[Abstract unavailable

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F 1 heads was constant. The angle between monomers in the dimer was 70°or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantumdot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.cryoelectron tomography | subtomogram averaging | membrane curvature | membrane potential | mitochondrial ultrastructure M itochondria, the powerhouses of eukaryotic cells, generate ATP, the universal energy carrier in all life forms. The F 1 F o ATP synthase uses the energy stored in the electrochemical proton gradient across the inner mitochondrial membrane to produce ATP from ADP and phosphate. The proton gradient is established by the respiratory chain complexes I, III, and IV, which pump protons out of the mitochondrial matrix into the cristae space while transferring electrons from the electron donors NADH, FADH, or succinate (via complex II) to the final electron acceptor O 2 . The F 1 F o ATP synthase and complex I (NADH dehydrogenase) are the largest membrane protein complexes in mitochondria, composed of more than 20 or 40 individual protein subunits, respectively (1, 2). The 600-kDa ATP synthase consists of the F o part in the membrane that works like a proton-driven turbine, and the catalytic F 1 part on the matrix side. The two parts are held together by a static peripheral stalk and a rotating central stalk that transmits the torque from the rotor unit in the membrane to the catalytic F 1 head (3, 4). Complex I is an L-shaped molecule of approximately 1 MDa. Its membrane arm has three or four proton-pumping modules, while the matrix arm catalyzes electron transfer from NADH to the hydrophobic electron acceptor ubiquinol (5). The structures of both complexes have been determined by X-ray crystallography, either partially in the case of the F 1 F o ATP synthase (6), or at low resolution in the case of mitochondrial complex I (7, 8), but their relative organization in the mitochondrial inner membrane is largely unknown.The two large complexes occur at an approximate ratio of one molecule of complex I per 3.5 ATP...

The caseinolytic peptidase P (CLPP) is conserved from bacteria to humans. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder together with the chaperone CLPX. In spite of a known relevance for the mitochondrial unfolded protein response, its substrates and tissue-specific roles are unclear in mammals. Recessive CLPP mutations were recently observed in the human Perrault variant of ovarian failure and sensorineural hearing loss. Here, a first characterization of CLPP null mice demonstrated complete female and male infertility and auditory deficits. Disrupted spermatogenesis already at the spermatid stage and ovarian follicular differentiation failure were evident. Reduced pre-/post-natal survival and marked ubiquitous growth retardation contrasted with only light impairment of movement and respiratory activities. Interestingly, the mice showed resistance to ulcerative dermatitis. Systematic expression studies detected up-regulation of other mitochondrial chaperones, accumulation of CLPX and mtDNA as well as inflammatory factors throughout tissues. T-lymphocytes in the spleen were activated. Thus, murine Clpp deletion represents a faithful Perrault model. The disease mechanism probably involves deficient clearance of mitochondrial components and inflammatory tissue destruction.

Ageing of biological systems is accompanied by alterations in mitochondrial morphology, including a transformation from networks and filaments to punctuate units. The significance of these alterations with regard to ageing is not known. Here, we demonstrate that the dynamin-related protein 1 (Dnm1p), a mitochondrial fission protein conserved from yeast to humans, affects ageing in the two model systems we studied, Podospora anserina and Saccharomyces cerevisiae. Deletion of the Dnm1 gene delays the transformation of filamentous to punctuate mitochondria and retards ageing without impairing fitness and fertility typically observed in long-lived mutants. Our data further suggest that reduced mitochondrial fission extends life span by increasing cellular resistance to the induction of apoptosis and links mitochondrial dynamics, apoptosis and life-span control.

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Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian "respirasome" model (Schä gger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I 2 and I 2 III 2 ), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase-or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.

psbA in Synechocystis 6803 was found to belong to a small multigene family with three copies. The psbA gene family was inactivated in vitro by insertation of bacterial drug resistance markers. Inactivation of all three genes resulted in a transformant that is unable to grow photosynthetically but can be cultured photoheterotrophically. This mutant lacks oxygen evolving capacity but retains photosystem I activity. Room temperature measurements of chlorophyll a fluorescence induction demonstrated that the transformant exhibits a high fluorescence yield with little or no variable fluorescence. Immunoblot analyses showed complete loss of the psbA gene product (the DI polypeptide) from thylakoid membranes in the transformant. However, the extrinsic 33 kilodalton polypeptide of the water-splitting complex of photosystem II, is still present. The results indicate that assembly of a partial photosystem II complex may occur even in the absence of the intrinsic D1 polypeptide, a protein implicated as a crucial component of the photosystem II reaction center.