RESUMEN
A mutant of Neurospora crassa (cni-1) has been isolated that has two pathways of mitochondrial respiration. One pathway is sensitive to cyanide and antimycin A, the other is sensitive only to salicyl hydroxamic acid. Respiration can proceed through either pathway and both pathways together in this mutant account for greater than 90% of all mitochondrial respiration. The cni-1 mutation segregates as a nuclear gene in crosses to other strains of Neurospora. Absorption spectra of isolated mitochondria from cni-1 show typical b- and c-type cytochromes but the absorption peaks corresponding to cytochrome aa(3) are not detectable. Extraction of soluble cytochrome c-546 from these mitochondria followed by reduction with ascorbate reveals a new absorption peak at 426 nm that is not present in wild-type mitochondria. This peak may be due to an altered cytochrome oxidase with abnormal spectral properties. Mitochondria from cni-1 have elevated levels of succinate-cytochrome c reductase but reduced levels of nicotinamide adenine dinucleotide reduced form cytochrome c reductase and of cyanide- and azide-sensitive cytochrome c oxidase. These studies suggest that the cni-1 mutation results in the abnormal assembly of cytochrome c oxidase so that the typical cytochrome aa(3) spectrum is lost and the enzyme activity is reduced. As a consequence of this alteration, a cyanide-insensitive respiratory pathway is elaborated by these mitochondria which may serve to stimulate adenosine 5'-triphosphate production via substrate level phosphorylation by glycolysis and the Krebs cycle.
Asunto(s)
Cromosomas Bacterianos/metabolismo , Mitocondrias/metabolismo , Neurospora/metabolismo , Consumo de Oxígeno , Antimicina A/farmacología , Cruzamientos Genéticos , Cianuros/farmacología , Citocromos/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Genes , Ácidos Hidroxámicos/farmacología , Cinética , Mutación , NAD/metabolismo , Neurospora/efectos de los fármacos , Neurospora crassa/citología , Neurospora crassa/metabolismo , Salicilatos/farmacologíaRESUMEN
A method is described that permits the isolation of mutants that are defective in mitochondrial respiration. The techniques of inositol-less death and overlay with 2,3,5-triphenyl-2H-tetrazolium chloride are utilized to select for mutant colonies. Colonies that survive inositol-less death and fail to reduce the tetrazolium dye are then tested polarographically for cyanide-sensitive respiration. A preliminary characterization of three mutants obtained by this method is presented. The mutants have been characterized by their cyanide-sensitive respiration rate, growth rate, the state III respiration rate of isolated mitochondria, inhibition of the respiration of isolated mitochondria by cyanide and antimycin A, and cytochrome spectra. All of the mutants described differ from the parent strain in some of these aspects.