transcription, RNA processing, RNA editing, splicing, the control of RNA turnover and translation throughout eukaryotes. These families of proteins, although evolutionary independent, seem to share a common overall architecture. For all of them, proteins contain tandem arrays of repeated motifs. Each module is composed of two to three α-helices and their succession forms a super-helix. Here, we review the features characterising these protein families, in particular, their distribution, the identified functions and mode of action and propose that they might share similar substrate recognition mechanisms.
Assuntos
Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas de Plantas/genética , Plantas/genética , Motivos de Aminoácidos , Animais , Cloroplastos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Edição de RNA , Splicing de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In mitochondria, two mono heme c-type cytochromes are essential electron shuttles of the respiratory chain. They are characterized by the covalent attachment of their heme C to a CXXCH motif in the apoproteins. This post-translational modification occurs in the intermembrane space compartment. Dedicated assembly pathways have evolved to achieve this chemical reaction that requires a strict reducing environment. In mitochondria, two unrelated machineries operate, the rather simple System III in yeast and animals and System I in plants and some protozoans. System I is also found in bacteria and shares some common features with System II that operates in bacteria and plastids. This review aims at presenting how different systems control the chemical requirements for the heme ligation in the compartments where cytochrome c maturation takes place. A special emphasis will be given on the redox processes that are required for the heme attachment reaction onto apocytochromes c.
Assuntos
Oxirredução , Motivos de Aminoácidos , Animais , Apoproteínas/genética , Apoproteínas/metabolismo , Bactérias/genética , Bactérias/metabolismo , Citocromos c/química , Citocromos c/genética , Citocromos c/metabolismo , Heme/química , Heme/genética , Heme/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Plantas/genética , Plantas/metabolismo , Plastídeos/genética , Plastídeos/metabolismo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Three reading frames called ccmF(N1), ccmF(N2), and ccmF(c) are found in the mitochondrial genome of Arabidopsis. These sequences are similar to regions of the bacterial gene ccmF involved in cytochrome c maturation. ccmF genes are always absent from animal and fungi genomes but are found in mitochondrial genomes of land plant and several evolutionary distant eukaryotes. In Arabidopsis, ccmF(N2) despite the absence of a classical initiation codon is not a pseudo gene. The 3 ccmF genes of Arabidopsis are expressed at the protein level. Their products are integral proteins of the mitochondrial inner membrane with in total 11 to 13 predicted transmembrane helices. The conserved WWD domain of CcmF(N2) is localized in the inter membrane space. The 3 CcmF proteins are all detected in a high molecular mass complex of 500 kDa by Blue Native PAGE. Direct interaction between CcmF(N2) and both CcmF(N1) and CcmF(C) is shown with the yeast two-hybrid split ubiquitin system, but no interaction is observed between CcmF(N1) and CcmF(C). Similarly, interaction is detected between CcmF(N2) and apocytochrome c but also with apocytochrome c(1). Finally, CcmF(N1) and CcmF(N2) both interact with CCMH previously shown to interact as well with cytochrome c. This strengthens the hypothesis that CcmF and CCMH make a complex that performs the assembly of heme with c-type apocytochromes in plant mitochondria.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Citocromos c/química , Proteínas de Membrana/química , Proteínas Mitocondriais/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Animais , Códon , Genes de Plantas , Proteínas de Membrana/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Coelhos , Frações Subcelulares/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/químicaRESUMO
ABC transporters make a large and diverse family of proteins found in all phylae. AtCCMA is the nucleotide binding domain of a novel Arabidopsis mitochondrial ABC transporter. It is encoded in the nucleus and imported into mitochondria. Sub-organellar and topology studies find AtCCMA bound to the mitochondrial inner membrane, facing the matrix. AtCCMA exhibits an ATPase activity, and ATP/Mg(2+) can facilitate its dissociation from membranes. Blue Native PAGE shows that it is part of a 480-kDa complex. Yeast two-hybrid assays reveal interactions between AtCCMA and domains of CcmB, the mitochondria-encoded transmembrane protein of a conserved ABC transporter. All these properties designate the protein as the ortholog in plant mitochondria of the bacterial CcmA required for cytochrome c maturation. The transporter that involves AtCCMA defines a new category of eukaryotic ABC proteins because its transmembrane and nucleotide binding domains are encoded by separate genomes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Citocromos c/metabolismo , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Proteínas de Arabidopsis/biossíntese , Clonagem Molecular , Genoma de Planta , Magnésio/química , Mitocôndrias/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Glutationa/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Antocianinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Diploide , Ativação Enzimática , Fertilidade , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Glutarredoxinas , Modelos Biológicos , Mutação/genética , Oxirredução , Estresse Oxidativo , Oxirredutases/metabolismo , Fenótipo , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Pólen/metabolismo , Plântula/metabolismo , Sementes/metabolismo , Tiorredoxina Dissulfeto Redutase/genéticaRESUMO
The Arabidopsis thaliana genome contains two nearly identical genes which encode proteins showing similarity with the yeast metal chaperone Cox19p, involved in cytochrome c oxidase biogenesis. One of these genes (AtCOX19-1) produces two transcript forms that arise from an alternative splicing event and encode proteins with different N-terminal portions. Both AtCOX19 isoforms are imported into mitochondria in vitro and are found attached to the inner membrane facing the intermembrane space. The smaller AtCOX19-1 isoform, but not the larger one, is able to restore growth on non-fermentable carbon sources when expressed in a yeast cox19 null mutant. AtCOX19 transcript levels increase by treatment with copper or compounds that produce reactive oxygen species. Young roots and anthers are highly stained in AtCOX19-1::GUS plants. Expression in leaves is only observed when cuts are produced, suggesting an induction by wounding. Infection of plants with the pathogenic bacterium Pseudomonas syringae pv. tomato also induces AtCOX19 gene expression. The results suggest that AtCOX19 genes encode functional homologues of the yeast metal chaperone. Induction by biotic and abiotic stress factors may indicate a relevant role of this protein in the biogenesis of cytochrome c oxidase to replace damaged forms of the enzyme or a more general role in the response of plants to stress.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Proteínas Mitocondriais/genética , Processamento Alternativo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Cobre/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Teste de Complementação Genética , Glucuronidase/genética , Glucuronidase/metabolismo , Metais/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Mutação , Plantas Geneticamente Modificadas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pseudomonas syringae/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Estresse Mecânico , Leveduras/genética , Leveduras/metabolismoRESUMO
The maturation of c-type cytochromes requires the covalent ligation of the heme cofactor to reduced cysteines of the CXXCH motif of apocytochromes. In contrast to mitochondria of other eukaryotes, plant mitochondria follow a pathway close to that found in alpha- and gamma-proteobacteria. We identified a nuclear-encoded protein, AtCCMH, the Arabidopsis thaliana ortholog of bacterial CcmH/CycL proteins. In bacteria, CcmH and the thioredoxin CcmG are components of a periplasmic thio-reduction pathway proposed to maintain the apocytochrome c cysteines in a reduced state. AtCCMH is located exclusively in mitochondria. AtCCMH is an integral protein of the inner membrane with the conserved RCXXC motif facing the intermembrane space. Reduction assays show that the cysteine thiols in the RCXXC motif of AtCCMH can form a disulfide bond that can be reduced by enzymatic thiol reductants. A reduced form of AtCCMH can reduce the intra-disulfide bridge of a model peptide of apocytochrome c. When expressed in Escherichia coli, AtCCMH coimmunoprecipitates with the bacterial CcmF, a proposed component of the heme lyase. Blue-native PAGE of mitochondrial membrane complexes reveals the colocalization of AtCCMH and AtCcmF(N2) in a 500-kDa complex. Yeast two-hybrid assays show an interaction between the AtCCMH intermembrane space domain and A. thaliana apocytochrome c. A. thaliana ccmh/ccmh knockout plants show lethality at the torpedo stage of embryogenesis. Our results show that AtCCMH is an essential mitochondrial protein with characteristics consistent with its proposed apocytochrome c-reducing and heme lyase function.
Assuntos
Proteínas de Arabidopsis/metabolismo , Grupo dos Citocromos c/metabolismo , Citocromos c/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Motivos de Aminoácidos , Cisteína , Grupo dos Citocromos c/fisiologia , Membranas Intracelulares/química , Proteínas de Membrana/metabolismo , Oxirredução , Técnicas do Sistema de Duplo-HíbridoRESUMO
The Arabidopsis type II peroxiredoxin (PRXII) family is composed of six different genes, five of which are expressed. On the basis of the nucleotide and protein sequences, we were able to define three subgroups among the PRXII family. The first subgroup is composed of AtPRXII-B, -C, and -D, which are highly similar and localized in the cytosol. AtPRXII-B is ubiquitously expressed. More striking is the specific expression of AtPRXII-C and AtPRXII-D localized in pollen. The second subgroup comprises the mitochondrial AtPRXII-F, the corresponding gene of which is expressed constitutively. We show that AtPRXII-E, belonging to the last subgroup, is expressed mostly in reproductive tissues and that its product is addressed to the plastid. By in vitro enzymatic experiments, we demonstrate that glutaredoxin is the electron donor of recombinant AtPRXII-B for peroxidase reaction, but the donors of AtPRXII-E and AtPRXII-F have still to be identified.