Reconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas.

in Plant Journal : for Cell & Molecular Biology (2012)

Defects in complex I (NADH:ubiquinone oxidoreductase) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate ... [more ▼]

Defects in complex I (NADH:ubiquinone oxidoreductase) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate 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. We propose that the unicellular green alga Chlamydomonas could be used to study such 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 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. [less ▲]

Mitochondrial transformation and in vitro DNA delivery

in Genomics of Chloroplasts and Mitochondria (2012)

Characterization of complex I mutants in Chlamydomonas reinhardtii : Role of structural subunits and identification of assembly factors.

Poster (2010, July 10)

Characterization of complex I mutants in Chlamydomonas reinhardtii : Role of structural subunits and identification of assembly factors.

Scientific conference (2009)

High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes

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 ▲]