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...

Alternative (telomerase-independent) lengthening of telomeres mediated through homologous recombination is often accompanied by a generation of extrachromosomal telomeric circles (t-circles), whose role in direct promotion of recombinational telomere elongation has been recently demonstrated. Here we present evidence that t-circles in a natural telomerase-deficient system of mitochondria of the yeast Candida parapsilosis replicate independently of the linear chromosome via a rollingcircle mechanism. This is supported by an observation of (i) single-stranded DNA consisting of concatameric arrays of telomeric sequence, (ii) lasso-shaped molecules representing rolling-circle intermediates, and (iii) preferential incorporation of deoxyribonucleotides into telomeric fragments and t-circles. Analysis of naturally occurring variant t-circles revealed conserved motifs with potential function in driving the rolling-circle replication. These data indicate that extrachromosomal t-circles observed in a wide variety of organisms, including yeasts, plants, Xenopus laevis, and certain human cell lines, may represent independent replicons generating telomeric sequences and, thus, actively participating in telomere dynamics. Moreover, because of the promiscuous occurrence of t-circles across phyla, the results from yeast mitochondria have implications related to the primordial system of telomere maintenance, providing a paradigm for evolution of telomeres in nuclei of early eukaryotes.

The yeast species Candida parapsilosis, an opportunistic pathogen, exhibits genetic and genomic heterogeneity. To assess the polymorphism at the level of mitochondrial DNA (mtDNA), the organization of the mitochondrial genome in strains belonging to the three variant groups of this species was investigated. Although these analyses revealed a group-specific restriction fragment pattern of mtDNA, strains belonging to different groups appear to have similar genes in the same gene order. An extensive survey of C. parapsilosis isolates uncovered surprising alterations in the molecular architecture of their mitochondrial genome. A screening strategy for strains harbouring mtDNA with rearranged architecture showed that nearly all strains from groups I and III possess linear mtDNA molecules terminating with arrays of tandem repeat units, while most of the group II strains have a circular mitochondrial genome. In addition, it was found that linear genophores in mitochondria of strains from different groups differ in the sequence of the mitochondrial telomeric repeat unit. The occurrence of altered forms of mtDNA among C. parapsilosis strains opens up the unique possibility to address questions concerning the evolutionary origin and replication strategy of linear and circular genomes in mitochondria.

In anaerobically grown bacteria, transport of nitrite is catalyzed by an integral membrane protein of the form ate-nitrite transporter family, NirC, which in Salmonella typhimurium plays a critical role in intracellular virulence. We present a functional characterization of the S. typhimurium nitrite transporter StmNirC in native membrane vesicles as well as purified and reconstituted into proteoliposomes. Using an electrophysiological technique based on solid supported membranes, we show nitrite induced translocation of negative charges into proteoliposomes reconstituted with purified StmNirC. These data demonstrate the electrogenicity of StmNirC and its substrate specificity for nitrite. Monitoring changes in ΔpH on everted membrane vesicles containing overexpressed StmNirC using acridine orange as a pH indicator we demonstrate that StmNirC acts as a secondary active transporter. It promotes low affinity transport of nitrite coupled to H(+) antiport with a pH independent profile in the pH range from 6 to 8. In addition to nitrite also nitrate is transported by StmNirC, but with reduced flux and complete absence of proton antiport activity. Taken together, these data suggest a bispecific anion selectivity of StmNirC with an ion specific transport mode. This may play a role in regulating nitrite transport under physiological conditions.