Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH: ubiquinone reductase of Neurospora mitochondria.
The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH: ubiquinone reductase (Complex I) from Neurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain of N. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functional nuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH: ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.
da Silva
M V
MV
Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Portugal.
Alves
P C
PC
Duarte
M
M
Mota
N
N
Lobo-da-Cunha
A
A
Harkness
T A
TA
Nargang
F E
FE
Videira
A
A
D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000235:genetics; D002499:Centrifugation, Density Gradient; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004579:Electron Transport / Q000235:genetics; D042967:Electron Transport Complex I; D005800:Genes, Fungal; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D017354:Point Mutation; D015183:Restriction Mapping; D017421:Sequence Analysis
D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I