Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

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.

MULTIMERIC respiratory complexes I, III, and IV in the mitochondrial inner membrane generate the proton motive force that is critical for ATP production. Complex I, the largest respiratory complex, is a NADH-ubiquinone oxidoreductase with a hydrophilic peripheral arm protruding into the mitochondrial matrix and a membrane arm (Sazanov and Hinchliffe 2006; Efremov et al. 2010; Hunte et al. 2010). It is known that the assembly of multimeric enzymes is assisted by factors absent from the mature enzyme but nevertheless essential in promoting its assembly into an active form. In humans, complex I dysfunction is the cause of severe diseases (Distelmaier et al. 2009). Since only 40% of the nuclear mutations in complex Ilinked diseases occur in structural subunits of complex I, it is generally accepted that the other 60% represent defects in factors recruited to assemble or regulate the complex (Loeffen et al. 2000; Thorburn 2004).

In recent years, the use of model organisms has contributed to the identification of factors involved in the assembly of respiratory complexes. Most of these studies have been carried out in the model organism Saccharomyces cerevisiae, which has a broad spectrum of genetic and molecular tools facilitating research (Barrientos 2003). However, given that S. cerevisiae and its related species have lost complex I subunits (Gray et al. 2001), they cannot be used as a model for the study of complex I assembly. Instead, the lack of complex I in S. cerevisiae and other eukaryotes has been instrumental in revealing candidate complex I assembly factors through subtractive phylogenetic analyses. In conjunction with mitochondrial proteomic data from complex I-bearing organisms, these analyses were recently used to identify a number of candidate assembly factors (Pagliarini et al. 2008). Three of these factors, C8ORF38, C20ORF7, and FOXRED1, were found to be mutated in complex I-deficient patients (Pagliarini et al. 2008; Sugiana et al. 2008; Fassone et al. 2010). A limitation of subtractive phylogenetics is that assembly factors are assumed to have been systematically lost from organisms lacking complex I, hence excluding the possibility of finding conserved genes that have acquired dual or novel function. Therefore, there is still a need for a genetic approach to discover loci controlling complex I assembly on the basis of loss-of-function phenotypes.

Fungi, animals, and vascular plants have been used extensively as experimental systems for the study of complex I defects (Remacle et al. 2008). Although such models were invaluable in documenting the impacts of specific mutations in complex I function, they were not developed for the isolation of complex I mutants through forward genetics. …

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