title: 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.
authors.affiliation: Department of Molecular Genetics and Department of Molecular Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: M Rosario
authors.initials: MR
authors.lastname: Barbieri
authors.forename: Véronique
authors.initials: V
authors.lastname: Larosa
authors.forename: Cécile
authors.initials: C
authors.lastname: Nouet
authors.forename: Nitya
authors.initials: N
authors.lastname: Subrahmanian
authors.forename: Claire
authors.initials: C
authors.lastname: Remacle
authors.forename: Patrice P
authors.initials: PP
authors.lastname: Hamel
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000378:metabolism*; D042967:Electron Transport Complex I / Q000172:deficiency* / Q000235:genetics*; D042963:Electron Transport Complex II / Q000378:metabolism; D014450:Electron Transport Complex III / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D005838:Genotype; D015151:Immunoblotting; D016254:Mutagenesis, Insertional; D009154:Mutation; D010101:Oxygen Consumption
chemical_list: D042963:Electron Transport Complex II; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III