The mitochondrial biogenesis and adequate energy production are important for fetal growth and early postnatal adaptation. The aim of the study was to characterize mitochondrial DNA (mtDNA) content and expression patterns of POLG, TFAM, NRF1 , NRF2 and PGC1 family of regulated coactivators (PGC1A, PGC1B and PPRC1) involved in the mtDNA transcription, regulation and maintenance in human fetal tissues during second trimester of gestation. Further the mRNA expression profiles of selected cytochrome c oxidase (COX) subunits were analysed. Moreover enzyme activities of COX and citrate synthase (CS) and protein levels of COX subunits were analysed.DNA, RNA and proteins were isolated from 26 pairs of fetal liver and muscle samples obtained at autopsy after termination of pregnancy for genetic indications unrelated to OXPHOS deficiency between the 13th and 28th weeks of gestation. This work offers a broad view on the mtDNA content changes in two different tissues during the second trimester of gestation and in the corresponding tissues after birth. The important differences in expression of POLG, TFAM, NRF2 genes and family PGC1 coactivators were found between the fetal tissues. The significant tissue- specific changes in expression of selected COX subunits on mRNA level (COX4 and MTCO2) were observed. Further the considerable differences in enzyme activities of COX and CS are demonstrated between fetal and postnatal phases. In conclusion our study indicates that the fetal developing tissues might differ in the control of mitochondrial biogenesis depending on their energy demand and the age of gestation. Moreover the gene expression is changed mainly on transcriptional level through fetal period. This work was supported by grants GAUK35810 and MSM0021620806. doi:10.1016/j.bbabio.2010.04.328 13P.6 Oxidative protein folding in the intermembrane space of mitochondria Melanie Bien, Manuel Fischer, Sebastian Longen, Johannes M. Herrmann, Jan Riemer Institute of Cell Biology, Department of Biology, University of Kaiserslautern, Germany E-mail: jan.riemer@biologie.unil.de For a long time, the endoplasmic reticulum (ER) was considered to be the only compartment of the eukaryotic cell in which proteins are folded by dedicated enzymes in an oxidation-driven process. However, it became recently evident that eukaryotic cells harbor another oxidizing compartment, the small lumen between the outer and inner membranes of mitochondria—the intermembrane space (IMS). In mitochondria, protein oxidation can be used to drive protein translocation from the cytosol across the outer membrane. Moreover, redox reactions have been implied in many IMS- connected processes like apoptosis, aging and the regulation of the respiratory chain. Major players of the oxidative pathway in the IMS are the oxidoreductase Mia40 that oxidizes substrates and the sulfhydryl oxidase Erv1 that re-oxidizes Mia40. Erv1 derives its oxidative power from the respiratory chain via its interaction with cytochrome c. To detailedly analyse the mechanism of this oxidative pathway and the interplay of its components we reconstituted the complete process in vitro using purified cytochrome c, Erv1, Mia40 and the substrate Cox19. Hereby, we demonstrate that Erv1 dimerizes non-covalently, and that the subunits of this homodimer cooperate in intersubunit electron exchange. Moreover, we show that Mia40 promotes complete oxidation of the substrate Cox19. The efficient formation of disulfide bonds is hampered by the formation of long-lived, partially oxidized intermediates. The generation of these side products is efficiently counteracted by reduced glutathione. Thus, our findings suggest a role for a glutathione-dependent proof reading during oxidative protein folding by the mitochondrial disulfide relay. doi:10.1016/j.bbabio.2010.04.329 13P.7 The role of SenC in assembly of the cytochrome cbb 3 oxidase in Rhodobacter capsulatus Sebastian Schröder 1 , Grzegorz Pawlik 1 , Annette Peters 1 , Carmen Kulajta 1 , Fevzi Daldal 2 , Hans-Georg Koch 1 1 University of Freiburg, Institute for Biochemistry and Molecular Biology, Germany 2 University of Pennsylvania, Department of Biology, USA E-mail: sebastian.schroeder@biochemie.uni-freiburg.de As a member of the heme copper oxidase superfamily, the cbb 3 -type cytochrome oxidase (cbb 3 -Cox) is composed of four subunits. Based on their high oxygen affinity they are considered to facilitate bacterial growth at low oxygen concentrations. Many pathogenic bacteria like V. cholerae, H. pylori, C. jejuni and N. meningitidis seem to be dependent on the cbb 3 -Cox for colonizing the human host. SenC in Rhodobacter capsulatus (Sco1 in yeast and human) is an assembly protein which contains a copper binding motif and has been implicated so far in the assembly of the Cu A centre of aa 3 -type cytochrome oxidases. Sco1 also contains a thiol-disulfide oxidoreductase activity, which is discussed to keep the Cu-ligating residues in their correct redox-state. Although, the cbb 3 -Cox, which is the only cytochrome-c-oxidase in R. capsulatus, does not contain a Cu A centre, the senC knock-out strain shows a strongly reduced oxygen uptake activity. We could show that this is due to the absence of all four subunits CcoNOQP of the cbb 3 -Cox in the senC knock- out. Because the loss of senC does not cause a major change in the transcriptional level of the cbb 3 -Cox subunit CcoN, as shown by RT-PCR, the absence of the cbb 3 -Cox is due to an instability or a defect in the assembly process. Furthermore the cbb 3 -Cox can be restored by growing cells in the presence of increased Cu concentrations. This effect was copper specific and was not observed in the presence of iron or magnesium ions. To further elucidate the role of SenC in cbb 3 -Cox maturation, we analysed direct interactions of SenC with other proteins. On Blue Native PAGE SenC is running in complexes of 230 kDa, 70 kDa and 40 kDa. Interestingly, the cbb 3 -Cox also runs as a 230 kDa complex, which could indicate an interaction between SenC and the cbb 3 -Cox. To further show a contact to an individual subunit of the cbb 3 -Cox we performed copurification- and formaldehyde-crosslinking-experi- ments. While we could not detect interactions between SenC and the cbb 3 -Cox main subunit CcoN, which contains a heme b and a heme b 3 -CuB-center, we could copurify SenC with the subunit CcoP which is a c-type cytochrome and could also crosslink these two proteins with formaldehyde. These data indicate that SenC is a general assembly factor for cytochrome-oxidases and not only for cytochrome-oxidases which contain a Cu A centre. References [1] Swem D.L. et al (2005) J. Bacteriol. 187: 8081–8087. [2] Kulajta C. et al (2006) J. Mol. Biol. 355: 989–1004. [3] Peters A. et al (2008) J. Bacteriol. 190: 5576–5586. doi:10.1016/j.bbabio.2010.04.330 13P.8 Mitochondrial biogenesis in human osteosarcoma cells with chronic mitochondrial stress Marta Wojewoda, Jerzy Duszynski, Joanna Szczepanowska Nencki Institute of Experimental Biology, Polish Academy of Science, Warsaw, Poland E-mail: j.szczepanowska@nencki.gov.pl 109 Abstracts brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Elsevier - Publisher Connector