RESUMEN
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
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
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 AminoácidoRESUMEN
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
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
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.
Asunto(s)
Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , NADH Deshidrogenasa/metabolismo , Serina Endopeptidasas , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Grupo Citocromo c/metabolismo , ADN , Endopeptidasas/genética , Endopeptidasas/aislamiento & purificación , Sustancias Macromoleculares , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Homología de Secuencia de Ácido Nucleico , Solanum tuberosum/genética , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMEN
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.