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

Abstract

NARP (neuropathy, ataxia, and retinitis pigmentosa) and MILS (maternally inherited Leigh syndrome) are mitochondrial disorders associated with point mutations of the mitochondrial DNA (mtDNA) in the gene encoding the Atp6p subunit of the ATP synthase. The most common and studied of these mutations is T8993G converting the highly conserved leucine 156 into arginine. We have introduced this mutation at the corresponding position (183) of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. The "yeast NARP mutant" grew very slowly on respiratory substrates, possibly because mitochondrial ATP synthesis was only 10% of the wild type level. The mutated ATP synthase was found to be correctly assembled and present at nearly normal levels (80% of the wild type). Contrary to what has been reported for human NARP cells, the reverse functioning of the ATP synthase, i.e. ATP hydrolysis in the F 1 coupled to F 0 -mediated proton translocation out of the mitochondrial matrix, was significantly compromised in the yeast NARP mutant. Interestingly, the oxygen consumption rate in the yeast NARP mutant was decreased by about 80% compared with the wild type, due to a selective lowering in cytochrome c oxidase (complex IV) content. This finding suggests a possible regulatory mechanism between ATP synthase activity and complex IV expression in yeast mitochondria. The availability of a yeast NARP model could ease the search for rescuing mechanisms against this mitochondrial disease.

A Genetic Screen Targeted on the FO Component of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

“…Indeed, much more radical mutations at the interface between the c-ring and subunit a, like the a-L183R mutation located one helix turn from a-R186 (50), were found to have no effect on the assembly/stability of the ATP synthase. Although the a-L183R mutation severely compromises the functioning of the ATP synthase proton channel, the assembly/ stability of subunit a is unaffected (50). Altogether these findings suggest that the contact zone between subunit a and the c-ring only involves critical transient interactions confined to the region of the F O where protons are exchanged between the two subunits.…”

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

“…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.…”

“…The mutagenesis was performed on an EcoRI-BamHI fragment containing the last 38 codons of ATP6 cloned in pUC19 (plasmid pSDC9) [12]. The mutated fragment was liberated by restriction with EcoRI and SapI and ligated with pSDC14 [12] cut with the same enzymes to reconstruct a whole ATP6 gene with the S250P or L252P…”

The F1F0-ATP Synthase Complex Influences the Assembly State of the Cytochrome bc1-Cytochrome Oxidase Supercomplex and Its Association with the TIM23 Machinery

“…As ATP synthase assembly mutants often exhibit instability in their mtDNA (and hence coding capacity for key COX subunits), it could be argued that the reduced COX activity could merely be due to partial loss of the mtDNA in these strains. Reduced COX enzyme activity, however, was also recently reported in atp6 mutants, where retention of the mtDNA had been ensured by growing the cells under appropriate selective pressure, thereby suggesting that loss of the ATP synthase complex and/or enzyme activity can negatively impact the COX complex in a direct manner (40,41). Furthermore, the F 0 sector subunit Su g has also been reported to be required for maximal COX enzyme activity despite the fact that spectral analysis indicated that ⌬su g mitochondria display a normal cytochrome content (42).…”