title: Reconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas.
abstract: Defects in complex I (NADH:ubiquinone oxidoreductase (EC 1.6.5.3)) 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 may hide the biochemical effect of the mutation. We propose that the unicellular green alga Chlamydomonas could be used to study such mutations because (i) respiratory complex-deficient mutants are viable and mitochondrial mutations are found in the homoplasmic state, (ii) transformation of the mitochondrial genome is feasible, and (iii) Chlamydomonas complex I is similar to that of humans. To illustrate this proposal, we introduced a Leu157Pro substitution into the Chlamydomonas ND4 subunit of complex I in two 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 defects when present in the heteroplasmic state in a patient with chronic progressive external ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent assembly of whole complex I (950 kDa) and the NADH dehydrogenase activity of the peripheral arm of the complex is mildly affected. However, the NADH:duroquinone oxidoreductase activity is strongly reduced, suggesting that the substitution could affect binding of ubiquinone to the membrane domain. The in vitro defects correlate with a decrease in dark respiration and growth rate in vivo.
authors.affiliation: Genetics of Microorganisms, Department of Life Sciences, Institute of Botany, University of Liege, B-4000 Liege, Belgium.
authors.forename: Véronique
authors.initials: V
authors.lastname: Larosa
authors.forename: Nadine
authors.initials: N
authors.lastname: Coosemans
authors.forename: Patrick
authors.initials: P
authors.lastname: Motte
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bonnefoy
authors.forename: Claire
authors.initials: C
authors.lastname: Remacle
mesh_terms: D019943:Amino Acid Substitution; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism; D042967:Electron Transport Complex I / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D015894:Genome, Human; D054629:Genome, Mitochondrial; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism; D009154:Mutation; D009245:NADH Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D004272:DNA, Mitochondrial; C098483:NADH dehydrogenase subunit 4; D006861:Hydrogen Peroxide; D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I