Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

Abstract

Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.

Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C

“…The atp6-S250P mutation had no visible influence on yeast ATP synthase assembly/stability ( Fig.2A), indicating a partial functional impairment of the enzyme. There gives also relatively mild clinical phenotypes [31]. These findings reveal that a deficit in mitochondrial ATP production so modest as 30% is sufficient to impact human health.…”

“…We previously constructed yeast models of the pathogenic ATP6 mutations T8993G [12], T8993C [13], T9176G [14], T9176C [15] and T8851C [16]. The effects of these mutations on yeast ATP synthase correlated well with those observed in humans, which reflects the high level of evolutionary conservation within the regions of Atp6p affected by these mutations.…”

“…Indeed as previously shown through the analysis of numerous yeast ATP synthase mutants, the activity of this enzyme needs to be decreased by at least 80% to see an obvious respiratory growth defect, which indicates that ATP synthase is far from limiting for the proliferation of yeast cells producing ATP by oxidative phosphorylation [30,31]. However, when the rate of mitochondrial ATP production is diminished cells become more sensitive to chemical inhibition of ATP synthase with oligomycin [31], a compound that is presumed to target the F O because mutations in Atp9p and Atp6p can confer an increased resistance to it [32,33].…”

“…Thus, the higher in vivo sensitivity to oligomycin of mutants in which ATP synthase is partially compromised is due to the fact that less of the drug is required to reach the 20% oxidative phosphorylation threshold under which the production of ATP becomes limiting for growth [31]. We therefore tested the in vivo sensitivity to oligomycin of the mutant atp6-S250P.…”

A yeast-based assay identifies drugs active against human mitochondrial disorders

“…Because of the natural instability of heteroplasmy in yeast, homoplasmic populations in which 100% of mitochondria contain mutated mtDNA can be easily generated. Thus, yeast models of the five most common ATP6 mutations found in NARP patients (T8993G, T8993C, T9176G, T9176C, and T8851C) have been generated and characterized (8)(9)(10)(11). Other patients exhibiting ATP synthase deficiency have been found to carry mutations in two nuclear genes, ATP12 (12) and TMEM70 (13), which encode proteins that are required for ATP synthase assembly.…”