Transcriptomics and Proteomics of a Secondary Green Alga.
Perez, Emilie ; ; et al
Poster (2014, August)
Euglena gracilis is a secondary green alga related to trypanosomes that derives from a secondary endosymbiosis between a phagotrophic ancestor and a prasinophycean green alga. Our general objective is to ... [more ▼]
Euglena gracilis is a secondary green alga related to trypanosomes that derives from a secondary endosymbiosis between a phagotrophic ancestor and a prasinophycean green alga. Our general objective is to study the metabolic interactions established between the secondary plastid and the mitochondrion after the endosymbiotic event and to determine the phylogenetic origin of the genes encoding the proteins involved in the energetic pathways. As a first step, we analysed the subunit composition of the mitochondrial respiratory chain, both in silico and by targeted proteomics, to assess the extent of its similitude with the respiratory chain of Trypanosomatidae. We have shown that Euglena shares many additional subunits with trypanosomes, which suggests that these subunits are not especially associated to a parasitic lifestyle. As a second step, we sequenced the total transcriptome of Euglena and determined the phylogenetic origin of each predicted transcript using a database of about 1000 complete proteomes representing the diversity of life. These analyses confirmed that Euglena recruited its genes from a very diverse set of sources. As a third step, we performed a high-throughput analysis of the mitochondrial proteome of Euglena. Our MS/MS experiments, taking advantage of the availability of our transcriptome, mostly recovered mitochondrial proteins, which indicates that our mitochondrial extracts were quite pure. The identified proteins encompassed about 15 different mitochondrial pathways. We are now in the process of comparing the expression levels of both the transcripts and the corresponding proteins across a range of culture conditions selected to differently stimulate the mitochondrion and the plastid. [less ▲]Detailed reference viewed: 63 (15 ULg)
The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae.
Perez, Emilie ; ; et al
in Mitochondrion (2014)
The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential ... [more ▼]
The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential therapeutic target. Recently, numerous additional subunits of the respiratory-chain complexes have been described in Trypanosoma brucei and Trypanosoma cruzi. Since these subunits had apparently no counterparts in other organisms, they were interpreted as potentially associated with the parasitic trypanosome lifestyle. Here we used two complementary approaches to characterise the subunit composition of respiratory complexes in Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes. First, we developed a phylogenetic pipeline aimed at mining sequence databases for identifying homologs to known respiratory-complex subunits with high confidence. Second, we used MS/MS proteomics after two-dimensional separation of the respiratory complexes by Blue Native- and SDS-PAGE to both confirm in silico predictions and to identify further additional subunits. Altogether, we identified 41 subunits that are restricted to E. gracilis, T. brucei and T. cruzi, along with 48 classical subunits described in other eukaryotes (i.e. plants, mammals and fungi). This moreover demonstrates that at least half of the subunits recently reported in T. brucei and T. cruzi are actually not specific to Trypanosomatidae, but extend at least to other Euglenozoa, and that their origin and function are thus not specifically associated with the parasitic lifestyle. Furthermore, preliminary biochemical analyses suggest that some of these additional subunits underlie the peculiarities of the respiratory chain observed in Euglenozoa. [less ▲]Detailed reference viewed: 36 (13 ULg)
Inactivation of genes coding for mitochondrial Nd7 and Nd9 complex I subunits in Chlamydomonas reinhardtii. Impact of complex I loss on respiration and energetic metabolism.
; Larosa, Véronique ; et al
in Mitochondrion (2013)
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 58 (16 ULg)
Atypical subunit composition of the chlorophycean mitochondrial F1FO ATP synthase and role of Asa7 protein in stability and oligomycin resistance of the enzyme.
; ; et al
in Molecular Biology and Evolution (2010), 27(7), 1630-1644
Background. In yeast, mammals, and land plants, mitochondrial F(1)F(O) ATP synthase (complex V) is a remarkable enzymatic machinery which comprises about 15 conserved subunits. Peculiar among eukaryotes ... [more ▼]
Background. In yeast, mammals, and land plants, mitochondrial F(1)F(O) ATP synthase (complex V) is a remarkable enzymatic machinery which comprises about 15 conserved subunits. Peculiar among eukaryotes, complex V from Chlamydomonadales algae (order of chlorophycean class) has an atypical subunit composition of its peripheral stator and dimerization module, with 9 subunits of unknown evolutionary origin (Asa subunits). In vitro, this enzyme exhibits an increased stability of its dimeric form, and in vivo, Chlamydomonas reinhardtii cells are insensitive to oligomycins, which are potent inhibitors of proton translocation through the F(O) moiety. Methodology/Principal Findings. In this work, we showed that the atypical features of the Chlamydomonadales complex V enzyme are shared by the other chlorophycean orders. By biochemical and in silico analyses, we detected several atypical Asa subunits in Scenedesmus obliquus (Sphaeropleales) and Chlorococcum ellipsoideum (Chlorococcales). In contrast, Complex V has a canonical subunit composition in other classes of Chlorophytes (Trebouxiophyceae, Prasinophyceae, and Ulvophyceae) as well as in Streptophytes (land plants) and in Rhodophytes (red algae). Growth, respiration and ATP levels in Chlorophyceae were also barely affected by oligomycin concentrations that affect representatives of the other classes of Chlorophytes. We finally studied the function of the Asa7 atypical subunit by using RNA interference in C. reinhardtii. Although the loss of Asa7 subunit has no impact on cell bioenergetics or mitochondrial structures, it destabilizes in vitro the enzyme dimeric form and renders growth, respiration and ATP level sensitive to oligomycins. Conclusions/Significance. Altogether, our results suggest that the loss of canonical components of the Complex V stator happened at the root of chlorophycean lineage and was accompanied by the recruitment of novel polypeptides. Such a massive modification of Complex V stator features might have conferred novel properties, including the stabilization of the enzyme dimeric form and the shielding of the proton channel. In these respects, we discuss an evolutionary scenario for F(1)F(O) ATP synthase in the whole green lineage (i.e. Chlorophyta and Streptophyta). [less ▲]Detailed reference viewed: 54 (18 ULg)