The mtDNA-encoded ND6 subunit of mitochondrial NADH dehydrogenase is essential for the assembly of the membrane arm and the respiratory function of the enzyme

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Abstract
Seven of the ∼40 subunits of the mammalian respiratory NADH dehydrogenase (Complex I) are encoded in mitochondrial DNA (mtDNA). Their function is almost completely unknown. In this work, a novel selection scheme has led to the isolation of a mouse A9 cell derivative defective in NADH dehydrogenase activity. This cell line carries a near‐homoplasmic frameshift mutation in the mtDNA gene for the ND6 subunit resulting in an almost complete absence of this polypeptide, while lacking any mutation in the other mtDNA‐encoded subunits of the enzyme complex. Both the functional defect and the mutation were transferred with the mutant mitochondria into mtDNA‐less (ρ) mouse LL/2‐m21 cells, pointing to the pure mitochondrial genetic origin of the defect. A detailed biosynthetic and functional analysis of the original mutant and of the ρ cell transformants revealed that the mutation causes a loss of assembly of the mtDNA‐encoded subunits of the enzyme and, correspondingly, a reduction in malate/glutamate‐dependent respiration in digitonin‐permeabilized cells by ∼90% and a decrease in NADH:Q1 oxidoreductase activity in mitochondrial extracts by ∼99%. Furthermore, the ND6− cells, in contrast to the parental cells, completely fail to grow in a medium containing galactose instead of glucose, indicating a serious impairment in oxidative phosphorylation function. These observations provide the first evidence of the essential role of the ND6 subunit in the respiratory function of Complex I and give some insights into the pathogenic mechanism of the known disease‐causing ND6 gene mutations.