Volker Kruft

Redundancy, phylogeny and differential expression of Histoplasma capsulatum catalases.

Histoplasma capsulatum produces an extracellular catalase termed M antigen, which is similar to catalase B of Aspergillus and Emericella species. Evidence is presented here for two additional catalase isozymes in H. capsulatum. Catalase A is highly similar to a large-subunit catalase in Aspergillus and Emericella species, while catalase P is a small-subunit catalase protein with greatest similarity to known peroxisomal catalases of animals and Saccharomycotina yeasts. Complete cDNAs for the CATA and CATP genes (encoding catalases A and P, respectively) were isolated. The transcriptional expression of the H. capsulatum CATA, CATB (M antigen) and CATP genes was assessed by Northern blot hybridizations on total RNA. Results at the transcript levels for these genes are shown for three conditions: cell morphology (mycelial versus yeast phase cells), oxidative stress (in response to a challenge with H(2)O(2)) and carbon source (glucose vs glycerol). Collectively, these results demonstrated regulation of CATA by both cell morphology and oxidative stress, but not by carbon source, and regulation of CATB and CATP by carbon source but not cell morphology or oxidative stress. A phylogenetic analysis of presently available catalase sequences and intron residences was done. The results support a model for evolution of eukaryotic monofunctional catalase genes from prokaryotic genes.

The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain.

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.

Mitochondrial cytochrome c oxidase and succinate dehydrogenase complexes contain plant specific subunits

Respiratory oxidative phosphorylation represents a central functionality in plant metabolism, but the subunit composition of the respiratory complexes in plants is still being defined. Most notably, complex II (succinate dehydrogenase) and complex IV (cytochrome c oxidase) are the least defined in plant mitochondria. Using Arabidopsis mitochondrial samples and 2D Blue-native/SDS-PAGE, we have separated complex II and IV from each other and displayed their individual subunits for analysis by tandem mass spectrometry and Edman sequencing. Complex II can be discretely separated from other complexes on Blue-native gels and consists of eight protein bands. It contains the four classical SDH subunits as well as four subunits unknown in mitochondria from other eukaryotes. Five of these proteins have previously been identified, while three are newly identified in this study. Complex IV consists of 9–10 protein bands, however, it is more diffuse in Blue-native gels and co-migrates in part with the translocase of the outer membrane (TOM) complex. Differential analysis of TOM and complex IV reveals that complex IV probably contains eight subunits with similarity to known complex IV subunits from other eukaryotes and a further six putative subunits which all represent proteins of unknown function in Arabidopsis. Comparison of the Arabidopsis data with Blue-native/SDS-PAGE separation of potato and bean mitochondria confirmed the protein band complexity of these two respiratory complexes in plants. Two-dimensional Blue-native/Blue-native PAGE, using digitonin followed by dodecylmaltoside in successive dimensions, separated a diffusely staining complex containing both TOM and complex IV. This suggests that the very similar mass of these complexes will likely prevent high purity separations based on size. The documented roles of several of the putative complex IV subunits in hypoxia response and ozone stress, and similarity between new complex II subunits and recently identified plant specific subunits of complex I, suggest novel biological insights can be gained from respiratory complex composition analysis.

Trypanosoma brucei gBP21 AN ARGININE-RICH MITOCHONDRIAL PROTEIN THAT BINDS TO GUIDE RNA WITH HIGH AFFINITY

RNA editing in Trypanosoma brucei is a mitochondrial RNA processing reaction that results in the insertion and deletion of uridylate residues into otherwise untranslatable mRNAs. The process is directed by guide RNAs which function to specify the edited sequence. RNA editing in vitro requires mitochondrial protein extracts and guide RNAs have been identified as part of high molecular weight ribonucleoprotein complexes. Within the complexes, the RNAs are in close contact with several mitochondrial proteins and here we describe the isolation and cloning of a gRNA-interacting polypeptide from Trypanosoma brucei The protein was named gBP21 for uide RNA-inding rotein of kDa. gBP21 shows no homology to proteins in other organisms, it is arginine-rich and binds to gRNA molecules with a dissociation constant in the nanomolar range. The protein does not discriminate for differences in the primary structures of gRNAs and thus likely binds to higher order structural features common to all gRNA molecules. gBP21 binding does not perturb the overall structure of gRNAs but the gRNA/gBP21 ribonucleoprotein complex is more stable than naked guide RNAs. Although the protein is arginine-rich, the free amino acid or an arginine-rich peptide were not able to inhibit the association to the RNAs. In contrast, the gRNA-gBP21 complex formation was sensitive to potassium and ammonium cations, thus indicating a contribution of ionic contacts to the binding.

Cytochrome c1 from potato: a protein with a presequence for targeting to the mitochondrial intermembrane space.

SummaryHere 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.

New insights into the co-evolution of cytochrome c reductase and the mitochondrial processing peptidase.

The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme that forms part of the cytochrome creductase complex from higher plants. Mitochondria from mammals and yeast contain two homologous enzymes: (i) an active MPP within the mitochondrial matrix and (ii) an inactive MPP within the cytochromec reductase complex. To elucidate the evolution of MPP, the cytochrome c reductase complexes from lower plants were isolated and tested for processing activity. Mitochondria were prepared from the staghorn fern Platycerium bifurcatum, from the horsetail Equisetum arvense, and from the colorless algaePolytomella, and cytochrome c reductase complexes were purified by a micro-isolation procedure based on Blue-native polyacrylamide gel electrophoresis and electroelution. This is the first report on the subunit composition of a respiratory enzyme complex from a fern or a horsetail. The cytochrome creductase complexes from P. bifurcatum and E. arvense are shown to efficiently process mitochondrial precursor proteins, whereas the enzyme complex from Polytomella lacks proteolytic activity. An evolutionary model is suggested that assumes a correlation between the presence of an active MPP within the cytochromec reductase complex and the occurrence of chloroplasts.

The general mitochondrial processing peptidase from wheat is integrated into the cytochrome bc1-complex of the respiratory chain

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 β-subunit of the general mitochondrial processing peptidase, and the 51.5 and 51.0-kDa proteins the α-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.

Extended N‐terminal sequencing of proteins of the large ribosomal subunit from yeast mitochondria

We have determined the N‐termini of 26 proteins of the large ribosomal subunit from yeast mitochondria by direct amino acid micro‐sequencing. The N‐terminal sequences of proteins YmL33 and YmL38 showed a significant similarity to eubacterial ribosomal (r‐) proteins L30 and L14, respectively. In addition, several proteins could be assigned to their corresponding yeast nuclear genes. Based on a comparison of the protein sequences deduced from the corresponding DNA regions with the N‐termini of the mature proteins, the putative leader peptides responsible for mitochondrial matrix‐targeting were compiled. In most leader sequences a relative abundance of aromatic amino acids, preferentially phenylalanine, was found.

4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene

We have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete Mucor mucedo. The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (-) mating type of M. mucedo after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic M. mucedo DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of M. mucedo. PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned M. mucedo fragment showed that a fragment of similar length cross-hybridized in Blakeslea trispora (Choanephoraceae) as well as in Parasitella parasitica and Absidia glauca (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of Dictyostelium discoideum.

Molecular features, processing and import of the Rieske iron-sulfur protein from potato mitochondria

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.