Inactivation of genes encoding subunits of the peripheral and membrane arms of neurospora mitochondrial complex I and effects on enzyme assembly.
We have isolated and characterized the nuclear genes encoding the 12.3-kD subunit of the membrane arm and the 29.9-kD subunit of the peripheral arm of complex I from Neurospora crassa. The former gene was known to be located in linkage group I and the latter is now assigned to linkage group IV of the fungal genome. The genes were separately transformed into different N. crassa strains and transformants with duplicated DNA sequences were isolated. Selected transformants were then mated with other strains to generate repeat-induced point mutations in both copies of the genes present in the nucleus of the parental transformant. From the progeny of the crosses, we were then able to recover two individual mutants lacking the 12.3- and 29.9-kD proteins in their mitochondria, mutants nuo12.3 and nuo29.9, respectively. Several other subunits of complex I are present in the mutant organelles, although with altered stoichiometries as compared with those in the wild-type strain. Based on the analysis of Triton-solubilized mitochondrial complexes in sucrose gradients, neither mutant is able to fully assemble complex I. Our results indicate that mutant nuo12.3 separately assembles the peripheral arm and most of the membrane arm of the enzyme. Mutant nuo29.9 seems to accumulate the membrane arm of complex I and being devoid of the peripheral part. This implicates the 29.9-kD protein in an early step of complex I assembly.
Duarte
M
M
Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Portugal.
Sousa
R
R
Videira
A
A
D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D005800:Genes, Fungal; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D016296:Mutagenesis; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D017354:Point Mutation; D015183:Restriction Mapping; D014170:Transformation, Genetic
D016660:NAD(P)H Dehydrogenase (Quinone)