Proteomic and Functional Characterization of a Chlamydomonas reinhardtii Mutant Lacking the Mitochondrial Alternative Oxidase 1

Laboratory of Bioenergetics and Cellular Physiology, University of Liege, Belgium.
Journal of Proteome Research (Impact Factor: 4.25). 06/2010; 9(6):2825-38. DOI: 10.1021/pr900866e
Source: PubMed


In the present work, we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase 1 (AOX1). The AOX1-deficient strain displays a remarkable doubling of the cell volume and biomass without alteration of the generation time or change in total respiratory rate, with a significantly higher ROS production. To identify the molecular adaptation underlying these observations, we have carried out a comparative study of both the mitochondrial and the cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important quantitative modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways.

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Available from: Arnaud Blomme, Feb 07, 2015
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    • "As a direct comparison, in a mutant deprived of the alternative oxidase (AOX1) of the respiratory chain, although total respiration and growth were not impaired in mixotrophic conditions, a strong increase of intracellular ROS content was observed. By the same comparative proteomic approach, major modifications in the expression of proteins of primary metabolism were described in AOX1 mutant, namely a decrease of enzymes of the main catabolic pathways, an increase of enzymes involved in anabolic pathways and a strong up-regulation of the ROS scavenging system enzyme (Mathy et al., 2010). This suggests that the sensing of oxidative stress in the mitochondria would be a primary event that leads to the genetic control of the general metabolic pathways. "
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    ABSTRACT: In Chlamydomonas, unlike in flowering plants, genes coding for Nd7 (NAD7/49kDa) and Nd9 (NAD9/30kDa) core subunits of mitochondrial respiratory-chain complex I are nucleus-encoded. Both genes possess all the features that facilitate their expression and proper import of the polypeptides in mitochondria. By inactivating their expression by RNA interference or insertional mutagenesis, we show that both subunits are required for complex I assembly and activity. Inactivation of complex I impairs the cell growth rate, reduces the respiratory rate, leads to lower intracellular ROS production and lower expression of ROS scavenging enzymes, and is associated to a diminished capacity to concentrate CO2 without compromising photosynthetic capacity.
    Full-text · Article · Dec 2013 · Mitochondrion
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    • "References for the new data are indicated. (a) [1], (b) [17], (c) [68], (d) [69], (e) [70], (f) [71], (g) [72], (h) [73], (i) [74], (j) [75], (k) [76], (l) [77], (m) [22], (n) [29], (o) [78], (p) [58], (q) [79], (r) [34]. Fig. 1. "
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    ABSTRACT: Genetic manipulation of the unicellular green alga Chlamydomonas reinhardtii is straightforward. Nuclear genes can be interrupted by insertional mutagenesis or targeted by RNA interference whereas random or site-directed mutagenesis allows the introduction of mutations in the mitochondrial genome. This, combined with a screen that easily allows discriminating respiratory-deficient mutants, makes Chlamydomonas a model system of choice to study mitochondria biology in photosynthetic organisms. Since the first description of Chlamydomonas respiratory-deficient mutants in 1977 by random mutagenesis, many other mutants affected in mitochondrial components have been characterized. These respiratory-deficient mutants increased our knowledge on function and assembly of the respiratory enzyme complexes. More recently some of these mutants allowed the study of mitochondrial gene expression processes poorly understood in Chlamydomonas. In this review, we update the data concerning the respiratory components with a special focus on the assembly factors identified on other organisms. In addition, we make an inventory of different mitochondrial respiratory mutants that are inactivated either on mitochondrial or nuclear genes.
    Full-text · Article · Oct 2013 · Biochimie
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    • "Proteomic analysis revealed that Nda3 was located in the chloroplast (Terashima et al. 2010), but no function has been yet assigned to this enzyme. The location of the other NADH dehydrogenases is still unknown and proteomic studies performed on isolated mitochondria did not reveal any Nda enzymes (Atteia et al. 2009; Mathy et al. 2010). It is thus of interest to determine whether some of the type-II NAD(P)H dehydrogenases truly belong to mitochondria in Chlamydomonas and what would be their role. "
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    ABSTRACT: Type-II NAD(P)H dehydrogenases form a multigene family that comprise six members in the green microalga Chlamydomonas. To date, only one enzyme (Nda2) located in the chloroplast has been characterized in this alga and demonstrated to participate in the reduction of the plastoquinone pool. We present here the functional characterization of Nda1. The enzyme is located on the inner face of the inner mitochondrial membrane. Its downregulation leads to a slight decrease of NADH:ferricyanide activity and of dark whole cell respiration. To determine whether the reduction of Nda1 combined with the lack of complex I would affect mitochondrial processes, double mutants affected in both Nda1 and complex I were isolated. Respiration and growth rates in heterotrophic conditions were significantly altered in the double mutants investigated, suggesting that Nda1 plays a role in the oxidation of matrix NADH in the absence of complex I.
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