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Biomedical subjects

U K Schmitz

Publications and source records attributed to U K Schmitz.

At least 19 recordsLinked to original sources
PubMed2003

Alloplasmic male sterility in Brassica napus (CMS 'Tournefortii-Stiewe') is associated with a special gene arrangement around a novel atp9 gene.

To identify regions of the mitochondrial genome potentially involved in the expression of alloplasmic 'Tournefortii-Stiewe' cytoplasmic male sterility (CMS) in Brassica napus, transcripts of 25 mitochondrial genes were analysed in fertile and near isogenic male-sterile plants (BC(8) generation). Differences were detected in the transcription of genes for subunit 9 of ATP synthase (atp9), cytochrome b (cob) and subunit 2 of NADH dehydrogenase (nad2). Structural analysis of these gene regions revealed differences in genome organisation around atp9 between male-sterile and fertile plants. Three atp9 genes, two of which were hitherto unknown, are present in the mitochondria of CMS plants, and rearrangements upstream of one of these genes have generated a chimeric 193-codon ORF, designated orf193. This region is transcribed as a CMS specific bi-cistronic mRNA of 1.58 kb comprising orf193 and atp9. The level of the aberrant 1.58-kb transcript is reduced in plants restored to fertility by as yet uncharacterized nuclear genes. orf193 encodes a polypeptide of 22.7 kDa which exhibits partial sequence identity to the subunit 6 of the ATP synthase complex. However, as it forms an uninterrupted ORF with one of the newly discovered atp9 genes it may also be translated as a chimeric 30.2-kDa protein. It is likely that either or both gene products interfere with the function or assembly of the mitochondrial F(0)F(1)-ATP synthase, thus impairing the highly ATP-dependent process of pollen development. The novel molecular features of alloplasmic 'Tournefortii-Stiewe' CMS are discussed with respect to the other known mechanisms of CMS in B. napus.

Apoproteins↗
PubMed2001

Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis. Identification of multiple forms of TOM20.

The translocase of the outer mitochondrial membrane (TOM) complex is a preprotein translocase that mediates transport of nuclear-encoded mitochondrial proteins across the outer mitochondrial membrane. Here we report the purification of this protein complex from Arabidopsis. On blue-native gels the Arabidopsis TOM complex runs at 230 kD and can be dissected into subunits of 34, 23, 21, 8, 7, and 6 kD. The identity of four subunits could be determined by immunoblotting and/or direct protein sequencing. The 21- and the 23-kD subunits exhibit significant sequence homology to the TOM20 preprotein receptor from other organisms. Analysis by two-dimensional isoelectric focusing/Tricine sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the presence of further forms for Arabidopsis TOM20. All TOM20 proteins comprise a large cytoplasmically exposed hydrophilic domain, which is degraded upon trypsination of intact mitochondria. Clones encoding four different forms of Arabidopsis TOM20 were identified and sequenced. The deduced amino acid sequences are rather conserved in the N-terminal half and in the very C-terminal part, but include a highly variable glycine-rich region close to the C terminus. Implications on the function of plant TOM complexes are discussed. Based on peptide and nucleic acid sequence data, the primary structure for Arabidopsis TOM40 is presented.

Adenosine Triphosphatases↗
PubMed2000

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.

Chloroplasts↗
PubMed1999

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.

Animals↗
PubMed1998

Unique composition of the preprotein translocase of the outer mitochondrial membrane from plants.

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.

Adenosine Triphosphatases↗
PubMed1998

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 c reductase 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 cytochrome c 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 algae Polytomella, 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 c reductase 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 cytochrome c reductase complex and the occurrence of chloroplasts.

Amino Acid Sequence↗
PubMed1997

The 23-kDa light-stress-regulated heat-shock protein of chenopodium rubrum L. is located in the mitochondria.

The 23-kDa nuclear-encoded heat-shock protein (HSP) of Chenopodium rubrum L. is regulated by light at the posttranslational level. Higher light intensities are more effective in inducing the accumulation of the mature protein under heat-shock conditions. Based on this and other properties the protein was considered to belong to the group of small chloroplastic HSPs. However, we have now obtained the following evidence that this 23-kDa HSP is localized in the mitochondria: (i) Immunogold-labelled protein was almost exclusively restricted to the mitochondria in electron microscope thin sections. (ii) Using purified, isolated mitochondria from potato tubers the in-vitro-synthesized translation product of 31 kDa was readily transported into mitochondria where it was processed to the 23-kDa product. (iii) The protein could be detected by Western blotting in a preparation of washed mitochondria of Chenopodium, while under the same conditions no signal could be obtained in a preparation of isolated chloroplasts. (iv) Finally, sequence comparison with the published sequences of mitochondrial proteins by Lenne et al. (1995, Biochem J 311:805-813) and LaFayette et al. (1996, Plant Mol Biol 30:159-169) showed clearly that the 23-kDa protein is considerably more similar to these two proteins than to the group of plastid small HSPs. From these data we infer that mitochondria are involved in the response of the plants to high light stress under heat-shock conditions.

Amino Acid Sequence↗
PubMed1996

Subunit VII of ubiquinol:cytochrome-c oxidoreductase from Neurospora crassa is functional in yeast and has an N-terminal extension that is not essential for mitochondrial targeting.

cDNA clones encoding subunit VII of the Neurospora crassa bc1 complex (ubiquinol:cytochrome-c oxidoreductase), which is homologous with subunit VIII of the complex from yeast (encoded by QCR8), were identified on the basis of functional complementation of a yeast QCR8 deletion strain. The clones contain an open reading frame encoding a protein with a calculated molecular mass of 11.8 kDa. The N-terminal eight residues of the amino acid sequence deduced from the cDNA clones are absent from the mature protein, as revealed by direct sequencing of the isolated protein. To investigate the potential role of the N-terminal octapeptide in mitochondrial targeting, constructs were made encoding the precursor and the mature form of subunit VII from Neurospora. Incubation of isolated mitochondria with the two proteins revealed that the N-terminal extension of the precursor is removed on import. However, the presequence does not encode information for targeting, as the proteins encoded by both constructs can be imported into isolated mitochondria with equal efficiency. In contrast, the octapeptide seems to have functional importance: the defect in the yeast qcr8-null mutant is not complemented on transformation with the construct encoding mature subunit VII from N. crassa in a single-copy plasmid. We therefore speculate that the N-terminal extension plays a role in intramitochondrial sorting of N. crassa subunit VII. This is supported by the fact that the subunit VII precursor is processed by a protease other than the general mitochondrial processing peptidase. Interestingly, the presequence of N. crassa subunit VII has an amino acid composition similar to the octapeptides cleaved off by the mitochondrial intermediate peptidase.

Amino Acid Sequence↗
PubMed1996

New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria.

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.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

Cytochrome c reductase from potato does not comprise three core proteins but contains an additional low-molecular-mass subunit.

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.

Amino Acid Sequence↗
PubMed1995

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 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.

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

The N-terminal extension of the ADP/ATP translocator is not involved in targeting to plant mitochondria in vivo.

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.

Base Sequence↗