Abstract
We investigated the biochemical phenotype of the mtDNA T8993G point mutation in the ATPase 6 gene, associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in three patients from two unrelated families. All three carried >80% mutant genome in platelets and were manifesting clinically various degrees of the NARP phenotype. Coupled submitochondrial particles prepared from platelets capable of succinate-sustained ATP synthesis were studied using very sensitive and rapid luminometric and fluorescence methods. A sharp decrease (>95%) in the succinate-sustained ATP synthesis rate of the particles was found, but both the ATP hydrolysis rate and ATP-driven proton translocation (when the protons flow from the matrix to the cytosol) were minimally affected. The T8993G mutation changes the highly conserved residue Leu(156) to Arg in the ATPase 6 subunit (subunit a). This subunit, together with subunit c, is thought to cooperatively catalyze proton translocation and rotate, one with respect to the other, during the catalytic cycle of the F(1)F(0) complex. Our results suggest that the T8993G mutation induces a structural defect in human F(1)F(0)-ATPase that causes a severe impairment of ATP synthesis. This is possibly due to a defect in either the vectorial proton transport from the cytosol to the mitochondrial matrix or the coupling of proton flow through F(0) to ATP synthesis in F(1). Whatever mechanism is involved, this leads to impaired ATP synthesis. On the other hand, ATP hydrolysis that involves proton flow from the matrix to the cytosol is essentially unaffected.
Highlights
Both the neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)1 syndrome and the maternally inherited Leigh disease have been associated with the mtDNA T8993G point mutation in the ATPase 6 subunit gene of the mitochondrial ATP synthase (F1F0-ATPase), being the variable load of the mutant mtDNA associated with the different clinical expression [1,2,3]
We investigated the biochemical phenotype of the mtDNA T8993G point mutation in the ATPase 6 gene, associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in three patients from two unrelated families
Because we are not doing a strict correlation between the mutation load and the biochemical results in the single samples, we can affirm that our biochemical mtDNA T8993G Mutation and F1F0-ATPase Activity observations are related to a percentage, conservatively estimated, of at least 80% of T8993G mutant mtDNA in the same tissue sample investigated, the platelets
Summary
Both the neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome and the maternally inherited Leigh disease have been associated with the mtDNA T8993G point mutation in the ATPase 6 subunit gene (subunit a) of the mitochondrial ATP synthase (F1F0-ATPase), being the variable load of the mutant mtDNA (heteroplasmy) associated with the different clinical expression [1,2,3]. It catalyzes ADP phosphorylation using a proton electrochemical gradient generated by the electron transport chain (4 –7) It is a ubiquitous, evolutionary conserved enzyme composed by two main sectors: F1, a soluble catalytic sector comprising five different subunits that is bound through two stalks to F0 [8, 9], and the membrane sector that contains up to 10 different subunits [4, 10]. Recent experimental evidence (14 –16) supports a mechanical coupling between the F0 and F1 sectors based on the rotation of a central rotor, consisting of the smaller subunit(s), with respect to a hexagonal ring made up of the main polypeptides containing the catalytic sites Energy transduction in this model would occur through a rotation within the F0 sector powered by proton flow, which promotes ATP synthesis and release. The proton transport activity of the mutated enzyme has never been measured directly
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