Cellular and functional analysis of four mutations located in the mitochondrial ATPase6 gene

Abstract

The smallest rotary motor of living cells, F0F1-ATP synthase, couples proton flow-generated by the OXPHOS system-from the intermembrane space back to the matrix with the conversion of ADP to ATP. While all mutations affecting the multisubunit complexes of the OXPHOS system probably impact on the cell's output of ATP, only mutations in complex V can be considered to affect this output directly. So far, most of the F0F1-ATP synthase variations have been detected in the mitochondrial ATPase6 gene. In this study, the four most frequent mutations in the ATPase6 gene, namely L156R, L217R, L156P, and L217P, are studied for the first time together, both in primary cells and in cybrid clones. Arginine ("R") mutations were associated with a much more severe phenotype than Proline ("P") mutations, in terms of both biochemical activity and growth capacity. Also, a threshold effect in both "R" mutations appeared at 50% mutation load. Different mechanisms seemed to emerge for the two "R" mutations: the F1 seemed loosely bound to the membrane in the L156R mutant, whereas the L217R mutant induced low activity of complex V, possibly the result of a reduced rate of proton flow through the A6 channel.

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

“…8 and 17) before a clear growth defect on respiratory medium can be observed in yeast. Even more significant is the fact that these observations also correlate with what is known about the relative severity of the corresponding atp6-NARP mutations in human (2,18,19), which likely reflects a high level of evolutionary conservation within the region of subunit a affected by these mutations. These observations constitute a preliminary validation of the use of yeast to model human inherited mitochondrial diseases affecting ATP synthase.…”

Enhancement of mitochondrial ATP production by the Escherichia coli cytotoxic necrotizing factor 1

“…Complex V activity (in the direction of ATP synthesis) was measured in isolated mitochondria using reported spectrophotometric methods and, as substrate, either succinate (that energized mitochondria from complex II) or malate (that energized mitochondria from complex I). As a control, we routinely analyzed complex V activity by incubating mitochondria with oligomycin as reported by Vazquez‐Memije and coworkers . For the ATP synthesis activity, the experiments were performed 15 times using succinate and six times using malate.…”

Episodic weakness due to mitochondrial DNA MT-ATP6/8 mutations

“…The deleterious potential of mutations m.9176T.C and m. 8993T.C has been demonstrated in cybrids. 25,26 Mutation MT-ATP8 m.8403T.C p.Ile13Thr modifies an isoleucine that is conserved in all mammals and is almost always a hydrophobic amino acid in other species. It was considered deleterious by all usual prediction software (SIFT, GVGD, and Polyphen).…”