RESUMO
The single nuclear gene encoding the 17-kDa subunit VI of yeast ubiquinol: cytochrome c oxidoreductase has been inactivated by one-step gene disruption. Disruption was verified by Southern blot analysis of nuclear DNA and immunoblotting. Cells lacking the 17-kDa protein are still capable of growth on glycerol and they contain all other subunits of complex III at wild-type levels, implying that the 17-kDa subunit is not essential for either assembly of complex III, or its function. In vitro, electron transport activity of complex III of mutant cells is about 40% of the wild-type complex, but for the total respiratory chain no significant differences in activity was measured between mutant and wild type. The energy-transducing capacity of the complex is not reduced in the absence of the 17-kDa protein. In a relatively high proportion of the transformants, disruption of the 17-kDa gene was accompanied by the appearance of a second mutation causing a petite phenotype. In these cells which lack cytochrome b, the presence of the 17-kDa protein (after complementation) results in stabilization of cytochrome c1.
