TY - JOUR

T1 - Disruption of a Nuclear Gene Encoding a Mitochondrial Gamma Carbonic Anhydrase Reduces Complex I and Supercomplex I+III2 Levels and Alters Mitochondrial Physiology in Arabidopsis

AU - Perales, Mariano

AU - Eubel, Holger

AU - Heinemeyer, Jesco

AU - Colaneri, Alejandro

AU - Zabaleta, Eduardo

AU - Braun, Hans Peter

N1 - Funding information: Arabidopsis knockout line SALK_010194 was obtained from The Arabidopsis Biological Resource Center, Ohio State University. We thank Tom Elton, Nebraska University, US and Allan Rasmusson, Lund University, Sweden, for providing antibodies directed against AOX and NDA/NDB, Dagmar Lewejohann for expert technical assistance and Dieter Markowsky, Amersham Biosciences, for support concerning the DIGE system. The presented work was supported by the Deutsche Forschungsgemeinschaft (grant BR 1829-7/1), by ANPCyT (grants 05008 and 13432) and by a short term scholarship of the Deutsche Akademische Austauschdienst (DAAD) to M.P.

PY - 2005/7/8

Y1 - 2005/7/8

N2 - Mitochondrial NADH dehydrogenase (complex I) of plants includes quite a number of plant-specific subunits, some of which exhibit sequence similarity to bacterial γ-carbonic anhydrases. A homozygous Arabidopsis knockout mutant carrying a T-DNA insertion in a gene encoding one of these subunits (At1g47260) was generated to investigate its physiological role. Isolation of mitochondria and separation of mitochondrial protein complexes by Blue-native polyacrylamide gel electrophoresis or sucrose gradient ultracentrifugation revealed drastically reduced complex I levels. Furthermore, the mitochondrial I+III2 supercomplex was very much reduced in mutant plants. Remaining complex I had normal molecular mass, suggesting substitution of the At1g47260 protein by one or several of the structurally related subunits of this respiratory protein complex. Immune-blotting experiments using polyclonal antibodies directed against the At1g47260 protein indicated its presence within complex I, the I+III2 supercomplex and smaller protein complexes, which possibly represent subcomplexes of complex I. Changes within the mitochondrial proteome of mutant cells were systematically monitored by fluorescence difference gel electrophoresis using 2D Blue-native/SDS and 2D isoelectric focussing/SDS polyacrylamide gel electrophoresis. Complex I subunits are largely absent within the mitochondrial proteome. Further mitochondrial proteins are reduced in mutant plants, like mitochondrial ferredoxin, others are increased, like formate dehydrogenase. Development of mutant plants was normal under standard growth conditions. However, a suspension cell culture generated from mutant plants exhibited clearly reduced growth rates and respiration. In summary, At1g47260 is important for complex I assembly in plant mitochondria and respiration. A role of At1g47260 in mitochondrial one-carbon metabolism is supported by micro-array analyses.

AB - Mitochondrial NADH dehydrogenase (complex I) of plants includes quite a number of plant-specific subunits, some of which exhibit sequence similarity to bacterial γ-carbonic anhydrases. A homozygous Arabidopsis knockout mutant carrying a T-DNA insertion in a gene encoding one of these subunits (At1g47260) was generated to investigate its physiological role. Isolation of mitochondria and separation of mitochondrial protein complexes by Blue-native polyacrylamide gel electrophoresis or sucrose gradient ultracentrifugation revealed drastically reduced complex I levels. Furthermore, the mitochondrial I+III2 supercomplex was very much reduced in mutant plants. Remaining complex I had normal molecular mass, suggesting substitution of the At1g47260 protein by one or several of the structurally related subunits of this respiratory protein complex. Immune-blotting experiments using polyclonal antibodies directed against the At1g47260 protein indicated its presence within complex I, the I+III2 supercomplex and smaller protein complexes, which possibly represent subcomplexes of complex I. Changes within the mitochondrial proteome of mutant cells were systematically monitored by fluorescence difference gel electrophoresis using 2D Blue-native/SDS and 2D isoelectric focussing/SDS polyacrylamide gel electrophoresis. Complex I subunits are largely absent within the mitochondrial proteome. Further mitochondrial proteins are reduced in mutant plants, like mitochondrial ferredoxin, others are increased, like formate dehydrogenase. Development of mutant plants was normal under standard growth conditions. However, a suspension cell culture generated from mutant plants exhibited clearly reduced growth rates and respiration. In summary, At1g47260 is important for complex I assembly in plant mitochondria and respiration. A role of At1g47260 in mitochondrial one-carbon metabolism is supported by micro-array analyses.

KW - Arabidopsis

KW - Gamma carbonic anhydrase

KW - Mitochondria

KW - NADH dehydrogenase complex (complex I)

KW - Respiratory supercomplex

UR - http://www.scopus.com/inward/record.url?scp=20444398508&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2005.04.062

DO - 10.1016/j.jmb.2005.04.062

M3 - Article

C2 - 15935378

AN - SCOPUS:20444398508

VL - 350

SP - 263

EP - 277

JO - Journal of molecular biology

JF - Journal of molecular biology

SN - 0022-2836

IS - 2

ER -