Catalytic Activities of Mitochondrial ATP Synthase in Patients with Mitochondrial DNA T8993G Mutation in the ATPase 6 Gene Encoding Subunit a

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.

ATP6 Homoplasmic Mutations Inhibit and Destabilize the Human F1F0-ATP Synthase without Preventing Enzyme Assembly and Oligomerization

“…Among the possible consequences of ATP6 point mutations, impaired F 1 F 0 assembly (26,27), enzyme uncoupling (11,40), and proton conduction blockade through F 0 (6, 7, 9) have all been proposed. To distinguish between them, concluding evidence on the effect of NARP/MILS mutations on F 1 F 0 assembly was limited because the reported human F 1 F 0 structural studies have been done mostly with heteroplasmic cells and provided apparently opposing results (9,(25)(26)(27)).…”

“…Oligomycin Sensitivity in T8993G and T8993C MutantsBased on studies on heteroplasmic T8993G cells, some groups (11,40) propose that the pathogenic mechanism of NARP/ MILS is a partial uncoupling of F 0 -proton translocation from F 1 -ATP synthesis. We evaluated functional coupling between F 1 and F 0 sectors in the homoplasmic T8993G cybrids by assaying oligomycin sensitivity during ATP synthesis and hydrolysis (Fig.…”

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

“…The primary biochemical consequence of the T8993G mutation is a decrease in the rate of ATP synthesis. 11 Therefore, it was important to evaluate changes in both total cellular ATP content and in the rate of mitochondrial ATP synthesis following Ad.1891 infection. The improvement in the ability of JCP239 cells to utilize galactose as the sole carbon source upon Ad.1891 infection was accompanied by significant increases in total cellular ATP content and in the rate of ATP synthesis by mitochondria (Figures 5a and b).…”

“…10 T8993G mutations results in a substitution of a highly conserved hydrophobic leucine 156 in the transmembrane domain of the subunit a of F 1 F 0 -ATPase with a highly charged arginine, which is unfavorable thermodynamically. The mutant protein imparts upon F 1 F 0 -ATPase a defect in ATP synthesis, but not hydrolysis, 11 which may result from inefficient coupling between proton transport and ATP synthesis. 12 At the DNA level, the T8993G transversion generates a unique recognition site for SmaI and XmaI restriction endonucleases (REs), which is absent in wild type mtDNA and can be used for the diagnostic purposes.…”

A Yeast Model of the Neurogenic Ataxia Retinitis Pigmentosa (NARP) T8993G Mutation in the Mitochondrial ATP Synthase-6 Gene

“…50 to 90% (13)(14)(15)(16)(17)(18)(19)(20). However, the precise impact of the leucine to arginine pathogenic change on the ATP synthase is still unknown.…”