RESUMEN
Purification of mitochondria and mitochondrial protein complexes from green tissues is often severely impaired by the presence of chloroplasts and their proteins. Here we present a method which allows analysis of respiratory protein complexes from potato leaves. The procedure includes the preparation of an organellar fraction specifically enriched in mitochondria and the separation of organellar protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). For the first time mitochondrial and chloroplast protein complexes have been resolved simultaneously in a native gel. BN-PAGE allowed the separation of eleven bands, including the mitochondrial NADH-dehydrogenase, the bc1 complex and the mitochondrial F1-ATP synthase as well as the chloroplast F1-ATP synthase, the cytochrome b6f complex, the two photosystems and the light harvesting complex. The resolution of the protein complexes in the first dimension was good enough to allow identification of all subunits of individual complexes in the second dimension under denaturing conditions. Thus, BN-PAGE offers an opportunity to analyze mitochondrial and chloroplast protein complexes from a single preparation from very small amounts of tissue. The implications of our findings, for studies on protein expression and turnover in different tissues and developmental stages, are discussed.
Asunto(s)
Proteínas de Plantas/aislamiento & purificación , Cloroplastos/química , Electroforesis en Gel Bidimensional , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Mitocondrias/química , Hojas de la Planta/química , Proteínas de Plantas/química , Solanum tuberosum/químicaRESUMEN
Transport of most nuclear encoded mitochondrial proteins into mitochondria is mediated by heteropolymeric translocases in the membranes of the organelles. The translocase of the outer mitochondrial membrane (TOM) was characterized in fungi, and it was shown that TOM from yeast comprises nine different subunits. This publication is the first report on the preparation of the TOM complex from plant mitochondria. The protein complex from potato was purified by (a) blue native polyacrylamide gel electrophoresis and (b) by immunoaffinity chromatography. On blue native gels, the potato TOM complex runs close to cytochrome c oxidase at 230 kDa and hence only comprises about half of the size of fungal TOM complexes. Analysis of the TOM complex from potato by SDS-polyacrylamide gel electrophoresis allows separation of seven different subunits of 70, 36, 23, 9, 8, 7, and 6 kDa. The 23-kDa protein is identical to the previously characterized potato TOM20 receptor, as shown by in vitro assembly of this protein into the 230-kDa complex, by immunoblotting and by direct protein sequencing. Partial amino acid sequence data of the other subunits allowed us to identify sequence similarity between the 36-kDa protein and fungal TOM40. Sequence analysis of cDNAs encoding the 7-kDa protein revealed significant sequence homology of this protein to TOM7 from yeast. However, potato TOM7 has a N-terminal extension, which is very rich in basic amino acids. Counterparts to the TOM22 and TOM37 proteins from yeast seem to be absent in the potato TOM complex, whereas an additional low molecular mass subunit occurs. Functional implications of these findings are discussed.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Proteínas de Transporte de Membrana , Mitocondrias/enzimología , Solanum tuberosum/enzimología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Cromatografía de Afinidad/métodos , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Membranas Intracelulares/enzimología , Datos de Secuencia Molecular , Peso Molecular , Canales de Translocación SEC , Proteína SecA , Homología de Secuencia de AminoácidoRESUMEN
Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.
Asunto(s)
Mitocondrias/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Superficie Celular/metabolismo , Solanum tuberosum/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Clonación Molecular , Hongos/metabolismo , Biblioteca de Genes , Humanos , Inmunoglobulina G , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/química , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TripsinaRESUMEN
Recently a powerful electrophoresis method for the native preparation and characterization of the respiratory protein complexes of mitochondria from fungi and mammals has been developed, which employs Coomassie dyes to introduce charge shifts on proteins (Schägger and von Jagow (1991) Anal. Biochem. 199, 223-231). The procedure, which is called 'blue native-polyacrylamide gel electrophoresis' (BN-PAGE), was modified and introduced for the analysis of mitochondria from higher plants. BN-PAGE of mitochondrial protein from potato allows the separation of nine distinct protein complexes between 100 and 1000 kDa and reveals novel results for their composition, molecular mass and stoichiometry. For the first time soluble mitochondrial protein complexes, like the HSP60 complex (750 kDa) and a complex of 200 kDa, which includes a formate dehydrogenase, are analysed by BN-PAGE. Complex I from potato (1000 kDa) is about 100 kDa larger than the corresponding enzyme from beef and can be resolved into more than 30 different subunits on a second gel dimension. The F1F0 ATP synthase (580 kDa) and the cytochrome c oxidase (160 kDa) from potato seem to contain more subunits than hitherto reported. Direct sequencing of subunits revealed that the F1 part of the F1F0 ATP synthase lacks the oligomycin sensitivity conferring protein (OSCP), which was reported to be present in F1 parts of dicotyledonous plants, but contains the ATPase inhibitory protein. N-terminal sequences of 16 mitochondrial proteins were obtained, several of which are presented for the first time from a plant source. BN-PAGE allows the preparation of mitochondrial protein complexes from gram amounts of plant tissue, as the procedure only requires milligram amounts of organelles. This potential of BN-PAGE is demonstrated by the separation and characterization of the mitochondrial enzyme complexes from Arabidopsis thaliana. Further analysis of organellar protein complexes by BN-PAGE will allow the generation of 'protein maps' from different tissues and developmental stages or from mutant plants.
Asunto(s)
Mitocondrias/química , Proteínas de Plantas/química , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/enzimología , Arabidopsis/genética , Electroforesis en Gel de Poliacrilamida/métodos , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Conformación Proteica , Desnaturalización Proteica , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/aislamiento & purificación , Solanum tuberosum/química , Solanum tuberosum/genética , SolubilidadRESUMEN
Cytochrome c reductase from potato has been extensively studied with respect to its catalytic activities, its subunit composition, and the biogenesis of individual subunits. Molecular characterization of all 10 subunits revealed that the high-molecular-weight subunits exhibit striking homologies with the components of the general mitochondrial processing peptidase (MPP) from fungi and mammals. Some of the other subunits show differences in the structure of their targeting signals or in their molecular composition when compared to their counterparts from heterotrophic organisms. The proteolytic activity of MPP was found in the cytochrome c reductase complexes from potato, spinach, and wheat, suggesting that the integration of the protease into this respiratory complex is a general feature of higher plants.
Asunto(s)
Precursores Enzimáticos/química , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Mitocondrias/enzimología , NADH Deshidrogenasa/química , NADH Deshidrogenasa/metabolismo , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Precursores Enzimáticos/metabolismo , Cinética , Sustancias Macromoleculares , Metaloendopeptidasas/biosíntesis , Datos de Secuencia Molecular , NADH Deshidrogenasa/biosíntesis , Plantas/enzimología , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Peptidasa de Procesamiento MitocondrialRESUMEN
The mitochondrial ADP/ATP translocator, also called adenine nucleotide translocase (ANT), is synthesized in plants with an N-terminal extension which is cleaved upon import into mitochondria. In contrast, the homologous proteins of mammals or fungi do not contain such a transient amino terminal presequence. To investigate whether the N-terminal extension is needed for correct intracellular sorting in vivo, translational fusions were constructed of the translocator cDNA--with and without presequence--with the beta-glucuronidase (gus) reporter gene. The distribution of reporter enzymatic activity in the subcellular compartments of transgenic plants and transformed yeast cells was subsequently analysed. The results show that: (i) the plant translocator presequence is not necessary for the correct localization of the ANT to the mitochondria; (ii) the mitochondrial targeting information contained in the mature part of the protein is sufficient to overcome, to some extent, the presence of plastid transit peptides; and (iii) the presequence alone is not able to target a passenger protein to mitochondria in vivo.
Asunto(s)
Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Señales de Clasificación de Proteína/metabolismo , Solanum tuberosum/metabolismo , Secuencia de Bases , ADN de Plantas , Glucuronidasa/genética , Translocasas Mitocondriales de ADP y ATP/genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Solanum tuberosum/genéticaRESUMEN
The cytochrome-c reductase (EC 1.10.2.2) of the mitochondrial respiratory chain couples electron transport from ubiquinol to cytochrome c with proton translocation across the inner mitochondrial membrane. The enzyme from potato was shown to be composed of 10 subunits. Isolation and characterization of cDNA clones for the second smallest subunit reveal an open reading frame of 216 bp encoding a protein of 8.0 kDa. The protein exhibits similarities to a 7.2/7.3 kDa subunit of cytochrome-c reductase from bovine and yeast, that is localized on the intermembrane space side of the enzyme complex. It also shows similarity to a previously unidentified 7.8 kDa protein of cytochrome-c reductase from Euglena. The potato 8.0 kDa protein has a segmental structure, as its sequence can be divided into four parts, each comprising a central Arg-(Xaa)5-Val motif. N-terminal sequencing of the mature 8.0 kDa proteins indicates the absence of a cleavable mitochondrial targeting sequence. Import of the in vitro synthesized 8.0 kDa protein into isolated potato mitochondria confirms the lack of a presequence and reveals a dependence of the transport on the membrane potential delta psi across the inner mitochondrial membrane. These features are unique among the intermembrane space proteins known so far.
Asunto(s)
NADH Deshidrogenasa/química , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Clonación Molecular , ADN Complementario , ADN de Plantas , Potenciales de la Membrana , Datos de Secuencia Molecular , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The cytochrome c reductase complexes from fungi and mammals both contain a 14-kD protein (yeast, 14.4 kD; bovine, 13.4 kD) that does not directly participate in electron transfer but possibly is indirectly involved in the function of the complex and has a role in assembly of the multimeric enzyme. A subunit of comparable size was identified for the bc1 complex of higher plants. The 14-kD protein from potato (Solanum tuberosum) was specifically separated from the isolated protein complex in the presence of 6 M urea and is, therefore, assumed to be a peripheral component. Direct sequence analysis of the proteins from potato and wheat (Triticum aestivum) and isolation of corresponding cDNA clones for the subunit from potato revealed clear similarity to the equivalent proteins from yeast and bovine. The wheat 14-kD protein seems to occur in two isoforms. The 14-kD protein from plants is very hydrophilic, has a characteristic charge distribution, and contains no potential membrane-spanning helices. In vitro import of the radiolabeled 14-kD protein from potato into isolated mitochondria depends on the membrane potential across the inner mitochondrial membrane. The protein seems to lack a cleavable mitochondrial presequence, because it is not processed upon translocation. Possible intramolecular regions involved in targeting of the 14-kD protein to plant mitochondria are discussed.
Asunto(s)
NADH Deshidrogenasa/genética , Solanum tuberosum/enzimología , Solanum tuberosum/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN de Plantas/genética , Mitocondrias/enzimología , Datos de Secuencia Molecular , Peso Molecular , NADH Deshidrogenasa/química , NADH Deshidrogenasa/metabolismo , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Triticum/enzimología , Triticum/genéticaAsunto(s)
ADN de Plantas/genética , Isoenzimas/genética , Metaloendopeptidasas/genética , Solanum tuberosum/enzimología , Solanum tuberosum/genética , Clonación Molecular , ADN Complementario/genética , Isoenzimas/química , Metaloendopeptidasas/química , Datos de Secuencia Molecular , Conformación Proteica , Peptidasa de Procesamiento MitocondrialRESUMEN
Analysis of cytochrome c reductase from potato by Tricine/SDS/PAGE reveals 10 bands representing 10 different subunits. In comparison to glycine/SDS/PAGE one additional small protein becomes visible, whereas the three large core proteins are not resolved. The identity of the subunits was determined by immunoblotting and direct sequence determination. Sequence data for the novel small component were used to derive oligonucleotides for probing a potato cDNA-library and isolating corresponding clones. The newly identified subunit is a 6.7-kDa protein, that exhibits significant sequence similarity to a 8.5-kDa subunit of cytochrome c reductase from yeast and the 6.5-kDa iron-sulfur-protein-binding factor from the equivalent enzyme complex from beef. Also the potato 6.7-kDa subunit can be dissociated from the cytochrome c reductase complex together with the iron-sulfur protein. To address the question of whether three or two core subunits occur simultaneously in monomeric cytochrome c reductase complexes from potato, a peptide-specific antibody was generated. The antiserum is capable of discriminating between the 55-kDa and 53-kDa core proteins, which can be separated by glycine/SDS/PAGE and which were previously found to be structurally related. Immunoprecipitations of isolated cytochrome c reductase from potato using this antibody revealed an enzyme complex containing only two core proteins. The simultaneous occurrence of only two core subunits was confirmed by a comparison of the molecular masses of cytochrome c reductase from potato and beef by blue-native-gel electrophoresis. Hence the cytochrome c reductase complexes from potato, beef and yeast have a very conserved subunit composition. The evolutionary implications of these findings are discussed.
Asunto(s)
NADH Deshidrogenasa/química , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Bovinos , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Carne , Mitocondrias/enzimología , Datos de Secuencia Molecular , Peso Molecular , NADH Deshidrogenasa/genética , Pruebas de Precipitina , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de AminoácidoAsunto(s)
Complejo III de Transporte de Electrones/aislamiento & purificación , Endopeptidasas/química , Endopeptidasas/aislamiento & purificación , Mitocondrias/enzimología , Complejos Multienzimáticos/química , Complejos Multienzimáticos/aislamiento & purificación , NADH Deshidrogenasa/aislamiento & purificación , Solanum tuberosum/enzimología , Animales , Cromatografía de Afinidad/métodos , Grupo Citocromo c , Transporte de Electrón , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Endopeptidasas/metabolismo , Caballos , Indicadores y Reactivos , Sustancias Macromoleculares , Peso Molecular , Complejos Multienzimáticos/metabolismo , Miocardio , NADH Deshidrogenasa/química , NADH Deshidrogenasa/metabolismoRESUMEN
Cytochrome c reductase from potato comprises ten subunits with apparent molecular sizes between 55 and < 10 kDa. The subunit with the highest electrophoretic mobility on SDS-polyacrylamide gels was isolated and analysed by cyclic Edman degradation. Mixtures of degenerative oligonucleotides were derived from the obtained sequence data and used for the isolation of corresponding cDNA clones. The clones encode a protein of 72 amino acids which exhibits significant sequence identity with a 9.5 kDa subunit of cytochrome c reductase from bovine and a 11 kDa subunit of the enzyme complex from yeast. Comparison between the deduced amino acid sequence of the open reading frame and the sequence of the mature protein reveals that only the initiator methionine is absent in the functional subunit. Hence the protein has a calculated molecular mass of 8.2 kDa. Transcripts of the potato 8.2 kDa protein were not translated in reticulocyte lysates but in vitro translation worked efficiently with wheat germ lysate. Import of the radiolabelled protein into isolated mitochondria from potato seems to depend on a potential across the inner membrane and confirms the absence of a cleavable mitochondrial presequence.
Asunto(s)
NADH Deshidrogenasa/química , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Mitocondrias/enzimología , Datos de Secuencia Molecular , NADH Deshidrogenasa/síntesis química , NADH Deshidrogenasa/aislamiento & purificación , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Metaloendopeptidasas/genética , Mitocondrias/enzimología , Complejos Multienzimáticos/genética , NADH Deshidrogenasa/genética , Proteínas de Plantas/genética , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , ADN de Plantas/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Metaloendopeptidasas/química , Datos de Secuencia Molecular , Proteínas de Plantas/química , Ratas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Solanum tuberosum/genética , Especificidad de la Especie , Transcripción Genética , Levaduras/química , Peptidasa de Procesamiento MitocondrialRESUMEN
The mitochondrial outer membrane of eukaryotic cells contains a voltage-dependent anion channel termed porin. In the organisms studied so far only one type of porin has been identified at the protein level. Here we present a biochemical and molecular genetic analysis of two different porin polypeptides of M(r) 34,000 and 36,000 from the outer membranes of potato mitochondria (termed POM 34 and POM 36, respectively). N-terminal sequencing and the use of labeled oligonucleotide mixtures derived from these amino acid sequences allowed the isolation of cDNA clones encoding the 34- and 36-kDa proteins. They have similar steady state protein levels and share about 75% identical amino acids suggesting that they represent isoforms. In addition, a third cDNA clone coding for a slightly different isoform of the 36-kDa protein was characterized. The polypeptides encoded by the three cDNA clones share the highest degree of sequence identity with mitochondrial porins from fungi and mammals. Tentative models of the secondary structure of the 34- and 36-kDa proteins suggest the occurrence of a 16-stranded beta-barrel typical for bacterial and mitochondrial porins. Purification of the 34-kDa protein by hydroxyapatite chromatography allowed conductance measurements in artificial bilayers. The 34-kDa protein is a voltage-dependent, channel-forming component with single channel conductances of 3.5 and 2.0 nanosiemens in 1 M KCl. In spite of the striking functional similarities to mitochondrial porins from other organisms neither the 34- nor the 36-kDa proteins are able to complement the respiratory defect of a yeast por- mutant.
Asunto(s)
Mitocondrias/química , Porinas/aislamiento & purificación , Solanum tuberosum/química , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Porinas/química , Porinas/inmunología , Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Genes de Plantas , Proteínas Ribosómicas/genética , Solanum tuberosum/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citosol/metabolismo , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Ratas , Proteínas Ribosómicas/biosíntesis , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Solanum tuberosum/metabolismoRESUMEN
The 'Hinge' protein of cytochrome c reductase from fungi and mammals is thought to support electron transport from cytochrome c1 to cytochrome c and was reported to be one of the most acidic proteins known. Isolation and analysis of cDNA clones of the first 'Hinge' protein from a plant source reveals that it has a surplus of basic residues in potato. While the overall identity between the deduced amino acid sequence of the potato 'Hinge' protein and the proteins from yeast and bovine is in the range of 40%, the characteristic acidic domain is lacking. Therefore the numerous theories on the function of the mitochondrial 'Hinge' protein seem not to apply for the protein from potato. Also the atypical acidic presequence of the 'Hinge' protein from fungi and mammals is absent as revealed by N-terminal sequencing of the isolated potato 'Hinge' protein. Functional implications of these results for the 'Hinge' proteins from other organisms are discussed.
Asunto(s)
Mitocondrias/enzimología , NADH Deshidrogenasa/genética , Proteínas/genética , Solanum tuberosum/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Biblioteca de Genes , Datos de Secuencia Molecular , NADH Deshidrogenasa/química , Conformación Proteica , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína , Proteínas/química , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Solanum tuberosum/enzimologíaRESUMEN
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.
Asunto(s)
Complejo III de Transporte de Electrones , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/química , Solanum tuberosum/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Bovinos , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Genes de Plantas , Humanos , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/aislamiento & purificación , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Neurospora/metabolismo , Neurospora/ultraestructura , Procesamiento Proteico-Postraduccional , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Solanum tuberosum/ultraestructura , Transcripción Genética , Levaduras/química , Levaduras/metabolismoRESUMEN
Cytochrome-c reductase (EC 1.10.2.2.) from Solanum tuberosum L. comprises ten subunits with apparent molecular sizes of 55, 53, 51, 35, 33, 25, 14, 12, 11 and 10 kDa on 14% SDS-PAGE. The identity of the subunits was analysed by direct amino-acid sequencing via cyclic Edman degradation. A large-scale purification procedure for the enzyme complex based on affinity chromatography and gelfiltraton is described. All subunits were enzymatically fragmented and the generated peptides were separated by reverse-phase HPLC. Complete or partial sequence determination of 33 peptides comprising a total of nearly 500 amino acids showed, that cytochrome-c reductase from potato contains three respiratory proteins (cytochrome b, cytochrome c1, and the "Rieske" iron-sulfur protein), four small proteins with molecular sizes below 15 kDa (so-called Q-binding, hinge, cytochrome-c1-linked and core-linked proteins) and three proteins in the 50-kDa range which show similarity to members of the core/PEP/MPP protein family (core/processing enhancing protein/mitochondrial processing peptidase). In fact these subunits show highest sequence identity either to MPP or PEP, which is in line with earlier findings, that isolated cytochrome-c reductase from potato exhibits processing activity towards mitochondrial precursor proteins.
Asunto(s)
Mitocondrias/enzimología , NADH Deshidrogenasa/química , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Endopeptidasas/metabolismo , Mitocondrias/enzimología , NADH Deshidrogenasa/metabolismo , Compuestos de Sodio , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Bromuros/farmacología , ADN/química , ADN/aislamiento & purificación , Estabilidad de Enzimas , Escherichia coli/genética , Expresión Génica , Sustancias Macromoleculares , Peso Molecular , NADH Deshidrogenasa/química , NADH Deshidrogenasa/genética , Octoxinol , Polietilenglicoles/farmacología , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia , Sodio/farmacología , Cloruro de Sodio/farmacología , Yoduro de Sodio/farmacología , Solanum tuberosum/ultraestructuraRESUMEN
The structural organization of a nuclear gene encoding cytochrome c1 from potato was determined. The gene spans 5.1 kb and contains eight introns. All intron/exon junctions follow the GT/AG rule. Functional domains of the mature cytochrome c1 protein are located on separate exons. The presequence, which targets the cytochrome c1 precursor to the mitochondrion and to the correct intra-mitochondrial location, is encoded on the first four exons. The largest intron (2.8 kb) separates the information for mitochondrial targeting from the "intra-mitochondrial sorting domain" of the cytochrome c1 protein. In contrast to other organellar precursor proteins, there is no intron between the DNA sequence encoding the presequence and the mature protein. This may indicate that during evolution the genetic information for the prokaryotic cytochrome c1 was transferred to the nucleus together with the bacterial secretion signal which is structurally and functionally related to "intramitochondrial sorting domains".