Impact of Mutations Affecting ND Mitochondria-encoded Subunits on the Activity and Assembly of Complex I in Chlamydomonas. Implication for the Structural Organization of the Enzyme

Cardol, Pierre; Matagne, René-Fernand; Remacle, Claire

doi:10.1016/s0022-2836(02)00407-2

Cited by 115 publications

(106 citation statements)

References 55 publications

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“…CI (rotenone-sensitive NADH:duroquinone oxidoreductase), CII+III (succinate:cytochrome c oxidoreductase) and CIV (cyanide-sensitive cytochrome c oxidase) activities were measured as described in Cardol et al (2002) and Remacle et al (2001). CV (oligomycin-sensitive ATP synthase) activity was measured as described in Villavicencio-Queijeiro et al (2009) based on the method from Pullman et al (1960).…”

Section: Enzyme Activitiesmentioning

confidence: 99%

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

et al. 2014

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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.Liège, february 06 th 2014Dear Editor, Please find the fully revised version of our manuscript "The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae."We are grateful to you for having encouraged us to resubmit a modified manuscript. We have addressed all the comments raised by the reviewers on our first submission. A point to point answer has been submitted along with the manuscript. Main changes are : (i) additional explanations about the phylogenic/bioinformatic strategy including some phylogenetic trees (supplemental figures 1 and 2); (ii) all data that were previously not shown (mass spectrometry data, in vivo respiratory assays) are now included in the manuscript as supplemental figures/tables.We hope that we have now significantly improved our manuscript so you'll find it suitable for publication in the special issue of Mitochondrion dedicated to "Plant Mitochondria Biology". R: Primary MS data are now given in Supplemental Table 3 for each polypeptide for which the score was > 60. (BLAST alignments, Evalues, etc.) are actually presented in the paper to support the inference that a particular E. gracilis protein is a true homolog of a given kinetoplastid protein. The most important conclusion of t...

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Section: Enzyme Activitiesmentioning

confidence: 99%

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

et al. 2014

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“…Purified mitochondria were obtained according to (Cardol et al, 2002). Proteins amounts were determined by the Bradford method (Bradford, 1976).…”

Section: Protein Analysesmentioning

confidence: 99%

“…This provides an advantage to screen for complex I mutants, as they display robust growth in the light but slow growth under heterotrophic conditions . Several mutations in four of the complex I subunits (ND1, 4, 5, and 6) encoded by the mitochondrial genome have been described so far in Chlamydomonas Cardol et al, 2002;Larosa et al, 2012;Remacle et al, 2001;Remacle et al, 2006). Several complex I-deficient mutants (called amc for "assembly of mitochondrial complex I) of nuclear origin have also been isolated by using a mutagenesis approach (Barbieri et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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In Chlamydomonas, unlike in flowering plants, genes coding for Nd7 (NAD7/49 kDa) and Nd9 (NAD9/30 kDa) 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 CO 2 without compromising photosynthetic capacity.

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“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 16 [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] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 17 ((a) [48], (b) [45], (c) [47], (d) [43], (e) [44], (f) [39], (g) …”

Section: Resultsunclassified

“…Complex I mutants affected in subunits encoded by the mitochondrial genome were mostly obtained by random mutagenesis with intercalating dyes [46][47][48]. They…”

Section: Mutants Affected In Complex I or In Complexes I And Iiimentioning

confidence: 99%

Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: A review

et al. 2014

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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 respiratorydeficient 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. Opposed Reviewers:Claire Remacle -Génétique des microorganismes -Institut de Botanique B22 Boulevard du Rectorat, 27 -Université de Liège-B-4000 Liège, Belgique Tel +32 4 3663812 -Fax +32 4 3663840 e-mail: c.remacle@ulg.ac.be First, we wish to thank the two anonymous reviewers for their constructive comments. We found their suggestions very helpful to improve the quality of our manuscript. We have addressed all the points they raised and have modified the text accordingly. A detailed response to reviewers' comments is included below.We believe we have addressed all referees' comments and we hope this revised version is now suitable for publication in Biochimie.We are looking forward to hearing from you, With best regards, Cover LetterResponse to reviewers The text has been modified in order to give more background details on the methodology used to create and screen respiratory mutants in Chlamydomonas. The main modifications correspond to: Paragraph 2.3: -p8. A figure (Fig. 4) has been added in order to provide more information on dark-dier/slow growth screens for different classes of mutant.-p9. The mitochondrial transformation method used in Chlamydomonas has been detailed.Paragraph 2.4: -p10. The TTC-based screen has been explained.Finally, all along the text the methods used to create nuclear and mitochondrial mutants has been indicated. Also, the text could benefit from being proof-read by a native English speaker as the grammar is a little awkward in places. I highlight a couple of points below, but there are a number of other places where the English could be improved for clarity.The text has been proof-read by a good English speaker as requested and the text has been modified according to the comments.Minor points:1. Abstract, line 1: "the genetics" have been replaced by "genetic manipu...

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Impact of Mutations Affecting ND Mitochondria-encoded Subunits on the Activity and Assembly of Complex I in Chlamydomonas. Implication for the Structural Organization of the Enzyme

Cited by 115 publications

References 55 publications

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

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

Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: A review

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