Are the 'core' proteins of the mitochondrial bc1 complex evolutionary relics of a processing protease?

The so-called 'core' proteins of the respiratory cytochrome bc1 complex and the two subunits of the mitochondrial processing peptidase (MPP) are structurally similar but their evolutionary relationship remains a mystery. Here, we present a model suggesting that the core proteins originated from an ancient proteolytic enzyme that was integrated into the bc1 complex during early stages of endosymbiosis.

Molecular structure of the 8.0 kDa subunit of cytochrome-c reductase from potato and its delta psi-dependent import into isolated mitochondria.

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.

Primary structure, cell-free synthesis and mitochondrial targeting of the 8.2 kDa protein of cytochrome c reductase from potato.

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.

Primary structure and expression of a gene encoding the cytosolic ribosomal protein S4 from potato.

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.

The Cytochrome c Reductase Integrated Processing Peptidase from Potato Mitochondria Belongs to a New Class of Metalloendoproteases.

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.

The mitochondrial processing peptidase.

The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme which plays an essential role in mitochondrial protein import. It cleaves off the N-terminal targeting signals of nuclear encoded mitochondrial proteins upon their transport into the organelle. In mammals and yeast the enzyme is localized in the mitochondrial matrix while in plants it is integrated into a protein complex of the respiratory chain. As the activity of MPP is essential for the viability of eukaryotic cells it is conceivable that inhibitors of MPP which are specific for the soluble enzyme only present in fungi and animals may work as fungicides or insecticides.

Purification and sequencing of cytochrome b from potato reveals methionine cleavage of a mitochondrially encoded protein.

Several mitochondrial genes from a large number of different fungi, mammals and plants have been sequenced but little is known about the corresponding translation products. We have affinity purified cytochrome c reductase from potato mitochondria and isolated the mitochondrially encoded cytochrome b protein. Amino-terminal sequencing reveals that the polypeptide does not start with a methionine. Comparison of the amino acid sequence with the recently published sequence of the gene encoding the cytochrome b apoprotein suggests that the N-formylmethionine is removed. This result provides the first evidence for the presence of a deformylase and a methionine aminopeptidase in mitochondria.

The 'Hinge' protein of cytochrome c reductase from potato lacks the acidic domain and has no cleavable presequence.

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.

Resolution of mitochondrial and chloroplast membrane protein complexes from green leaves of potato on blue-native polyacrylamide gels.

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.

The presequence of cytochrome c1 from potato mitochondria is encoded on four exons.

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".

The bifunctional cytochrome c reductase/processing peptidase complex from plant mitochondria.

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.

The protein-import apparatus of plant mitochondria.

The import and assembly of mitochondrial proteins synthesized in the cytosol is mediated by the protein-import apparatus which plays a central role in mitochondrial biogenesis. Ten years ago only some components of the protein-import apparatus from fungi and mammals were characterized, but today its major components have been analyzed at the molecular level also in plants. Interestingly there are specific features which distinguish the protein-import apparatus of plants from that of fungi and mammals. Here we give an overview of all known components of the protein-import apparatus from plants and focus on its differences in comparison to heterotrophic eukaryotes.

Molecular features and mitochondrial import pathway of the 14-kilodalton subunit of cytochrome c reductase from potato.

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.

A receptor for protein import into potato mitochondria.

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.

A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo.

To date, the presequence of the mitochondrial beta-subunit of ATPase from tobacco is the only signal sequence that has been shown to target a foreign protein into plant mitochondria in vivo. Here we report that the presequence of a yeast mitochondrial protein directs bacterial beta-glucuronidase (GUS) specifically into the mitochondrial compartment of transgenic tobacco plants. Fusions between the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene from yeast and the GUS gene have been introduced into tobacco plants and yeast cells. In both systems, proteins containing the complete yeast mitochondrial presequence are efficiently imported in the mitochondria. Measurements of GUS activity in different subcellular fractions indicate that there is no substantial misrouting of the chimeric proteins in plant cells. In vitro synthesized GUS fusion proteins have a higher molecular weight than those found inside yeast and tobacco mitochondria, suggesting a processing of the precursors during import. Interestingly, fusion proteins translocated across the mitochondrial membranes of tobacco have the same size as those that are imported into yeast mitochondria. We conclude that the processing enzyme in plant mitochondria may recognize a proximate or even the same cleavage site within the mitochondrial tryptophanyl-tRNA-synthetase presequence as the matrix protease from yeast.

Polymorphism and gene arrangement among plastomes of ten Epilobium species.

Plastid DNAs of ten different Epilobium species from four continents have been analysed using the restriction endonucleases BamHI, BglI, BglII, EcoRI, PstI, PvuII and SalI. With respect to the position of cleavage sites of those enzymes, each species has a specific plastome. Fragment patterns of different species from the same continent show a higher degree of similarity than those from different continents. Physical maps of the circular plastid DNA molecule have been constructed for each of the ten species by localising the cleavage sites of the enzymes BglI, PvuII and SalI. As in most other higher plants, the plastid DNA of Epilobium is segmentally organized into two inverted repeats separated by a large and a small single copy region. In heterologous hybridization experiments using radioactively labelled gene probes, the positions of structural genes coding for the rRNAs and for seven polypeptides have been determined. In contrast to its closest relative, Oenothera, the gene arrangement of Epilobium plastomes has the same order as in spinach. This indicates that changes in gene arrangement may be genus-specific and not the result of one or several events affecting all members of a plant family.

Dwarfism and male sterility in interspecific hybrids of Epilobium : 2. Expression of mitochondrial genes and structure of the mitochondrial DNA.

Mitochondrial DNA (mtDNA) and transcriptional patterns of mitochondrial genes have been examined in dwarf, normal, fertile and male sterile Epilobium hybrids. No alterations or rearrangements of mitochondrial DNA could be detected in the developmentally disturbed hybrids. They exhibit restriction patterns of mtDNA that correspond exactly to those of their female parents. However, the transcription of at least one mitochondrial gene is significantly altered in the male sterile hybrid E. hirsutum x montanum. In normal plants, one mRNA of 1.6 kb hybridizes to the cytochrome c oxidase subunit II gene, while in male sterile plants a transcript of this size is lacking and instead a major transcript of 2.0 kb and two smaller ones occur. The transcript pattern of the F1 ATPase alpha subunit (atpA) gene exhibits slight alterations in sterile plants also. Since these hybrids have the same cytoplasm as normal plants, an incompatibility between the nuclear and the mitochondrial genotype may be responsible for the altered mitochondrial gene expression. No alteration of the transcripts of the mitochondrial genes tested could be detected in dwarf hybrids. The coincidence of male sterility with an altered transcription pattern of mitochondrial genes suggests that the mitochondria are involved in the occurrence of this phenotype.