MOLECULAR REMEDY OF COMPLEX-I DEFECTS - ROTENONE-INSENSITIVE INTERNALNADH-QUINONE OXIDOREDUCTASE OF SACCHAROMYCES-CEREVISIAE MITOCHONDRIA RESTORES THE NADH OXIDASE ACTIVITY OF COMPLEX-I-DEFICIENT MAMMALIAN-CELLS

The NDI1 gene encoding rotenone-insensitive internal NADH-quinone oxid oreductase of Saccharomyces cerevisiae mitochondria was cotransfected into the complex I-deficient Chinese hamster CCL16-B2 cells. Stable ND I1-transfected cells were obtained by screening with antibiotic G418, The NDI1 gene was shown to be expressed in the transfected cells. The expressed Ndi1 enzyme was recognized to be localized to mitochondria b y immunoblotting and confocal immunofluorescence microscopic analyses. Using digitonin-permeabilized cells, it was shown that the transfecte d cells, but not nontransfected control cells, exhibited the electron transfer activities with glutamate/malate as the respiratory substrate . The activities were inhibited by flavone, antimycin A, and KCN but n ot by rotenone, Added NADH did not serve as the substrate, suggesting that the expressed Ndi1 enzyme was located on the matrix side of the i nner mitochondrial membranes. Furthermore, although nontransfected cel ls could not survive in a medium low in glucose (0.6 mM), which is a s ubstrate of glycolysis, the NDI1-transfected cells were able to grow i n the absence of added glucose. When glycolysis is slow, either at low glucose concentrations or in the presence of galactose, respiration i s required for cells to survive. The mutant cells do not survive at lo w glucose or in galactose, but they can be rescued by Ndi1, These resu lts indicated that the S. cerevisiae Ndi1 was expressed functionally i n CCL16-B2 cells and catalyzed electron transfer from NADH in the matr ix to ubiquinone-10 in the inner mitochondrial membranes. It is conclu ded that the NDI1 gene provides a potentially useful tool for gene the rapy of mitochondrial diseases caused by complex I deficiency.