A Forward Genetic Screen Identifies Mutants Deficient for Mitochondrial Complex I Assembly in Chlamydomonas reinhardtii

Abstract

Mitochondrial complex I is the largest multimeric enzyme of the respiratory chain. The lack of a model system with facile genetics has limited the molecular dissection of complex I assembly. Using Chlamydomonas reinhardtii as an experimental system to screen for complex I defects, we isolated, via forward genetics, amc1-7 nuclear mutants (for assembly of mitochondrial complex I) displaying reduced or no complex I activity. Blue native (BN)-PAGE and immunoblot analyses revealed that amc3 and amc4 accumulate reduced levels of the complex I holoenzyme (950 kDa) while all other amc mutants fail to accumulate a mature complex. In amc1, -2, -5-7, the detection of a 700 kDa subcomplex retaining NADH dehydrogenase activity indicates an arrest in the assembly process. Genetic analyses established that amc5 and amc7 are alleles of the same locus while amc1-4 and amc6 define distinct complementation groups. The locus defined by the amc5 and amc7 alleles corresponds to the NUOB10 gene, encoding PDSW, a subunit of the membrane arm of complex I. This is the first report of a forward genetic screen yielding the isolation of complex I mutants. This work illustrates the potential of using Chlamydomonas as a genetically tractable organism to decipher complex I manufacture.

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

“…Amplification of hygromycin B resistance cassette (HygR, encoded by the APH7 gene) from the pHyg3 plasmid, electroporation of 3A+ or 4C cells with 100 ng of HygR cassette, and subsequent selection of hygromycine resistant transformants were carried out as described in (Barbieri et al, 2011). The transmission pattern of the dark -and Hyg R loci in crosses were determined by random analysis of the meiotic products.…”

“…A previous forward genetic screen allowed to isolate seven amc nuclear mutants (for assembly of mitochondrial complex I) displaying reduced or no complex I activity (Barbieri et al, 2011). To uncover additional AMC loci, we undertook another screen for nuclear mutants defective in complex I (see 2.3.2).…”

“…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). In C. reinhardtii, RNA interference is an alternative powerful tool to knock-down the expression of specific nuclear genes (Schroda, 2006).…”

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

“…The growth phenotype was inherited in a Mendelian fashion demonstrating the nuclear location of the mutation. More recently, insertional mutagenesis (e.g., [38]) or RNA interference technique (e.g., [39], [40]) were used in order to target nuclear genes involved in respiration (see below for the description of the mutants).…”

“…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. Mutants Seven complex I mutants affected in subunits encoded by the nuclear genome were isolated: four knock-down mutants corresponding to ND3, ND4L, ND7 and 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 10 ND9 subunits were obtained by RNA interference [39] and three knock-out mutants in the NUOB10 (PDSW subunit), NDUFS3 (ND9 subunit) and NUOP4 genes by insertional mutagenesis [38].…”