Disruption of CA2 gene causes a dramatic changes in complex I assembly and respiration in Arabidopsis cell cultures

PERALES, M.; EUBEL, H.; HEINEMEYER, H.; COLANERI, A.; EDUARDO JULIAN ZABALETA; BRAUN, H.

JOURNAL OF MOLECULAR BIOLOGY

Elsevier Ltd

Año: 2005 vol. 350 p. 263 - 277

0022-2836

Mitochondrial NADH dehydrogenase (complex I) of plants includes quite a number of plant-specific subunits, some of which exhibit sequence similarity to bacterial g-carbonic anhydrases. A homozygous Arabidopsisg-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 ICIII2 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 ICIII2 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 ICIII2 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 ICIII2 CIII2 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 ICIII2CIII2 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 microarray analyses.