Disruption of the gene coding for the 21.3-kDa subunit of the peripheral arm of complex I from Neurospora crassa.
A 21.3-kDa subunit of the peripheral arm of complex I from Neurospora is encoded by a single chromosomal gene, nuo-21.3b. It is located on linkage group V of the fungal genome, linked to inl. We have isolated and characterized a genomic clone containing this nuclear gene. A DNA fragment containing a portion of the coding region of the gene and upstream flanking sequences was introduced by transformation into a wild-type strain of Neurospora crassa. A single copy transformant was selected and crossed with another strain, leading to inactivation of nuo-21.3b through repeat-induced point mutations. We have analyzed random progeny from this cross and isolated two mutant strains lacking the 21.3-kDa subunit of complex I. One of them was further characterized. Our results suggest that most, if not all, other subunits of complex I are present in the mitochondria of the mutant and assembled in a structure similar to complex I, representing delta 25% of the amounts of enzyme found in the wild type. Nevertheless, a major intermediate, containing proteins of the peripheral arm of complex I, accumulates in the mutant mitochondria. This indicates that the absence of the 21.3-kDa polypeptide results in a disturbed assembly of the enzyme complex, suggesting a role for this polypeptide. We observed similar rates of rotenone-sensitive NADH:ubiquinone oxido-reductase activity in mitochondrial membranes from the mutant and wild-type strains and discuss the possibility that this electron transfer is independent of the more hydrophobic part of complex I. Transformation of the mutant with the intact gene and flanking sequences restored the wild-type phenotype.
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Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Portugal.
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D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D008040:Genetic Linkage; D008928:Mitochondria / Q000201:enzymology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics; D010641:Phenotype; D017354:Point Mutation
D010446:Peptide Fragments; D016660:NAD(P)H Dehydrogenase (Quinone)