- Email: [email protected]
Genetically-encoded ATP biosensor for low temperatures
S156
Abstracts
to activated carboxyl groups of QDs surface [3], as well as by specific interaction between ZnS and ZnS-specific protein tags [4]. Hybrid structures were characterised by several methods, including chromatography (for overall size and stability), spectrophotometry and fluorymetry (for luminescent properties and the enzyme activity) as well as microscopy (for surface topography, size and stability). Conjugation did not significantly change fluorescent properties of QDs. What is of high importance, FNR activity was preserved. Determined parameters of enzyme kinetics (Km, kcat) indicate that an active site is not altered, although a substrate binding may be partially hampered. Our novel nanohybrids may serve in studies of in vivo/in vitro localization of FNR or its interaction with other proteins, as well as in examination of electron flow between QD and redox-active proteins. Acknowledgement Experiments were partially performed in NanoFun laboratories, POIG.02.02.00-00-025/09. Financial support was received from the National Science Centre (grant no. N202 130039) and the Foundation for Polish Science, Homing PLUS Programme, co-financed by EU under POIG. References [1] J.P. Benz, M. Lintala, J. Soll, M. Mulo, B. Bölter, Trends Plant Sci. 15 (2010) 608–613. [2] U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, T. Nann, Nat. Methods 5 (2008) 763–775. [3] Y. Xing, Q. Chaudry, C. Shen, K.Y. Kong, H.E. Zhau, L.W. Chung, J.A. Petros, R.M. O'Regan, M.V. Yezhelyev, Y.V. Simons, M.D. Wang, S. Nie, Nat. Protoc. 2 (2007) 1152–1165. [4] U.O.S. Seker, H.V. Demir, Molecules 16 (2011) 1426–1451. doi:10.1016/j.bbabio.2012.06.408
amine to ammonium, thus functionally replacing NrfA [6]. We produced the εHAO from Campylobacter consisus recombinantly in E. coli with the aid of the pEC86 plasmid [7], and then solved the atomic structure at a resolution of 2.2 Å by means of single crystal X-ray diffraction. The structure reveals some unique features hitherto not observed in other OCCs. In particular the formation of the active site and the coordination of heme group 7 with methionine as distal ligand differ from all previously characterized OCCs. In addition we could identify nitrite and hydroxyl-amine as substrates for reduction, yet with activities considerably lower as the aforementioned nitrite reductases[1,3]. References [1] O. Einsle, et al., Nature 400 (6743) (1999) 476–480. [2] N. Igarashi, et al., Nat. Struct. Mol. Biol. 4 (4) (1997) 276–284. [3] T.V. Tikhonova, et al., BBA 1764 (4) (2006) 715–723. [4] P. Lukat, et al., Biochemistry 47 (7) (2008) 2080–2086. [5] M.G. Klotz, et al., Environ. Microbiol. 10 (11) (2008) 3150–3163. [6] M. Kern, J. Simon, BBA - Bioenergetics 1787 (6) (2009) 646–656. [7] E. Arslan, et al., Biochem. Biophys. Res. Commun. 251 (3) (1998) 744–747. doi:10.1016/j.bbabio.2012.06.409
21P6 Genetically-encoded ATP biosensor for low temperatures H. Imamura, T. Tsuyama, J. Kishikawa, H. Noji, A. Kakizuka, K. Yokoyama, T. Uemura Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan Department of Applied Chemistry, The University of Tokyo, Tokyo 113-8656, Japan E-mail: [email protected]
21P5 Structure of a novel octaheme cytochrome c from Campylobacter concisus Bianca Hermann1, Florian Kemper1, Michael Braun1, Simon Netzera1, Melanie Dietrich1, Melanie Kern2, Jörg Simon2, Daniel Wohlwend1, Oliver Einsle1 1 Lehrstuhl für Biochemie, Institut für organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany 2 Technische Universität Darmstadt, Institut für Biologie, 64287 Darmstadt, Germany E-mail: [email protected] The family of multiheme cytochromes c (MCC) comprises diverse electron carriers and redox enzymes playing key roles in several metabolic pathways. The few structurally characterized MCCs all contain conserved heme-packing motifs, albeit their primary structures are largely unrelated [1,2,3]. As prototype MCC enzyme serves NrfA [1], a pentaheme cytochrome c nitrite reductase with a wide substrate range [4]. Some other known MCCs belong to the subfamily of octaheme cytochromes c (OCC) [5], such as octaheme cytochrome c nitrite reductase (ONR) [3] or hydroxylamine oxidoreductase (HAO) [2]. Notably, the latter is the only OCC known to date to function as an oxidase. Remarkably, some ε-proteobacteria, including various Campylobacter species, are described as nitrite ammonifiers but lacking an NrfA homologue. Instead, these organisms all harbor an uncharacterized class of OCCs termed εHAOs, derived from their originally postulated function as hydroxylamine oxidoreductases. These εHAOs have been hypothesized to be the sought after candidates for catalyzing the full reduction of nitrite via hydroxyl-
We have previously reported genetically-encoded FRET biosensors for ATP, named ATeam [1,2]. ATeam is a powerful tool to monitor ATP levels inside living mammalian cells with high temporal and spatial resolutions. One of the major drawbacks of the original biosensors is that their affinity to ATP is very sensitive to temperature changes. Although dissociation constant (Kd) of the original biosensor, AT1.03, at 37 °C is 3.3 mM, Kd at 24 °C is less than 0.6 mM, far below physiological ATP concentrations. This means at low temperatures (around 24 °C) the FRET signal of AT1.03 biosensor must be saturated with the physiological concentrations of ATP, making it difficult to detect a slight change of ATP levels. Because body temperatures of many model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, are the same with ambient temperatures (20–25 °C), it does not seem that the use of AT1.03 biosensor is suitable for these organisms or cells from them. In this study, we constructed mutants of AT1.03 by substituting amino acid residues at the ATP binding domain, the e subunit of FoF1ATPase. We found that one of the mutants (AT1.03NL) showed much lower affinity to ATP than the original AT1.03; Kd was 2.1 mM at 24 °C and 1.4 mM at 20 °C. To examine if AT1.03NL is actually effective in ATP imaging at low temperatures, we expressed AT1.03NL and AT1.03 in S2 cell, which originated from D. melanogaster and was cultured at 25 °C. If the FRET signal is saturated, it will not respond quickly to a metabolic challenge. When AT1.03-expressing cells were treated with 2-deoxyglucose and oligomycin A, there was a lag before the FRET signal started to decrease. This suggests that the FRET signal of AT1.03 is mostly saturated in S2 cells. On the other hand, the FRET signal of AT1.03NL-expressing cells started to decrease immediately after addition of inhibitors. Thus, the FRET signal of AT1.03NL is not
Abstracts
S157
saturated. Collectively, new AT1.03NL biosensor is more suitable for ATP imaging at low temperatures, and it may be useful for in vivo ATP imaging in model organisms with low body temperatures.
[2] L. Schaefer, A. Ballabio, H.Y. Zoghbi, Genomics 34 (1996) 166–172. [3] Q.P. Schwarz, T.C. Cox, Genomics 79 (2002) 51–57.
[1] Imamura H, Huynh Nhat KP, Togawa H, Saito K, Iino R, Kato-Yamada Y, Nagai T, Noji H (2009) Proc. Natl. Acad. Sci. U. S. A. 106: 15651-15656. [2] Nakano M, Imamura H, Nagai T, Noji H (2011) ACS Chem. Biol. 6: 709-715.
doi:10.1016/j.bbabio.2012.06.411
doi:10.1016/j.bbabio.2012.06.410
21P7 Severely impaired respiratory chain causes multisystem apoptosis-driven developmental defects, a new mitochondrial phenotype in vertebrates A. Indrieri, V. Van Rahden, V. Tiranti, I. Conte, J. Quartararo, M. Morleo, D. Iaconis, R. Tammaro, G. Chesi, M. Cermola, R. Tatè, I. Maystadt, S. Demuth, A. Zvulunov, I. D'Amato, P. Goffrini, I. Ferrero, P. Bovolenta, K. Kutsche, M. Zeviani, B. Franco Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany Unit of Molecular Neurogenetics, The Foundation “Carlo Besta” Institute of Neurology, Milan, Italy Departments of Genetics, Biology of Microorganisms, Anthropology and Evolution, University of Parma, Italy Integrated Microscopy Facility, Institute of Genetics and Biophysics “Adriano Buzzati Traverso”, CNR, Naples, Italy Centre de Genetique, France E-mail: [email protected] Intrinsic (mitochondrial) programmed cell death (PCD), plays an essential homoeostatic role, by selecting bioenergetically proficient cells suitable for normal tissue and organ development. In spite of intensive investigation on cellular and animal models, the impact of this crucial execution pathway in human disease remains mechanistically undefined. In particular, the link between apoptosis and mitochondrial disorders, i.e. primary defects of oxidative phosphorylation (OXPHOS), has not been persuasively demonstrated. On the other hand, a clearly developmental phenotype, Microphthalmia with Linear Skin lesions syndrome (MLS), is associated with mutations in HCCS [1], the gene encoding the holo-cytochrome c-type synthase, which incorporates catalytically active heme-c moieties in the mitochondrial respiratory chain (MRC) [2,3]. Notably, HCCS mutations are present in a subset of MLS patients, the remaining ones being still undefined at the molecular level. To gain mechanistic insight on the molecular pathogenesis of the developmental defect in MLS, we first showed that, similar to MLS patients, HCCS knockdown results in eye and brain abnormalities in medaka fish (Oryzias latipes). Next, we demonstrated that these defects are caused by increased PCD via apoptosome independent caspase-9 activation, triggered by MRC impairment and overproduction of reactive oxygen species (ROS). Based on these results, we then screened MRC-related genes in HCCSnegative MLS patients, and found deleterious mutations in a new gene related to MRC. These data indicate an essential role for the mitochondrial respiratory chain in organogenesis and define a new group of mitochondrial diseases hallmarked by apoptosis-driven abnormal development. References [1] I. Wimplinger, M. Morleo, G. Rosenberger, D. Iaconis, U. Orth, et al., Am. J. Hum. Genet. 79 (2006) 878–889.
21P8 The influence of STAT proteins on Dictyostelium discoideum bioenergetics A. Kicinska, W. Jarmuszkiewicz Adam Mickiewicz University, Department of Bioenergetics, Umultowska 89, PL-61-614, Poznan, Poland E-mail: [email protected] The amoeboid protozoan Dictyostelium discoideum is a powerful model system for studying cytokinesis, cell motility, phagocytosis, chemotaxis, signal transduction, and cell differentiation during development. Most of its life D. discoideum amoebae undergo the vegetative life cycle as separate, independent cells but, when starved, the cells interact to form multicellular structures. D. discoideum, is one of the simplest organisms using STAT (signal transducers and activators of transcription) mediated phosphotyrosineregulated signaling. STAT proteins are one of the important mediators of in metazoan cells. These proteins are components of signal transduction pathways regulating cellular differentiation, proliferation, immune response, cell fate, cell migration and programmed cell death in multicellular organisms. Additionally, some of the members of STAT protein family, namely mammalian STAT3 and STAT5, were found to be targeted to mitochondria [1, 2]. It was shown that, in addition to their nuclear transcriptional role, they regulate the metabolic function of mitochondria. So far, this dual function of STAT3 was found only in mammalian cells. The present work describes a role of STAT proteins in mitochondrial bioenergetic regulation of D. discoideum. We study bioenergetic properties of these organelles, namely, mitochondrial respiration with different oxidizable substrates, mitochondrial membrane potential and oxidative phosphorylation yield in wild type and STAT knockout D. discoideum cells. The role of STAT protein homologs (DdSTATB and Dd-STATC) in regulation of mitochondrial bioenergetics has been investigated. On the contrary to previous findings on mammalian cells, we could not detect the difference between the activities of electron transport chain Complexes I and II in the Dd-STATB protein knockout and wild type mitochondria. However the activity of Complex IV was significantly reduced in the absence of Dd-STATB protein. This work is founded by the Foundation for Polish Science, Pomost/2010-1/3 grant. References [1] J. Wegrzyn, Science 323 (2009) 793–797. [2] F.Y. Chueh, K.F. Leong, C.L. Yu, Biochem. Biophys. Res. Commun. 402 (2010) 778–783. doi:10.1016/j.bbabio.2012.06.412
21P9 Expression of the gene cluster for chlorate metabolism in the chlorate-respiring bacterium Ideonella dechloratans M. Hellberg Lindqvist, T. Nilsson, M. Rova Karlstad University, Department of Chemistry and Biomedical Sciences, SE 651 88 Karlstad, Sweden E-mail: [email protected]
Abstracts
to activated carboxyl groups of QDs surface [3], as well as by specific interaction between ZnS and ZnS-specific protein tags [4]. Hybrid structures were characterised by several methods, including chromatography (for overall size and stability), spectrophotometry and fluorymetry (for luminescent properties and the enzyme activity) as well as microscopy (for surface topography, size and stability). Conjugation did not significantly change fluorescent properties of QDs. What is of high importance, FNR activity was preserved. Determined parameters of enzyme kinetics (Km, kcat) indicate that an active site is not altered, although a substrate binding may be partially hampered. Our novel nanohybrids may serve in studies of in vivo/in vitro localization of FNR or its interaction with other proteins, as well as in examination of electron flow between QD and redox-active proteins. Acknowledgement Experiments were partially performed in NanoFun laboratories, POIG.02.02.00-00-025/09. Financial support was received from the National Science Centre (grant no. N202 130039) and the Foundation for Polish Science, Homing PLUS Programme, co-financed by EU under POIG. References [1] J.P. Benz, M. Lintala, J. Soll, M. Mulo, B. Bölter, Trends Plant Sci. 15 (2010) 608–613. [2] U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, T. Nann, Nat. Methods 5 (2008) 763–775. [3] Y. Xing, Q. Chaudry, C. Shen, K.Y. Kong, H.E. Zhau, L.W. Chung, J.A. Petros, R.M. O'Regan, M.V. Yezhelyev, Y.V. Simons, M.D. Wang, S. Nie, Nat. Protoc. 2 (2007) 1152–1165. [4] U.O.S. Seker, H.V. Demir, Molecules 16 (2011) 1426–1451. doi:10.1016/j.bbabio.2012.06.408
amine to ammonium, thus functionally replacing NrfA [6]. We produced the εHAO from Campylobacter consisus recombinantly in E. coli with the aid of the pEC86 plasmid [7], and then solved the atomic structure at a resolution of 2.2 Å by means of single crystal X-ray diffraction. The structure reveals some unique features hitherto not observed in other OCCs. In particular the formation of the active site and the coordination of heme group 7 with methionine as distal ligand differ from all previously characterized OCCs. In addition we could identify nitrite and hydroxyl-amine as substrates for reduction, yet with activities considerably lower as the aforementioned nitrite reductases[1,3]. References [1] O. Einsle, et al., Nature 400 (6743) (1999) 476–480. [2] N. Igarashi, et al., Nat. Struct. Mol. Biol. 4 (4) (1997) 276–284. [3] T.V. Tikhonova, et al., BBA 1764 (4) (2006) 715–723. [4] P. Lukat, et al., Biochemistry 47 (7) (2008) 2080–2086. [5] M.G. Klotz, et al., Environ. Microbiol. 10 (11) (2008) 3150–3163. [6] M. Kern, J. Simon, BBA - Bioenergetics 1787 (6) (2009) 646–656. [7] E. Arslan, et al., Biochem. Biophys. Res. Commun. 251 (3) (1998) 744–747. doi:10.1016/j.bbabio.2012.06.409
21P6 Genetically-encoded ATP biosensor for low temperatures H. Imamura, T. Tsuyama, J. Kishikawa, H. Noji, A. Kakizuka, K. Yokoyama, T. Uemura Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan Department of Applied Chemistry, The University of Tokyo, Tokyo 113-8656, Japan E-mail: [email protected]
21P5 Structure of a novel octaheme cytochrome c from Campylobacter concisus Bianca Hermann1, Florian Kemper1, Michael Braun1, Simon Netzera1, Melanie Dietrich1, Melanie Kern2, Jörg Simon2, Daniel Wohlwend1, Oliver Einsle1 1 Lehrstuhl für Biochemie, Institut für organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany 2 Technische Universität Darmstadt, Institut für Biologie, 64287 Darmstadt, Germany E-mail: [email protected] The family of multiheme cytochromes c (MCC) comprises diverse electron carriers and redox enzymes playing key roles in several metabolic pathways. The few structurally characterized MCCs all contain conserved heme-packing motifs, albeit their primary structures are largely unrelated [1,2,3]. As prototype MCC enzyme serves NrfA [1], a pentaheme cytochrome c nitrite reductase with a wide substrate range [4]. Some other known MCCs belong to the subfamily of octaheme cytochromes c (OCC) [5], such as octaheme cytochrome c nitrite reductase (ONR) [3] or hydroxylamine oxidoreductase (HAO) [2]. Notably, the latter is the only OCC known to date to function as an oxidase. Remarkably, some ε-proteobacteria, including various Campylobacter species, are described as nitrite ammonifiers but lacking an NrfA homologue. Instead, these organisms all harbor an uncharacterized class of OCCs termed εHAOs, derived from their originally postulated function as hydroxylamine oxidoreductases. These εHAOs have been hypothesized to be the sought after candidates for catalyzing the full reduction of nitrite via hydroxyl-
We have previously reported genetically-encoded FRET biosensors for ATP, named ATeam [1,2]. ATeam is a powerful tool to monitor ATP levels inside living mammalian cells with high temporal and spatial resolutions. One of the major drawbacks of the original biosensors is that their affinity to ATP is very sensitive to temperature changes. Although dissociation constant (Kd) of the original biosensor, AT1.03, at 37 °C is 3.3 mM, Kd at 24 °C is less than 0.6 mM, far below physiological ATP concentrations. This means at low temperatures (around 24 °C) the FRET signal of AT1.03 biosensor must be saturated with the physiological concentrations of ATP, making it difficult to detect a slight change of ATP levels. Because body temperatures of many model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, are the same with ambient temperatures (20–25 °C), it does not seem that the use of AT1.03 biosensor is suitable for these organisms or cells from them. In this study, we constructed mutants of AT1.03 by substituting amino acid residues at the ATP binding domain, the e subunit of FoF1ATPase. We found that one of the mutants (AT1.03NL) showed much lower affinity to ATP than the original AT1.03; Kd was 2.1 mM at 24 °C and 1.4 mM at 20 °C. To examine if AT1.03NL is actually effective in ATP imaging at low temperatures, we expressed AT1.03NL and AT1.03 in S2 cell, which originated from D. melanogaster and was cultured at 25 °C. If the FRET signal is saturated, it will not respond quickly to a metabolic challenge. When AT1.03-expressing cells were treated with 2-deoxyglucose and oligomycin A, there was a lag before the FRET signal started to decrease. This suggests that the FRET signal of AT1.03 is mostly saturated in S2 cells. On the other hand, the FRET signal of AT1.03NL-expressing cells started to decrease immediately after addition of inhibitors. Thus, the FRET signal of AT1.03NL is not
Abstracts
S157
saturated. Collectively, new AT1.03NL biosensor is more suitable for ATP imaging at low temperatures, and it may be useful for in vivo ATP imaging in model organisms with low body temperatures.
[2] L. Schaefer, A. Ballabio, H.Y. Zoghbi, Genomics 34 (1996) 166–172. [3] Q.P. Schwarz, T.C. Cox, Genomics 79 (2002) 51–57.
[1] Imamura H, Huynh Nhat KP, Togawa H, Saito K, Iino R, Kato-Yamada Y, Nagai T, Noji H (2009) Proc. Natl. Acad. Sci. U. S. A. 106: 15651-15656. [2] Nakano M, Imamura H, Nagai T, Noji H (2011) ACS Chem. Biol. 6: 709-715.
doi:10.1016/j.bbabio.2012.06.411
doi:10.1016/j.bbabio.2012.06.410
21P7 Severely impaired respiratory chain causes multisystem apoptosis-driven developmental defects, a new mitochondrial phenotype in vertebrates A. Indrieri, V. Van Rahden, V. Tiranti, I. Conte, J. Quartararo, M. Morleo, D. Iaconis, R. Tammaro, G. Chesi, M. Cermola, R. Tatè, I. Maystadt, S. Demuth, A. Zvulunov, I. D'Amato, P. Goffrini, I. Ferrero, P. Bovolenta, K. Kutsche, M. Zeviani, B. Franco Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany Unit of Molecular Neurogenetics, The Foundation “Carlo Besta” Institute of Neurology, Milan, Italy Departments of Genetics, Biology of Microorganisms, Anthropology and Evolution, University of Parma, Italy Integrated Microscopy Facility, Institute of Genetics and Biophysics “Adriano Buzzati Traverso”, CNR, Naples, Italy Centre de Genetique, France E-mail: [email protected] Intrinsic (mitochondrial) programmed cell death (PCD), plays an essential homoeostatic role, by selecting bioenergetically proficient cells suitable for normal tissue and organ development. In spite of intensive investigation on cellular and animal models, the impact of this crucial execution pathway in human disease remains mechanistically undefined. In particular, the link between apoptosis and mitochondrial disorders, i.e. primary defects of oxidative phosphorylation (OXPHOS), has not been persuasively demonstrated. On the other hand, a clearly developmental phenotype, Microphthalmia with Linear Skin lesions syndrome (MLS), is associated with mutations in HCCS [1], the gene encoding the holo-cytochrome c-type synthase, which incorporates catalytically active heme-c moieties in the mitochondrial respiratory chain (MRC) [2,3]. Notably, HCCS mutations are present in a subset of MLS patients, the remaining ones being still undefined at the molecular level. To gain mechanistic insight on the molecular pathogenesis of the developmental defect in MLS, we first showed that, similar to MLS patients, HCCS knockdown results in eye and brain abnormalities in medaka fish (Oryzias latipes). Next, we demonstrated that these defects are caused by increased PCD via apoptosome independent caspase-9 activation, triggered by MRC impairment and overproduction of reactive oxygen species (ROS). Based on these results, we then screened MRC-related genes in HCCSnegative MLS patients, and found deleterious mutations in a new gene related to MRC. These data indicate an essential role for the mitochondrial respiratory chain in organogenesis and define a new group of mitochondrial diseases hallmarked by apoptosis-driven abnormal development. References [1] I. Wimplinger, M. Morleo, G. Rosenberger, D. Iaconis, U. Orth, et al., Am. J. Hum. Genet. 79 (2006) 878–889.
21P8 The influence of STAT proteins on Dictyostelium discoideum bioenergetics A. Kicinska, W. Jarmuszkiewicz Adam Mickiewicz University, Department of Bioenergetics, Umultowska 89, PL-61-614, Poznan, Poland E-mail: [email protected] The amoeboid protozoan Dictyostelium discoideum is a powerful model system for studying cytokinesis, cell motility, phagocytosis, chemotaxis, signal transduction, and cell differentiation during development. Most of its life D. discoideum amoebae undergo the vegetative life cycle as separate, independent cells but, when starved, the cells interact to form multicellular structures. D. discoideum, is one of the simplest organisms using STAT (signal transducers and activators of transcription) mediated phosphotyrosineregulated signaling. STAT proteins are one of the important mediators of in metazoan cells. These proteins are components of signal transduction pathways regulating cellular differentiation, proliferation, immune response, cell fate, cell migration and programmed cell death in multicellular organisms. Additionally, some of the members of STAT protein family, namely mammalian STAT3 and STAT5, were found to be targeted to mitochondria [1, 2]. It was shown that, in addition to their nuclear transcriptional role, they regulate the metabolic function of mitochondria. So far, this dual function of STAT3 was found only in mammalian cells. The present work describes a role of STAT proteins in mitochondrial bioenergetic regulation of D. discoideum. We study bioenergetic properties of these organelles, namely, mitochondrial respiration with different oxidizable substrates, mitochondrial membrane potential and oxidative phosphorylation yield in wild type and STAT knockout D. discoideum cells. The role of STAT protein homologs (DdSTATB and Dd-STATC) in regulation of mitochondrial bioenergetics has been investigated. On the contrary to previous findings on mammalian cells, we could not detect the difference between the activities of electron transport chain Complexes I and II in the Dd-STATB protein knockout and wild type mitochondria. However the activity of Complex IV was significantly reduced in the absence of Dd-STATB protein. This work is founded by the Foundation for Polish Science, Pomost/2010-1/3 grant. References [1] J. Wegrzyn, Science 323 (2009) 793–797. [2] F.Y. Chueh, K.F. Leong, C.L. Yu, Biochem. Biophys. Res. Commun. 402 (2010) 778–783. doi:10.1016/j.bbabio.2012.06.412
21P9 Expression of the gene cluster for chlorate metabolism in the chlorate-respiring bacterium Ideonella dechloratans M. Hellberg Lindqvist, T. Nilsson, M. Rova Karlstad University, Department of Chemistry and Biomedical Sciences, SE 651 88 Karlstad, Sweden E-mail: [email protected]