A Structural Model of the Cytochrome c Reductase/Oxidase Supercomplex from Yeast Mitochondria

Mitochondrial respiratory chain complexes are arranged in supercomplexes within the inner membrane. Interaction of cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) was investigated in Saccharomyces cerevisiae. Projection maps at 15 Å resolution of supercomplexes III2 + IV1 and III2 + IV2 were obtained by electron microscopy. Based on a comparison of our maps with atomic x-ray structures for complexes III and IV we present a pseudo-atomic model of their precise interaction. Two complex IV monomers are specifically attached to dimeric complex III with their convex sides. The opposite sides, which represent the complex IV dimer interface in the x-ray structure, are open for complex IV-complex IV interactions. This could lead to oligomerization of III2 + IV2 supercomplexes, but this was not detected. Instead, binding of cytochrome c to the supercomplexes was revealed. It was calculated that cytochrome c has to move less than 40 Å at the surface of the supercomplex for electron transport between complex III2 and complex IV. Hence, the prime function of the supercomplex III2 + IV2 is proposed to be a scaffold for effective electron transport between complexes III and IV.

Entwicklung einer internetbasierten Anwendung für Wirtschaftlichkeitsberechnungen im ökologischen Marktfruchtbau

Eine Internetanwendung der Bayerischen Landesanstalt für Landwirtschaft ermöglicht Wirtschaftlichkeitsberechnungen für 16 Verfahren des ökologischen Marktfruchtbaus. Der Nutzer wird durch umfangreiche Datenhinterlegung und einen übersichtlichen Programmaufbau unterstützt. Die Kalkulationen sind nach dem Prinzip der Leistungs-Kosten-Rechnung aufgebaut und führen zur Ermittlung von Deckungs- und Gewinnbeiträgen. In einem Zusatzmodul werden weitere Kenngrößen wie Äquivalenzpreise und -erträge, Grenzpachtpreise und Fruchtfolgedeckungsbeiträge ausgewiesen. Die Anwendung wird regelmäßig aktualisiert und laufend erweitert

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed. © 2011 The Author(s)

Biophysical and structural characterization of proton-translocating NADH-dehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica

AbstractMitochondrial proton-translocating NADH-dehydrogenase (complex I) is one of the largest and most complicated membrane bound protein complexes. Despite its central role in eukaryotic oxidative phosphorylation and its involvement in a broad range of human disorders, little is known about its structure and function. Therefore, we have started to use the powerful genetic tools available for the strictly aerobic yeast Yarrowia lipolytica to study this respiratory chain enzyme. To establish Y. lipolytica as a model system for complex I, we purified and characterized the multisubunit enzyme from Y. lipolytica and sequenced the nuclear genes coding for the seven central subunits of its peripheral part. Complex I from Y. lipolytica is quite stable and could be isolated in a highly pure and monodisperse state. One binuclear and four tetranuclear iron–sulfur clusters, including N5, which was previously known only from mammalian mitochondria, were detected by EPR spectroscopy. Initial structural analysis by single particle electron microscopy in negative stain and ice shows complex I from Y. lipolytica as an L-shaped particle that does not exhibit a thin stalk between the peripheral and the membrane parts that has been observed in other systems

Clustering and dynamics of cytochrome bd-I complexes in the Escherichia coli plasma membrane in vivo

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Multiple deficiencies of mitochondrial DNA- and nuclear-encoded subunits of respiratory NADH dehydrogenase detected with peptide- and subunit-specific antibodies in mitochondrial myopathies

AbstractAntibodies have been raised against synthetic peptides corresponding to several computer-predicted epitopes of three mtDNA-encoded subunits, ND4, ND5 and ND6, of the human respiratory chain NADH dehydrogenase (Complex I). Antibodies were characterized by a sensitive immunoblotting assay using proteins from human skeletal muscle mitochondria and by immunoprecipitation of radio-labeled HeLa cell mitochondrial translation products. Only antibodies against two of six selected peptides of the ND4 subunit, i.e., the C-terminal peptide and an internal peptide close to the C-terminus, reacted in both assays with the subunit. Antibodies raised against an internal peptide close to the N-terminus of the ND5 subunit and antibodies raised against an internal epitope of the ND6 subunit also reacted in both the immunoblotting and immunoprecipitation assays. The antibodies described above and other Complex I subunit- or holoenzyme-specific antibodies were used to investigate the subunit deficiencies of the respiratory NADH dehydrogenase in the skeletal muscle of patients affected by mitochondrial myopathies associated with Complex I defects. The reduction in enzyme activity correlated in an immunoblot assay with a decrease of four mtDNA-encoded subunits of the enzyme, as well as with a decrease of other subunits of Complex I encoded in the nDNA. The present work provides the first evidence of a decrease in NADH dehydrogenase subunits encoded in the mitochondrial genome in myopathy patients

Organophosphate hydrolase is a lipoprotein and interacts with pi-specific transport system to facilitate growth of <i>brevundimonas diminuta</i> using op insecticide as source of phosphate

Organophosphate Hydrolase (OPH), encoded by the organophosphate degradation (opd) island, hydrolyzes the triester bond found in a variety of organophosphate insecticides and nerve agents. OPH is targeted to the inner membrane of Brevundimonas diminuta in a pre-folded conformation by the Twin arginine transport (Tat) pathway. The OPH signal peptide contains an invariant cysteine residue at the junction of the signal peptidase (Spase) cleavage site along with a well conserved lipobox motif. Treatment of cells producing native OPH with the signal peptidase II inhibitor globomycin resulted in accumulation of most of the pre-OPH in the cytoplasm with negligible processed OPH detected in the membrane. Substitution of the conserved lipobox cysteine to serine resulted in release of OPH into the periplasm, confirming that OPH is a lipoprotein. Analysis of purified OPH revealed that it was modified with the fatty acids palmitate and stearate. Membrane-bound OPH was shown to interact with the outer membrane efflux protein TolC and with PstS, the periplasmic component of the ABC transporter complex (PstSACB) involved in phosphate transport. Interaction of OPH with PstS appears to facilitate transport of Pigenerated from organophosphates due to the combined action of OPH and periplasmically located phosphatases. Consistent with this model, opd null mutants of B. diminuta failed to grow using the organophosphate insecticide methyl parathion as sole source of phosphate

Membrane Topology of the Lactococcal Bacteriocin ATP-binding Cassette Transporter Protein LcnC. Involvement of LcnC in Lactococcin A Maturation

Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors with N-terminal leader peptides different from those present in preproteins exported by the general sec-dependent (type II) secretion pathway. These bacteriocins utilize a dedicated (type I) secretion system for externalization. The secretion apparatus for the lactococcins A, B, and M/N (LcnA, B, and M/N) from Lactococcus lactis is composed of the two membrane proteins LcnC and LcnD. LcnC belongs to the ATP-binding cassette transporters, whereas LcnD is a protein with similarities to other accessory proteins of type I secretion systems. This paper shows that the N-terminal part of LcnC is involved in the processing of the precursor of LcnA. By making translational fusions of LcnC to the reporter proteins β-galactosidase (LacZ) and alkaline phosphatase (PhoA*), it was shown that both the N- and C-terminal parts of LcnC are located in the cytoplasm. As the N terminus of LcnC is required for LcnA maturation and is localized in the cytoplasm, we conclude that the processing of the bacteriocin LcnA to its mature form takes place at the cytosolic side of the cytoplasmic membrane.

A novel intermembrane space–targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding

A nine-residue intermembrane-targeting signal brings the active Cys of substrate proteins into contact with Mia40 oxidase for folding and import into mitochondria