Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed r8 cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in r8 cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP 4± for ADP 3± by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete F o F 1 -ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin-permeabilized mammalian r8 cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in r8 cells and an incomplete F o F 1 -ATP synthase was also required. The maximum value of this potential was <110 mV in permeabilized cells and <67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete F o F 1 -ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear-encoded proteins but <65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of r8 mitochondria showed that the azide-sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete F o F 1 -ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete F o F 1 -ATP synthase in maintaining the mitochondrial membrane potential in r8 cells.Keywords: mammalian r8 cells; mitochondrial membrane potential; adenine nucleotide carrier; F o F 1 -ATP synthase; mitochondrial DNA.Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of respiratory proton pumps and the F o F 1 -ATP synthase, which are all essential for oxidative phosphorylation [1±4]. Cells cultured with ethidium bromide lose their mtDNA and consequently these cells (termed r8 cells) cannot carry out oxidative phosphorylation, require pyruvate and uridine for growth and have glycolysis as their only source of ATP [2±4]. Even so, mitochondria are still essential for r8 cells because of vital metabolic pathways catalysed by mitochondrial proteins encoded by nuclear DNA [4,5]. Import of nuclear-encoded proteins into mitochondria requires a membrane potential, but in r8 cells this must arise by a mechanism other t...

White clover (Trifolium repens L.) plants from the cultivars Grasslands Huia and Grasslands Tahora have been transformed using Agrobacterium-mediated T-DNA transfer. Transgenic plants regenerated directly from cells of the cotyledonary axil. To transform white clover, shoot tips from 3 day old seedlings were co-cultivated with A. tumefaciens strain LBA4404 carrying the plasmid vector pPE64. This vector contains the neomycin phosphotransferase II gene (nptII) and β-glucuronidase reporter gene (gus) both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 42 days after transfer onto selective media. Integration of the nptII and gus genes into the white clover genome was confirmed using Southern blotting, and histochemical analysis indicated that the gus gene was expressed in a variety of tissues. In reciprocal crosses between a primary transformant and a non-transformed plant the introduced gus gene segregated as a single dominant Mendelian trait.

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Most secretory proteins, including antithrombin (AT), are synthesized with a signal peptide, which is cleaved before the mature protein is exported from the cell. The signal peptide is important in the process whereby nascent protein is recognized as requiring subsequent modification within the endoplasmic reticulum (ER). We have identified a novel mutation, 2436T→C L(-10)P, which affects the central hydrophobic domain of the AT signal peptide, in a proband presenting with venous thrombotic disease and type I AT deficiency. We investigated the basis of the phenotype by examining expression in mammalian cells of a range of variant AT cDNAs with mutations affecting the –10 residue. Glycosylated AT was secreted from COS-7 cells transfected with wild-type AT, –10L deletion, -10V or -10M variants, but not variants with P, T, R, or G at -10. Cell-free expression of wild-type and variant AT cDNAs was then performed in the presence of canine pancreatic microsomes, as a substitute for ER. Variant AT proteins with P, T, R, or G at residue –10 did not undergo posttranslational glycosylation, and their susceptibility to trypsin digestion suggested they had not been translocated into microsomes. Our results suggest that the ability of AT signal peptide to direct the protein to ER for cotranslational processing events appears to be critically dependent on maintaining the hydrophobic nature of the region including residue –10. The investigations have defined impaired cotranslational processing as a hitherto unrecognized cause of hereditary AT deficiency.

Most secretory proteins, including antithrombin (AT), are synthesized with a signal peptide, which is cleaved before the mature protein is exported from the cell. The signal peptide is important in the process whereby nascent protein is recognized as requiring subsequent modification within the endoplasmic reticulum (ER). We have identified a novel mutation, 2436T→C L(-10)P, which affects the central hydrophobic domain of the AT signal peptide, in a proband presenting with venous thrombotic disease and type I AT deficiency. We investigated the basis of the phenotype by examining expression in mammalian cells of a range of variant AT cDNAs with mutations affecting the –10 residue. Glycosylated AT was secreted from COS-7 cells transfected with wild-type AT, –10L deletion, -10V or -10M variants, but not variants with P, T, R, or G at -10. Cell-free expression of wild-type and variant AT cDNAs was then performed in the presence of canine pancreatic microsomes, as a substitute for ER. Variant AT proteins with P, T, R, or G at residue –10 did not undergo posttranslational glycosylation, and their susceptibility to trypsin digestion suggested they had not been translocated into microsomes. Our results suggest that the ability of AT signal peptide to direct the protein to ER for cotranslational processing events appears to be critically dependent on maintaining the hydrophobic nature of the region including residue –10. The investigations have defined impaired cotranslational processing as a hitherto unrecognized cause of hereditary AT deficiency.

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The SEED (Student Enterprise Experience in Dunedin) programme was developed as a four-week, intensive entrepreneurial ‘boot camp’ to provide a small group of participants with a highly experiential business course. Using pre-course and post-course surveys, the authors measured the entrepreneurial ability, knowledge and intentions of the sixteen SEED participants to determine whether the programme was successful as an educational format. The results showed that perceived entrepreneurial ability and knowledge significantly improved, while intent increased but not significantly. Almost all participants intended to start a business both before and after the course, while twice as many (six) indicated that they would undertake further formal study in entrepreneurship after the course compared to before. The data suggest that SEED is filling a niche in entrepreneurial development and support for budding entrepreneurs, with the course also facilitating local business development.

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