Rotenone‐insensitive internal NADH‐quinone oxidoreductase of Saccharomyces cerevisiae mitochondria: the enzyme expressed in Escherichia coli acts as a member of the respiratory chain in the host cells

Abstract

The NDI1 gene encodes the internal rotenone‐insensitive NADH‐quinone oxidoreductase localized in the inner mitochondrial membranes of Saccharomyces cerevisiae. The T7 tag‐fused mature NDI1 was overexpressed in Escherichia coli. The overexpressed NDI1 was exclusively found in the membrane fraction. The NDI1‐overexpressed membranes showed significantly increased activities of NADH oxidase and NADH‐ubiquinone‐1 (UQ₁) reductase when compared with the control membranes. Flavone, which is a specific inhibitor of the S. cerevisiae NDI1, inhibited almost completely NADH oxidase and NADH‐UQ₁ reductase activities of NDI1‐overexpressed membranes but scarcely inhibited these activities of the control membranes. In addition, the NADH oxidase activity of the NDI1‐overexpressed membranes was also inhibited by KCN as well as the control membranes. These results indicate that the overexpressed NDI1 worked as a member of the respiratory chain in the host cells, even though E. coli membranes are different from S. cerevisiae inner mitochondrial membranes in terms of quinones and lipid composition.