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.
ABSTRACT Mitochondrial NADH dehydrogenase (complex I) of plants includes quite a number of plant-specific subunits, some of which exhibit sequence similarity to bacterial gamma-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.
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ABSTRACT: Posttranslational modifications are essential regulators of protein functions as they can modify enzyme activities or protein-molecule interactions by changing the charge state or chemical properties of their target amino acid. The acetyl moiety of the central energy metabolite acetyl-CoA can be transferred to the ε-amino group of lysine, a process known as lysine acetylation which is implicated in the regulation of key metabolic enzymes in various organisms. Since plant mitochondria are of great importance for plant growth and development and as they house key enzymes of oxidative phosphorylation and photorespiration, it is essential to investigate the occurrence of lysine acetylation in this organelle. Here we characterised the plant mitochondrial acetylome of Arabidopsis mitochondria by LC-MS/MS analysis. In total 120 lysine-acetylated mitochondrial proteins containing 243 acetylated sites were identified. These proteins were mapped into functional categories showing that many proteins with essential functions from the tricaboxylic cycle and the respiratory chain are lysine-acetylated, as well as proteins involved in photorespiration, amino acid and protein metabolism, and redox regulation. Immuno-detection of mitochondrial proteins revealed that many lysine-acetylated proteins reside in native protein complexes. Furthermore, in vitro experiments demonstrated that lysine acetylation can occur non-enzymatically in Arabidopsis mitochondria at physiological matrix pH.Mitochondrion 04/2014; · 3.52 Impact Factor
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ABSTRACT: The mitochondrial NADH dehydrogenase complex (complex I) of the respiratory chain has several remarkable features in plants: (i) particularly many of its subunits are encoded by the mitochondrial genome, (ii) its mitochondrial transcripts undergo extensive maturation processes (e.g. RNA editing, trans-splicing), (iii) its assembly follows unique routes, (iv) it includes an additional functional domain which contains carbonic anhydrases and (v) it is, indirectly, involved in photosynthesis. Comprising about 50 distinct protein subunits, complex I of plants is very large. However, an even larger number of proteins are required to synthesize these subunits and assemble the enzyme complex. This review aims to follow the complete “life cycle” of plant complex I from various molecular perspectives. We provide arguments that complex I represents an ideal model system for studying the interplay of respiration and photosynthesis, the cooperation of mitochondria and the nucleus during organelle biogenesis and the evolution of the mitochondrial oxidative phosphorylation system.Mitochondrion 11/2014; · 3.52 Impact Factor
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ABSTRACT: The supramolecular organisation of the four respiratory chain complexes as well as the FOF1-ATP synthase (complex V), comprising together the oxidative phosphorylation (OXPHOS) system, has been a matter of debate since the beginning of their biochemical investigation some decades ago. The still generally accepted view found in the textbooks is the “random collision model” regarding the five oxidative phosphorylation complexes as independent entities in the mitochondrial inner membrane. Meanwhile, the experimental evidences favouring the existence of specific respiratory supercomplexes of complexes I, III and IV as well as of ATP synthase oligomers in most eukaryotic mitochondria provide a strong collection of arguments which is not less valid than that supporting the existence of individual respiratory complexes in vivo. Of particular note is the recent determination of single particle structures of OXPHOS supercomplexes from mammals, plants, algae and yeast. Although the number of studies supporting the existence of respiratory supercomplexes has been increasing at an accelerating pace, the functional significance of respiratory supercomplexes like conceivable substrate channeling is poorly understood. Besides enzymatic advantages, an emerging crucial role appears to be the assembly/stabilisation of individual complexes. In particular, the biogenesis and/or stabilisation of complex I, the largest and most complicated respiratory complex, seem to rely on the presence of other proteins (in particular complex III and/or IV), which may integrate complex I into supramolecular structures in the inner membrane. Herein a concise but broad overview about the recent advances in the research on respiratory supercomplexes and ATP synthase oligomers is presented, attempting to put the existence of oxidative phosphorylation superstructures into perspective with the overall supramolecular organisation of interrelated mitochondrial pathways and the sophisticated subcompartmentalisation of mitochondria.Complex I and Alternative NADH Dehydrogenases, Edited by M.I. González-Siso, 01/2007: chapter Oxidative phosphorylation supercomplexes in various eukaryotes: A paradigm change gains critical momentum.: pages 179-213; Transworld Research Network, Kerala, India., ISBN: 978-81-7895-300-7