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Page de résumé pour ULgetd-05142012-092120

Auteur : Larosa, Véronique
E-mail de l'auteur : Veronique.Larosa@ulg.ac.be
URN : ULgetd-05142012-092120
Langue : Anglais/English
Titre : Characterization of Chlamydomonas reinhardtii complex I mutants: the green alga at the crossroad of nuclear and mitochondrial transformation.
Intitulé du diplôme : Doctorat en sciences
Département : FS - Département des sciences de la vie
Jury :
Nom : Titre :
Bassi, Roberto Membre du jury/Committee Member
Baurain, Denis Membre du jury/Committee Member
Boutry, Marc Membre du jury/Committee Member
Cardol, Pierre Membre du jury/Committee Member
Hanikenne, Marc Membre du jury/Committee Member
Franck, Fabrice Président du jury/Committee Chair
Remacle, Claire Promoteur/Director
Mots-clés :
  • Mitochondria/Mitochondrie
  • Complex I/ComplexeI
  • Chlamydomonas
  • Genetic engineering/Transformation génétique
Date de soutenance : 2012-05-29
Type d'accès : Public/Internet
Résumé :

Respiratory complex I (proton-pumping NADH:ubiquinone oxidoreductase) is a very large

multiprotein complex found in most energy converting membranes of bacteria and eukaryotes.

Composed of 14 subunits in bacteria, this complex is much larger in eukaryotes since it comprises more

than 40 subunits in human and the green alga Chlamydomonas reinhardtii, which are encoded by both

nuclear and mitochondrial genomes. It is well recognized that the assembly of multimeric enzymes is

assisted by factors that are not part of the mature enzyme but nevertheless essential in promoting its

assembly into an active form. The number of assembly factors identified for complex I is growing every

year but still stays low since only 9 factors were identified among eukaryotes. It is thus highly probable

that additional assembly factors, encoded by the nuclear genome, are needed for the assembly. This

hypothesis is supported by the fact that in 60% of patients presenting a complex I pathology, mutations

cannot be found in the structural or assembly factors known for complex I. Moreover, in patients

suffering from a mitochondrial disease, the pathogenicity of a given human mitochondrial mutation can

be difficult to analyze because the mitochondrial genome harbors large numbers of polymorphic base

changes that have no pathogenic significance. In addition, mitochondrial mutations are usually found in

the heteroplasmic state, which could hide the biochemical effect of the mutation. Human mitochondrial

mutations can be reconstructed in the yeast S. cerevisiae except for complex I mutations because S.

cerevisiae is lacking complex I and oxidizes NADH via a monomeric type-II NADH dehydrogenase. As a

substitute, bacterial systems have been used to reconstruct human pathogenic mutations but are not

ideal because the membrane domain of eukaryotic complex I is much more complex than the

corresponding arm from bacterial complex I (28 subunits versus 7 subunits).

The first goal of my thesis thus focused on the identification of new assembly factors of complex I,

using Chlamydomonas reinhartii, a green alga, as an experimental system (realized in collaboration with

Prof. Hamel, Ohio State University - USA). For that purpose, an insertion library was built that was

subsequently screened for complex I mutants. I’ve contributed to the construction of the insertion

mutant library using a hygromycin (an aminoglycoside type antibiotic) resistance cassette and to the

screening of complex I mutants, based on the fact that these mutants present a reduced growth in

heterotrophic conditions (dark + acetate as carbon source). Complex I deficiency was found in 6 nuclear

mutants and named amc1 and amc 3 to 7 (for assembly of mitochondrial complex I). My work was to

characterize them biochemically and to compare them to amc2 mutant, a nuclear complex I mutant

previously isolated. These mutants displayed reduced or no complex I activity. Blue native (BN)-PAGE

and immunoblot analyses revealed that amc3, and -4 accumulated reduced levels of the complex I

holoenzyme (950 kDa) while all other amc mutants amc1, -2, -5 to -7 failed to accumulate a mature

complex. Instead, a 700 kDa subcomplex retaining NADH dehydrogenase activity was observed. Genetic

analyses established that amc5 and amc7 are alleles of the same locus while amc1–4 and amc6 defined

distinct complementation groups. So far, our goal was not completely reached because only the locus

defined by the amc5 and amc7 alleles corresponding to the NUOB10 gene, encoding PDSW, a subunit of

the membrane arm of complex I, was identified. Unfortunately, loci defined by the others alleles could

not be easily identified because mutations were not tagged with the resistance cassette. But other

techniques to retrieve mutations were planned such as whole-genome sequencing. Nowadays, this

technique becomes easy and affordable using Illumina's sequencing technology. Like this, in

collaboration with Prof. Denis Baurain (University of Liege), the entire genome of amc4 and amc8

(coming from another insertion library) mutants were sequenced. I’m currently comparing the entire

genomes of these two mutants and trying to localize the gene involved in the complex I deficiency.

The second goal of my project was to propose that the unicellular green alga Chlamydomonas

could be an attractive eukaryotic system to study heteroplasmic and uncharacterized human

mitochondrial mutations because (1) respiratory-deficient mutants are viable and mitochondrial

mutations are found in the homoplasmic state, (2) transformation of the mitochondrial genome is

feasible, (3) Chlamydomonas complex I is close to that of humans. To illustrate that, we have introduced

a Leu157Pro substitution in the Chlamydomonas ND4 subunit of complex I of two different recipient

strains by biolistic transformation, demonstrating that site-directed mutagenesis of the Chlamydomonas

mitochondrial genome is possible. This substitution did not apparently lead to any respiratory enzyme

defect when it is present in the heteroplasmic state in a patient presenting chronic progressive external

ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent the

assembly of the 950 kDa whole complex I which conserves nearly all the NADH dehydrogenase activity of

the peripheral arm. However, the NADH:duroquinone oxidoreductase activity is strongly reduced,

suggesting that the substitution could affect ubiquinone fixation to the membrane domain. The in vitro

defects are correlated in vivo with a decrease in dark respiration and growth rate.

A side project to the mitochondrial transformation was to analyze ends of the Chlamydomonas

mitochondrial genome, a small linear molecule of 15.8 kb. Ends are made of 500 pb inverted repeats,

with 3’end-OH single stranded overhangs of 40 nt. In addition, three repetitions of 86 nt sequence are

present in the genome: one at each end that comprises the first 40 single-stranded overhangs and 46 nt

of doubled-stranded DNA as well as one internal. We identified GG quadruplex in these 86 nucleotides

that could serve to form a T-loop, known to initiate telomeric replication in human chromosomes. In

addition, a PIF helicase that recognizes and stabilizes GG quadruplex is present in the nuclear genome.

Prediction location using MITOPROT is mitochondria. Sequencing circular molecules from the

mitochondrial genome led us to propose that the 86 nt repetition of the left end of the genome (and not

the 86 nt of the right extremity proposed previously) together with the internal repetition are involved in

the formation of the T-loop. To investigate the role of this extremity, a series of mitochondrial

transformations with fragments which comprise or not the 86 nt repetition of the left end of the genome

was performed. Transformation efficiency dropped dramatically when the single 3’OH extension of this

repetition was not present. In addition, all the transformants exhibited a restored left end, which

suggests a copy correction mechanism from the right end. Altogether, these results highlight the role of

the single 3’OH extension of the left telomere in transformation and probably replication.

Autre version :
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[Public/Internet] VeroniqueLarosaPART1.PDF 7.21 Mb 00:17:09 00:00:38
[Public/Internet] VeroniqueLarosaPART2.PDF 20.36 Mb 00:48:27 00:01:48

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