Abstract
The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.
Highlights
The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA-encoded subunit ND4
If the host cells are complex I-deficient because of a nuclear or mitochondrial DNA (mtDNA) mutation, functional expression of the NDI1 could be useful to correct the complex I defect. That this is the case for a complex I deficiency associated with a nuclear gene mutation has recently been demonstrated by experiments showing that the S. cerevisiae NDI1 gene, transfected into Chinese hamster cells carrying a deletion in the gene for the essential nuclear-encoded MWFE subunit, can restore the respiratory NADH dehydrogenase activity in the host cells [10]
Because most human mitochondrial diseases associated with defective complex I activity, which have been reported so far, are due to mutations in the mitochondrial genome [13], it was important to analyze the function of the yeast NDI1 gene and its encoded protein in human cells carrying mutations in mtDNA-encoded complex I subunits
Summary
The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. That this is the case for a complex I deficiency associated with a nuclear gene mutation has recently been demonstrated by experiments showing that the S. cerevisiae NDI1 gene, transfected into Chinese hamster cells carrying a deletion in the gene for the essential nuclear-encoded MWFE subunit, can restore the respiratory NADH dehydrogenase activity in the host cells [10].
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