Proteomic and functional characterization of a Chlamydomonas reinhardtii mutant lacking the mitochondrial alternative oxidase 1.
ABSTRACT 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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ABSTRACT: Isocitrate lyase is one of the key enzymes of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO2 is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data demonstrate that isocitrate lyase is required for growth in darkness with acetate (heterotrophic conditions) as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). In these latter conditions, reduced acetate assimilation and concomitant reduced respiration are found and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by (14) N/(15) N labelling has been performed and more than 1,600 proteins have been identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis paralleled with a shift of the TCA cycle towards amino acid synthesis, which is accompanied by an increase in free amino acids. The diminishment of glyoxylate cycle, gluconeogenesis as well as the decrease in enzymes involved in β-oxidation of fatty acids in the icl mutant are likely major factors that contribute to the remodeling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase and increased resistance to paraquat. This article is protected by copyright. All rights reserved.The Plant Journal 11/2013; · 6.58 Impact Factor
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ABSTRACT: The metabolism of microalgae is so flexible that it is not an easy task to give a comprehensive description of the interplay between the various metabolic pathways. There are however constraints that govern central carbon metabolism in Chlamydomonas that are revealed by the compartmentalization and regulation of the pathways and their relation to key cellular processes such as cell motility, division, carbon uptake and partitioning, external and internal rhythms and nutrient stress. Both photosynthetic and mitochondrial electron transfer provide energy for metabolic processes and how energy transfer impacts metabolism and vice versa is a means of exploring the regulation and function of these pathways. A key example is the specific chloroplast localization of glycolysis/gluconeogenesis and how it impacts the redox poise and ATP budget of the plastid in the dark. To compare starch and lipids as carbon reserves, their value can be calculated in terms of NAD(P)H and ATP. As microalgae are now considered as a potential renewable feedstock, we examine current work on the subject and also explore the possibility of rerouting metabolism towards lipid production.Eukaryotic Cell 03/2013; · 3.59 Impact Factor
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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.Mitochondrion 12/2013; · 4.03 Impact Factor