RESUMO
The primary structure of a cDNA clone encoding the S4 protein from the small subunit of 80S ribosomes from potato was determined. Cytosolic ribosomal protein S4 is hydrophilic and has a prevalence for positively charged residues. In potato it is 264 amino acids long and contains a putative nuclear targeting signal close to the N-terminus. Having 65-69% identical amino acids cytosolic ribosomal protein S4 from mammals, fungi and plants belongs to the highly conserved proteins. The S4 gene is transcribed in all potato tissues analysed and has a relatively high expression level in comparison to nuclear genes encoding mitochondrial proteins.
Assuntos
Genes de Plantas , Proteínas Ribossômicas/genética , Solanum tuberosum/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Citosol/metabolismo , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Ratos , Proteínas Ribossômicas/biossíntese , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Solanum tuberosum/metabolismoRESUMO
The general mitochondrial processing peptidase that removes the N-terminal targeting signals from proteins imported into mitochondria forms part of a respiratory protein complex in potato (Solanum tuberosum L.). We have termed this complex the "cytochrome c reductase/processing peptidase complex" and show that it acts on a variety of precursor proteins from different intramitochondrial locations. In potato, biochemical methods fail to separate the ubiquinol cytochrome c oxidoreductase function from the function of the processing protease. On the other hand, inhibition of electron flow with antimycin A or myxothiazol does not affect processing activity. The integration into an oligomeric protein complex causes the unique properties of the processing enzyme. It is fully active at high pH and in the presence of high salt. It does not need externally added metal ions, but it is inhibited by EDTA and 1,10-phenanthroline. Other protease inhibitors have no effect on the processing activity. Taken together, the molecular genetic and physiological results indicate that the mitochondrial processing protease does not belong to the thermolysin superfamily of metalloproteinases but may be a member of a new class of metalloendoproteases.
RESUMO
The ADP/ATP translocator is an abundant protein of the mitochondrial inner membrane, which in fungi and mammals is synthesized without a presequence. Here we report that the translocator from potato has an amino-terminal extension which may function in mitochondrial targeting. Several cDNA clones encoding the nucleotide sequence of the ADP/ATP translocator have been isolated from potato leaf and tuber cDNA libraries constructed in lambda phages. Only one class of cDNA clones was found but possibly different translocator genes are expressed in other tissues. High levels of transcripts for the translocator are found in all tissues analysed. Sequence determination of the complete insert of one of the clones reveals a long open reading frame of 1158 bp encoding a protein of 386 amino acids corresponding to a calculated molecular weight of 42 kDa. In contrast, the ADP/ATP translocator proteins from fungi and mammals are significantly smaller. Comparison of the Neurospora translocator with the potato protein shows about 75% sequence homology, being confined to the region after amino acid 85 of the potato polypeptide. Antibodies directed against the fungal translocator recognize a protein of 30 kDa in the inner membrane of potato mitochondria, suggesting that the mature protein has a similar size as the translocators from fungi and mammals. Thus, the additional segment of the potato ADP/ATP translocator forms an amino-terminal extension which may be involved in the import of the protein into plant mitochondria.
Assuntos
Translocases Mitocondriais de ADP e ATP/genética , Solanum tuberosum/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Northern Blotting , Western Blotting , Clonagem Molecular , DNA/isolamento & purificação , Fungos/enzimologia , Expressão Gênica , Humanos , Mitocôndrias/enzimologia , Translocases Mitocondriais de ADP e ATP/metabolismo , Dados de Sequência Molecular , Precursores de Proteínas/metabolismo , Mapeamento por Restrição , Alinhamento de Sequência , Solanum tuberosum/enzimologiaRESUMO
Cytochrome c reductase from potato is a bifunctional protein complex located in the inner mitochondrial membrane, which is involved in respiratory electron transport and processing of mitochondrial precursor proteins. The three largest subunits of the complex share the highest degree of sequence identity with the alpha- and beta-subunits of the soluble processing peptidase (MPP) from fungi and mammals. Evidence is provided that another substoichiometric polypeptide of the cytochrome c reductase complex resembles the alpha-subunit of MPP. A cDNA clone corresponding to the second alpha-MPP protein (alpha-II MPP) encodes a polypeptide of 504 amino acids which is 84% identical to alpha-I MPP. The two different alpha-MPP polypeptides have similar sizes on SDS-polyacrylamide gels but can be distinguished with an antibody raised against a decapeptide that is specific for alpha-II MPP. The presequences of both alpha-subunits of MPP are proteolytically removed by the integrated processing enzyme complex indicating that it acts on the targeting signals of its own precursor proteins. Gene-specific oligonucleotides reveal that the genes encoding alpha-subunit I and alpha-subunit II of MPP are differentially expressed in all tissues analysed but the transcript levels do not vary between tissues.
Assuntos
Metaloendopeptidases/genética , Mitocôndrias/enzimologia , Complexos Multienzimáticos/genética , NADH Desidrogenase/genética , Proteínas de Plantas/genética , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , DNA de Plantas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Metaloendopeptidases/química , Dados de Sequência Molecular , Proteínas de Plantas/química , Ratos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Solanum tuberosum/genética , Especificidade da Espécie , Transcrição Gênica , Leveduras/química , Peptidase de Processamento MitocondrialRESUMO
Here we report the primary structure of potato cytochrome c1, a nuclear-encoded subunit of complex III. Using heterologous antibodies directed against cytochrome c1 from yeast two types of clones were isolated from an expression library, suggesting that at least two different genes are present and expressed in the genome. Northern blot analysis reveals that slightly varying levels of cytochrome c1 transcripts are present in all potato tissues analysed. A 1304 bp insert of one of the cDNA clones (pC13II) encodes the entire 320 amino acids of the precursor protein corresponding to a molecular weight of 35.2 kDa. As revealed by direct amino acid sequence determination of the cytochrome c1 protein another cDNA clone (pC18I) encodes the major form of cytochrome c1 present in potato tuber mitochondria. Western blots of subfractionated potato mitochondria show that the mature protein present in the membrane fraction is smaller than the pC13II encoded protein synthesized in Escherichia coli. The transient presequence of the protein is 77 amino acids long and has a bipartite polarity profile characteristic of presequences involved in targeting to the intermembrane space of fungal mitochondria. It consists of a positively charged NH2-terminal part which resembles "matrix targeting domains" and an adjacent hydrophobic region showing sequence similarities to "intramitochondrial sorting domains". The amino-terminal region of potato cytochrome c1 is the first presequence of a plant protein of the mitochondrial intermembrane space to be determined and may be useful in the study of intramitochondrial sorting in plants.
Assuntos
Citocromos c1/genética , Membranas Intracelulares/enzimologia , Mitocôndrias/enzimologia , Proteínas de Plantas/genética , Solanum tuberosum/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Bovinos , Clonagem Molecular , Citocromos c1/biossíntese , Humanos , Mitocôndrias/química , Dados de Sequência Molecular , Proteínas de Plantas/biossíntese , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Solanum tuberosum/enzimologia , Transcrição GênicaRESUMO
In potato, cytochrome c reductase, a protein complex of the respiratory chain, exhibits processing activity toward mitochondrial precursor proteins. One of the two cooperating components of the processing peptidase was shown to be identical with subunit III of the complex. Here we report that two additional proteins of the complex (subunit I and II) share 40-50% sequence identity with the processing enhancing protein, the other component of the processing enzyme from fungi and mammals. Thus the composition and structure of the complex integrated processing peptidase seems to be different from its fungal and mammalian counterparts. Cytochrome c reductase from potato is extraordinarily stable, and separation of subunit III from the complex leads to aggregation of the remaining subcomplex and irreversible loss of processing activity. Expression of the three high molecular weight subunits of the complex allowed purification of each individual protein. Neither the individual subunits nor their combinations are active in in vitro processing assays suggesting that they may need the structural support of the complex for activity. In contrast to mitochondrial processing peptidases from other organisms, the purified potato enzyme is active in the presence of high salt (above 1 M NaCl) and works efficiently without addition of metal ions. These data indicate that potato cytochrome c reductase is a bifunctional protein complex with unique features. Possibly, there is a more general evolutionary relationship between cytochrome c reductases and mitochondrial processing peptidases than hitherto assumed.
Assuntos
Endopeptidases/metabolismo , Mitocôndrias/enzimologia , NADH Desidrogenase/metabolismo , Compostos de Sódio , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Brometos/farmacologia , DNA/química , DNA/isolamento & purificação , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Substâncias Macromoleculares , Peso Molecular , NADH Desidrogenase/química , NADH Desidrogenase/genética , Octoxinol , Polietilenoglicóis/farmacologia , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência , Sódio/farmacologia , Cloreto de Sódio/farmacologia , Iodeto de Sódio/farmacologia , Solanum tuberosum/ultraestruturaRESUMO
The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase.
Assuntos
Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , NADH Desidrogenase/metabolismo , Serina Endopeptidases , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Grupo dos Citocromos c/metabolismo , DNA , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Substâncias Macromoleculares , Proteínas de Membrana/genética , Dados de Sequência Molecular , NADH Desidrogenase/genética , NADH Desidrogenase/isolamento & purificação , Processamento de Proteína Pós-Traducional , Homologia de Sequência do Ácido Nucleico , Solanum tuberosum/genética , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMO
The bc1-complex (EC 1.10.2.2.) from Triticum aestivum L. was purified by cytochrome-c affinity chromatography and gel filtration using either etiolated seedlings or wheat-germ extract as starting material. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated enzyme revealed ten bands, which were analysed by immunoblotting and direct amino-acid sequencing. The enzyme from wheat is the first bc1-complex that is reported to contain four core proteins (55.5, 55.0, 51.5 and 51.0 kDa). In addition, the wheat bc1-complex comprises cytochrome b (35 kDa), cytochrome c1 (33 kDa) the "Rieske" iron-sulphur protein (25 kDa) and three small subunits < 15 kDa. This composition differs from the one reported in fungi, mammals and potato. Partial sequence determination of the large subunits suggests that the 55.5- and 55.0-kDa-proteins represent the beta-subunit of the general mitochondrial processing peptidase, and the 51.5- and 51.0-kDa proteins the alpha-subunit of this enzyme. The bc1-complex from wheat efficiently processes mitochondrial precursor proteins as shown in an in-vitro processing assay. In control experiments the isolated bc1-complexes from potato, yeast, Neurospora and beef, all purified by the same isolation procedure, were also tested for processing activity. Only the protein complexes from plants contain the general mitochondrial processing peptidase. The composition of the wheat bc1-complex sheds new light on the co-evolution of the processing peptidase and the middle segment of the respiratory chain.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Metaloendopeptidases/metabolismo , Triticum/enzimologia , Sequência de Aminoácidos , Cromatografia de Afinidade , Cromatografia em Gel , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , NADH Desidrogenase/isolamento & purificação , Homologia de Sequência de Aminoácidos , Peptidase de Processamento MitocondrialRESUMO
The mitochondrial iron-sulfur protein (also termed Rieske iron-sulfur protein) of cytochrome c reductase was purified from potato tubers and identified with heterologous antibodies. The sequences of the N-terminus of this 25 kDa protein and of an internal peptide were determined to design oligonucleotide mixtures for screening a cDNA library. One class of cDNA clones containing an open reading frame of 265 amino acids was isolated. The encoded protein contains the peptide sequences of the 25 kDa protein and shares about 50% sequence identity with the Rieske iron-sulfur proteins from fungi and around 43% with those from mammals. In vitro transcription and translation of the cDNA reveals that the iron-sulfur protein is made as a larger precursor of 30 kDa which is processed by the cytochrome c reductase/processing peptidase complex from potato. The processing product obtained after in vitro processing has the same size as the mature protein imported into isolated mitochondria. The presequence, which targets the protein to the organelle, is 53 amino acids long and has molecular features different from those found in presequences of fungal iron-sulfur proteins, which are processed in two steps. Our results indicate that, unlike in yeast and Neurospora, the presequence of the iron-sulfur protein from potato is removed by a single processing enzyme in one step.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/química , Solanum tuberosum/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Bovinos , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Genes de Plantas , Humanos , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/isolamento & purificação , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Neurospora/metabolismo , Neurospora/ultraestrutura , Processamento de Proteína Pós-Traducional , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Solanum tuberosum/ultraestrutura , Transcrição Gênica , Leveduras/química , Leveduras/metabolismoRESUMO
The aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole-plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose-6-phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.
Assuntos
Elementos Antissenso (Genética)/farmacologia , Carbono/metabolismo , Glucose-6-Fosfato/metabolismo , Fosfoglucomutase/antagonistas & inibidores , Plastídeos/enzimologia , Solanum tuberosum/metabolismo , Sequência de Bases , Transporte Biológico , Citosol/metabolismo , Primers do DNA , DNA Complementar , Glicólise , Organelas/metabolismo , Fenótipo , Plantas Geneticamente Modificadas/metabolismoRESUMO
The amino-terminal transit sequences of two preproteins destined for the chloroplast inner envelope membrane show similarities to mitochondrial presequences in the prevalence of positive charges and the potential formation of an amphipathic alpha-helix. We studied if these preproteins could be imported into mitochondria and found a low, yet significant import into isolated plant mitochondria. The plant mitochondria were previously shown not to import precursors of chloroplast stromal or thylakoidal proteins. To analyze the specificity of import into mitochondria we used the established import systems of fungal mitochondria. The envelope preproteins were efficiently imported into Saccharomyces cerevisiae or Neurospora crassa mitochondria. Their import showed the characteristics of specific mitochondrial protein uptake, including a requirement for the main receptor MOM19 (mitochondrial outer membrane protein of 19 kDa) and a membrane potential across the inner membrane, and depended on the presence of the chloroplast transit sequence. We conclude that some chloroplast transit sequences contain sufficient information for specific interaction with mitochondrial import receptors (at least from fungal sources).
Assuntos
Ascomicetos/metabolismo , Compartimento Celular , Cloroplastos/fisiologia , Mitocôndrias/metabolismo , Proteínas de Plantas/metabolismo , Precursores de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transporte Biológico , Membranas Intracelulares/química , Potenciais da Membrana , Proteínas de Transporte da Membrana Mitocondrial , Neurospora crassa/metabolismo , Proteínas de Plantas/genética , Precursores de Proteínas/genética , Saccharomyces cerevisiae/metabolismo , Especificidade da EspécieRESUMO
A 68-kDa heat-stress protein (HSP68) has been purified from cell-suspension cultures of tomato (Lycopersicon peruvianum L.). Antibodies raised against HSP68 cross-react with the Escherichia coli heat-stress protein DnaK. HSP68 was found to be a hydrophilic, ATP-binding protein. Immunological analysis of subcellular fractions and immunogold-labelling of ultrathin sections showed consistently that HSP68 is localized in the mitochondrial matrix. In-vitro translation experiments indicated that HSP68 is synthesized as a precursor protein. Immunoscreening of cDNA libraries from tomato and potato (Solanum tuberosum L.) led to the isolation of corresponding cDNA clones. The deduced amino-acid sequences show strong relationships to the DnaK-like proteins from bacteria and organelles of eukaryotic cells. The protein HSP68 is constitutively expressed, but its synthesis is increased during heat stress in all cells of higher plants investigated so far.