Respiratory chain complex I is essential for sexual development in neurospora and binding of iron sulfur clusters are required for enzyme assembly.
We have cloned and disrupted in vivo, by repeat-induced point mutations, the nuclear gene coding for an iron sulfur subunit of complex I from Neurospora crassa, homologue of the mammalian TYKY protein. Analysis of the obtained mutant nuo21.3c revealed that complex I fails to assemble. The peripheral arm of the enzyme is disrupted while its membrane arm accumulates. Furthermore, mutated 21.3c-kD proteins, in which selected cysteine residues were substituted with alanines or serines, were expressed in mutant nuo21. 3c. The phenotypes of these strains regarding the formation of complex I are similar to that of the original mutant, indicating that binding of iron sulfur centers to protein subunits is a prerequisite for complex I assembly. Homozygous crosses of nuo21.3c strain, and of other complex I mutants, are unable to complete sexual development. The crosses are blocked at an early developmental stage, before fusion of the nuclei of opposite mating types. This phenotype can be rescued only by transformation with the intact gene. Our results suggest that this might be due to the compromised capacity of complex I-defective strains in energy production.
Duarte
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M
Instituto de Biologia Molecular e Celular, University of Porto, 4150-180 Porto, Portugal.
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D000818:Animals; D003001:Cloning, Molecular; D042967:Electron Transport Complex I; D008322:Mammals; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D017354:Point Mutation; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D021122:Protein Subunits; D011994:Recombinant Proteins; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I