Abstract :
[en] 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 to 7 nuclear mutants (for assembly of mitochondrial complex I) displaying reduced or no Complex I activity. 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, 6, 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 to 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.
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