Yeast as Model to Study the Pathogenic Mechanisms of Mutations in Mitochondrial MT-ATP6 Gene
Résumé
Mutations in the mitochondrial MT-ATP6 gene lead to a deficit or a lack of ATP, the energy rich molecule synthesized in mitochondria by ATP synthase, and in consequences to mitochondrial diseases. Their number is constantly increasing due to the NGS methods used now more often in diagnosis of patients. Evaluating their functional consequences and pathogenicity is not an easy task especially when they are found in only a limited number of patients and when they coexist in cells and tissues with the wild type mitochondrial DNA (heteroplasmy), which is quite frequent. Taking advantage of the amenability of Saccharomyces cerevisiae to mitochondrial genetic transformation and strong instability of heteroplasmy in this organism, we constructed overall more than twenty yeast strains bearing the equivalent mutations in MT-ATP6 gene to those identified in patients. These mutations affect well conserved residues of the subunit a/Atp6 of ATP synthase, a protein essential for moving protons across the mitochondrial inner membrane coupled to ATP synthesis. Their consequences for functioning and biogenesis of the yeast ATP synthase were analyzed using the biochemical and molecular biology methods. Thanks’ to the advent of recently published structures of yeast ATP synthase we analyzed the consequences of the substitutions introduced by mutations in silico and proposed the mechanism of pathogenicity for five mutations at molecular level. The selection of genetic suppressors of some of these mutations, located in the distal regions of Atp6 protein, which restored the enzyme activity, holds promise for designing the small molecules that could be used in the therapy of these untreatable diseases. We herein report the properties of yeast models of nine MT-ATP6 gene mutations recently identified in patients presenting with various disorders: m.8843T>C, m.8950G>A, m.9016A>G, m.9025G>A, m.9029A>G, m.9058A>G, m.9139G>A, and m.9160T>C. Significant deficits in ATP production were observed in yeast models of m.9025G>A and m.9029A>G, providing evidence that these two mutations are pathogenic, while the six others had very mild if any effect on mitochondrial function, suggesting they have not, at least alone, the potential to compromise human health.