Inactivation of the gene encoding the 14-kDa subunit VII of yeast ubiquinol. Cytochrome c oxidoreductase and analysis of the resulting mutant.
The single nuclear gene encoding the 14-kDa subunit VII of yeast ubiquinol:cytochrome c oxidoreductase has been inactivated by one-step gene disruption, as verified by Southern blot analysis and immunoblotting. The resulting mutant has no ubiquinol:cytochrome c oxidoreductase activity and is respiratory-deficient. Immunoblotting shows that cells lacking the 14-kDa protein, also have lowered steady-state levels of other subunits of complex III, the nuclear-encoded 11-kDa subunit VIII, the Rieske Fe-S protein and the mitochondrially encoded cytochrome b. No cytochrome b can be detected spectrally. The steady-state levels of the transcripts from genes encoding these proteins are not reduced, implying that the mutation exerts its pleiotropic effects at a post-transcriptional level. The residual amounts of subunits of complex III are recovered in the mutant mitochondria, suggesting that import is unaffected. The results strongly suggest that the 14-kDa protein plays an essential role in the biosynthesis of the complex, most probably at the level of assembly. Field-inversion gel electrophoresis was used to separate chromosomes of HR2 wild type and the (14-kDa-protein) degrees mutant, after which the gene encoding the 14-kDa protein was located on chromosome IV by Southern blot analysis.
Schoppink
P J
PJ
Department of Molecular Cell Biology, University of Amsterdam, The Netherlands.
Berden
J A
JA
Grivell
L A
LA
D015152:Blotting, Northern; D014450:Electron Transport Complex III / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005800:Genes, Fungal; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013367:Submitochondrial Particles / Q000201:enzymology
D046911:Macromolecular Substances; D014450:Electron Transport Complex III