title: Neurospora strains harboring mitochondrial disease-associated mutations in iron-sulfur subunits of complex I.
abstract: We subjected the genes encoding the 19.3-, 21.3c-, and 51-kDa iron-sulfur subunits of respiratory chain complex I from Neurospora crassa to site-directed mutagenesis to mimic mutations in human complex I subunits associated with mitochondrial diseases. The V135M substitution was introduced into the 19.3-kDa cDNA, the P88L and R111H substitutions were separately introduced into the 21.3c-kDa cDNA, and the A353V and T435M alterations were separately introduced into the 51-kDa cDNA. The altered cDNAs were expressed in the corresponding null-mutants under the control of a heterologous promoter. With the exception of the A353V polypeptide, all mutated subunits were able to promote assembly of a functional complex I, rescuing the phenotypes of the respective null-mutants. Complex I from these strains displays spectroscopic and enzymatic properties similar to those observed in the wild-type strain. A decrease in total complex I amounts may be the major impact of the mutations, although expression levels of mutant genes from the heterologous promoter were sometimes lower and may also account for complex I levels. We discuss these findings in relation to the involvement of complex I deficiencies in mitochondrial disease.
authors.affiliation: Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal.
authors.forename: Margarida
authors.initials: M
authors.lastname: Duarte
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schulte
authors.forename: Alexandra V
authors.initials: AV
authors.lastname: Ushakova
authors.forename: Arnaldo
authors.initials: A
authors.lastname: Videira
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D007506:Iron-Sulfur Proteins / Q000235:genetics* / Q000378:metabolism; D028361:Mitochondrial Diseases / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009492:Neurospora crassa / Q000235:genetics* / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism
chemical_list: D007506:Iron-Sulfur Proteins; D024101:Mitochondrial Proteins; D012331:RNA, Fungal; D042967:Electron Transport Complex I