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See detailOn the evolution and expression of Chlamydomonas reinhardtii nucleus-encoded transfer RNA genes
Cognat, Valerie; Deragon, Jean*-Marc; Vinogradova, Elizaveta et al

in Genetics (2008), 179(1), 113-123

In Chlamydomonas reinhardtii, 259 tRNA genes were identified and classified into 49 tRNA isoaccepting families. By constructing phylogenetic trees, we determined the evolutionary history for each tRNA ... [more ▼]

In Chlamydomonas reinhardtii, 259 tRNA genes were identified and classified into 49 tRNA isoaccepting families. By constructing phylogenetic trees, we determined the evolutionary history for each tRNA gene family. The majority of the IRNA sequences are more closely related to their plant counterparts than to animals ones. Northern experiments also permitted LIS to show that at least one member of each IRNA isoacceptor family is transcribed and correctly processed in vivo. A short stretch of T residues known to be a signal for termination of polymerase III transcription was found downstream of most IRNA genes. It allowed us to propose that the vast majority of the IRNA genes are expressed and to confirm that numerous IRNA genes separated by short spacers are indeed cotranscribed. Interestingly, in silico analyses and hybridization experiments show that the cellular IRNA abundance is correlated with the number of tRTNA genes and is adjusted to the codon usage to optimize translation efficiency. Finally, we studied the origin of SINEs, short interspersed elements related to tRNAs, whose presence in Chlamydomonas is exceptional. Phylogenetic analysis strongly suggests that tRNA(Asp)-related SINEs originate front a prokaryotic-type IRNA either horizontally transferred from a bacterium or originally present in mitochondria or chloroplasts. [less ▲]

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See detailEukaryotic complex I: functional diversity and experimental systems to unravel the assembly process
Remacle, Claire ULg; Barbieri, Rosario; Cardol, Pierre ULg et al

in Molecular Genetics & Genomics (2008), 280

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See detailManipulating the mitochondrial genome in the green alga Chlamydomonas reinhardtii
Remacle, Claire ULg

Scientific conference (2007, May)

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See detailGenetic transformation of Saccharomyces cerevisiae and Chlamydomonas reinhardtii mitochondria
Bonnefoy, Nathalie; Remacle, Claire ULg; Fox, Thomas D

in Methods in Cell Biology (2007), 80

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See detailND3 and ND4L subunits of mitochondrial complex I, both nucleus encoded in Chlamydomonas reinhardtii, are required for activity and assembly of the enzyme
Cardol, Pierre ULg; Lapaille, Marie ULg; Minet, P. et al

in Eukaryotic Cell (2006), 5(9), 1460-1467

Made of more than 40 subunits, the rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. In vascular plants ... [more ▼]

Made of more than 40 subunits, the rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. In vascular plants, fungi, and animals, at least seven complex I subunits (ND1, -2, -3, -4, -4L, -5, and -6; ND is NADH dehydrogenase) are coded by mitochondrial genes. The role of these highly hydrophobic subunits in the enzyme activity and assembly is still poorly understood. In the unicellular green alga Chlamydomonas reinhardtii, the ND3 and ND4L subunits are encoded in the nuclear genome, and we show here that the corresponding genes, called NUO3 and NUO11, respectively, display features that facilitate their expression and allow the proper import of the corresponding proteins into mitochondria. In particular, both polypeptides show lower hydrophobicity compared to their mitochondrion-encoded counterparts. The expression of the NUO3 and NUO11 genes has been suppressed by RNA interference. We demonstrate that the absence of ND3 or ND4L polypeptides prevents the assembly of the 950-kDa whole complex I and suppresses the enzyme activity. The putative role of hydrophobic ND subunits is discussed in relation to the structure of the complex I enzyme. A model for the assembly pathway of the Chlamydomonas enzyme is proposed. [less ▲]

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See detailHigh-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes
Remacle, Claire ULg; Cardol, Pierre ULg; Coosemans, Nadine ULg et al

in Proceedings of the National Academy of Sciences of the United States of America (2006), 103(12), 4771-4776

Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb ... [more ▼]

Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Delta nd4 mutation causes a misassembly of complex 1, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex 1. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex 1, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex 1. [less ▲]

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See detailThe mitochondrial ATP synthase of chlorophycean algae contains eight subunits of unknown origin involved in the formation of an atypical stator-stalk and in the dimerization of the complex
Vazquez-Acevedo, Miriam; Cardol, Pierre ULg; Cano-Estrada, Araceli et al

in Journal of Bioenergetics & Biomembranes (2006), 38(5-6), 271-282

Mitochondrial F1FO-ATP synthase of Chlamydomonas reinhardtii and Polytomella sp. is a dimer of 1,600,000 Da. In Chlamydomonas the enzyme lacks the classical subunits that constitute the peripheral stator ... [more ▼]

Mitochondrial F1FO-ATP synthase of Chlamydomonas reinhardtii and Polytomella sp. is a dimer of 1,600,000 Da. In Chlamydomonas the enzyme lacks the classical subunits that constitute the peripheral stator-stalk as well as those involved in the dimerization of the fungal and mammal complex. Instead, it contains eight novel polypeptides named ASA1 to 8. We show that homologs of these subunits are also present in the chlorophycean algae Polytomella sp. and Volvox carterii. Blue Native Gel Electrophoresis analysis of mitochondria from different green algal species also indicates that stable dimeric mitochondrial ATP synthases may be characteristic of all Chlorophyceae. One additional subunit, ASA9, was identified in the purified mitochondrial ATP synthase of Polytomella sp. The dissociation profile of the Polytomella enzyme at high-temperatures and cross-linking experiments finally suggest that some of the ASA polypeptides constitute a stator-stalk with a unique architecture, while others may be involved in the formation of a highly-stable dimeric complex. The algal enzyme seems to have modified the structural features of its surrounding scaffold, while conserving almost intact the structure of its catalytic subunits. [less ▲]

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See detailProteomic and genetic analysis of Chlamydomonas complex I
Remacle, Claire ULg

Conference (2005, May)

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See detailFunctional distribution and dynamics of Arabidopsis SR splicing factors in living plant cells
Tillemans, Vinciane ULg; Dispa, Laurence ULg; Remacle, Claire ULg et al

in Plant Journal (The) (2005), 41(4), 567-582

Serine/arginine-rich (SR) proteins constitute an important class of splicing regulators in higher eukaryotes that share a modular structure consisting of one or two N-terminal RNA recognition motif (RRM ... [more ▼]

Serine/arginine-rich (SR) proteins constitute an important class of splicing regulators in higher eukaryotes that share a modular structure consisting of one or two N-terminal RNA recognition motif (RRM) domains and a C-terminal RS-rich domain. Herein, we have investigated the in vivo functional distribution of Arabidopsis SR factors. Agrobacterium-mediated transient transformation revealed nuclear speckled distribution and the overall colocalization of fluorescent protein (FP)-tagged SR factors in both tobacco and Arabidopsis cells. Their overall colocalization in larger nucleoplasmic domains was further observed after transcriptional and phosphorylation/dephosphorylation inhibition, indicating a close functional association between SR factors, independent of their phosphorylation state. Furthermore, we demonstrated in vivo the conserved role of the RS and RRM domains in the efficient targeting of Arabidopsis SR proteins to nuclear speckles by using a series of structural domain-deleted mutants of atRSp31 and atRSZp22. We suggest additional roles of RS domain such as the shuttling of atRSZp22 between nucleoplasm and nucleolus through its phosphorylation level. The coexpression of deletion mutants with wild-type SR proteins revealed potential complex associations between them. Fluorescence recovery after photobleaching demonstrated similar dynamic properties of SR factors in both tobacco transiently expressing cells and Arabidopsis transgenics. Cell cycle phase-dependent organization of FP-tagged SR proteins was observed in living tobacco BY-2 cells. We showed that atRSp31 is degraded at metaphase by fluorescence quantification. SR proteins also localized within small foci at anaphase. These results demonstrate interesting related features as well as potentially important differences between plant and animal SR proteins. [less ▲]

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See detailThe mitochondrial oxidative phosphorylation proteome of Chlamydomonas reinhardtii deduced from the genome sequencing project
Cardol, Pierre ULg; Gonzalez-Halphen, Diego; Reyes-Prieto, Adrian et al

in Plant Physiology (2005), 137(2), 447-459

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See detailStructural organization of mitochondrial human complex I: role of the ND4 and ND5 mitochondria-encoded subunits and interaction with prohibitin
Bourges, I.; Ramus, C.; de Camaret, B. M. et al

in Biochemical Journal (2004), 383(Part 3), 491-499

Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of ... [more ▼]

Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of these subunits have significant pathogenic consequences in humans. However, the way these events affect complex I assembly is poorly documented. To understand the effects of particular mutations in ND subunits on complex I assembly, we studied four human cell lines: ND4 non-expressing cells, ND5 non-expressing cells, and rhodegrees cells that do not express any ND subunits, in comparison with normal complex I control cells. In control cells. all the seven analysed nuclear-encoded complex I subunits Were found to be attached to the mitochondrial inner membrane, except for the 24 kDa subunit, which was nearly equally partitioned between the membranes and the matrix. Absence of a single ND subunit, or even all the seven ND subunits, caused no major changes in the nuclear-encoded complex I subunit content of mitochondria. However, in cells lacking ND4 or ND5, very low amounts of 24 kDa subunit were found associated with the membranes, whereas most of the other nuclear-encoded subunits remained attached. In contrast, membrane association of most of the nuclear subunits was significantly reduced in the absence of all seven ND proteins. Immunopurification detected several subcomplexes. One of these, containing the 23, 30 and 49 kDa subunits, also contained prohibitin. This is the first description of prohibitin interaction with complex I subunits and suggests that this protein might play a role in the assembly or degradation of mitochondrial complex I. [less ▲]

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See detailHigher plant-like subunit composition of mitochondrial complex I from Chlamydomonas reinhardtii: 31 conserved components among eukaryotes
Cardol, Pierre ULg; Vanrobaeys, F.; Devreese, B. et al

in Biochimica et Biophysica Acta-Bioenergetics (2004), 1658(3), 212-224

The rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. Notably the bovine enzyme comprises up to 46 subunits ... [more ▼]

The rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. Notably the bovine enzyme comprises up to 46 subunits, while 27 subunits could be considered as widely conserved among eukaryotic complex I. By combining proteomic and genomic approaches, we characterized the complex I composition from the unicellular green alga Chlamydomonas reinhardtii. After purification by blue-native polyacrylamide gel electrophoresis (BN-PAGE), constitutive subunits were analyzed by SDS-PAGE coupled to tandem mass spectrometry (MS) that allowed the identification of 30 proteins. We compared the known complex I components from higher plants, mammals, nematodes and fungi with this MS data set and the translated sequences from the algal genome project. This revealed that the Chlamydomonas complex I is likely composed of 42 proteins, for a total molecular mass of about 970 kDa. In addition to the 27 typical components, we have identified four new complex I subunit families (bovine ESSS, PFFD, B16.6, B12 homologues), extending the number of widely conserved eukaryote complex I components to 31. In parallel, our analysis showed that a variable number of subunits appears to be specific to each eukaryotic kingdom (animals, fungi or plants). Protein sequence divergence in these kingdom-specific sets is significant and currently we cannot exclude the possibility that homology between them exists, but has not yet been detected. (C) 2004 Elsevier B.V. All rights reserved. [less ▲]

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See detailImpact of a mutation in the mitochondrial LSU rRNA gene from Chlamydomonas reinhardtii on the activity and the assembly of respiratory-chain complexes
Remacle, Claire ULg; Gloire, Geoffrey ULg; Cardol, Pierre ULg et al

in Current Genetics (2004), 45(5), 323-330

Two substitutions A1090G and A1098C (together called the m mutation) located in the conserved GTPase domain of the mitochondrial LSU rRNA gene were recently shown to weakly compensate for the phenotypical ... [more ▼]

Two substitutions A1090G and A1098C (together called the m mutation) located in the conserved GTPase domain of the mitochondrial LSU rRNA gene were recently shown to weakly compensate for the phenotypical effect of a -1T frameshift mutation in the mitochondrial cox1 gene of C. reinhardtii. In order to analyze the impact of the m mutation on the mitochondrial translational machinery, a strain carrying the m mutation but wild-type for the cox1 gene was isolated. We found that the growth and the respiratory rate of the m mutant were affected and that the activities of complexes I, III, and IV, all containing mitochondria-encoded subunits, were lowered. In contrast the activities of complex II and of the alternative oxidase, both encoded exclusively by the nuclear genome, were not modified. The steady-state levels of complex I enzyme and of several components of the respiratory complexes I, III, and IV were also reduced in the mutant. We moreover showed that m did not suppress other frameshift or UGA stop mutations which affect mitochondrial genes. [less ▲]

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