Lack of Complex I Activity in Human Cells Carrying a Mutation in MtDNA-encoded ND4 Subunit Is Corrected by theSaccharomyces cerevisiae NADH-Quinone Oxidoreductase (NDI1) Gene

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/glutamatedependent 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.

Viral degradasome hijacks mitochondria to suppress innate immunity

“…The primary source of ATP in these cells is glycolysis. In the control "WT" cell line, C4T-aav, the NADH dehydrogenase activity was fully restored by the introduction of the homologous Saccharomyces cerevisiae NDI1 gene [48]. In these two cell lines, mitochondrial length was essentially identical (Figures 6A and 6B) and so was NS degradative activity, as measured by using STAT2 and RIG-I as substrates ( Figure 6C).…”

“…As such reductions likely affect mitochondrial ATP synthesis, we inquired whether or not the mitochondrial ATP synthesis plays a role in NSD function. We took advantage of the human C4T cell line [48], in which a frameshift mutation in the mitochondrial DNA (mtDNA)-encoded ND4 gene resulted in no assembly of NADH dehydrogenase, and a complete loss of enzyme activity, respiratory function and mitochondrial ATP synthesis. The primary source of ATP in these cells is glycolysis.…”

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“…More restrictively, human EAAT4 is expressed only by cerebellar neurons. Further investigation into the neuronal vesicular Glu transporter uncovered VGLUT1, which is only expressed in glutamatergic neurons 119,120 .…”

Targeting the NAD7 Subunit to Mitochondria Restores a Functional Complex I and a Wild Type Phenotype in the Nicotiana sylvestris CMS II Mutant Lacking nad7

“…The mitochondrial membranes of the yeast Saccharomyces cerevisiae are devoid of complex I and only contain the alternative NDH-2 enzyme (27). The S. cerevisiae NDI1 gene has been used successfully to transfect a complex I-deficient Chinese hamster cell line (28) and a human cell line (29), restoring respiration capacity. Therefore, plant and fungi mutants with a deficient complex I are potentially viable.…”