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

Abstract

SUMMARYDefects 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.

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

“…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).…”

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

“…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) …”

“…More recently, mitochondrial transformation was set up with the aim of performing site-directed mutagenesis of the respiratory subunits encoded by the mitochondrial genome (e.g., [43], [44]). Therefore C. reinhardtii is the only photosynthetic organism where reverse genetics is possible in mitochondria.…”

“…In addition, mitochondrial transformation by biolistic device allowed obtaining mutants for the nd4 gene (Δnd4, L157P ND4) [43,44]. The basis for the isolation of such transformants relies on the fact that, as stated before, complex I mutants are able to grow in the dark, albeit slower than the wild-type strain.…”

Genetic tools and techniques for Chlamydomonas reinhardtii

“…Since the nuclear genome is haploid in vegetative cells, recessive mutations do not occur, and selection for mutations of specific genes can be straightforward. As mentioned before, the genomes of chloroplast and mitochondria are present in multiple copies per cell, and heteroplasmy therefore can here be an issue (Remacle et al 2006); however, isolation of homoplasmic cell lines is possible (Larosa et al 2012). …”