The pathogenic m.8993 T > G mutation in mitochondrial ATP6 gene prevents proton release from the subunit c-ring rotor of ATP synthase.

The human ATP synthase is an assembly of 29 subunits of 18 different types, of which only two (a and 8) are encoded in the mitochondrial genome. Subunit a, together with an oligomeric ring of c-subunit (c-ring), forms the proton pathway responsible for the transport of protons through the mitochondrial inner membrane, coupled to rotation of the c-ring and ATP synthesis. Neuromuscular diseases have been associated to a number of mutations in the gene encoding subunit a, ATP6. The most common, m.8993 T > G, leads to replacement of a strictly conserved leucine residue with arginine (aL156R). We previously showed that the equivalent mutation (aL173R) dramatically compromises respiratory growth of Saccharomyces cerevisiae and causes a 90% drop in the rate of mitochondrial ATP synthesis. Here, we isolated revertants from the aL173R strain that show improved respiratory growth. Four first-site reversions at codon 173 (aL173M, aL173S, aL173K and aL173W) and five second-site reversions at another codon (aR169M, aR169S, aA170P, aA170G and aI216S) were identified. Based on the atomic structures of yeast ATP synthase and the biochemical properties of the revertant strains, we propose that the aL173R mutation is responsible for unfavorable electrostatic interactions that prevent the release of protons from the c-ring into a channel from which protons move from the c-ring to the mitochondrial matrix. The results provide further evidence that yeast aL173 (and thus human aL156) optimizes the exit of protons from ATP synthase, but is not essential despite its strict evolutionary conservation.

The atypical cadherin fat directly regulates mitochondrial function and metabolic state.

Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. Here, we show Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation. Proteolytic cleavage releases a soluble 68 kDa fragment (Ft(mito)) that is imported into mitochondria. Ft(mito) binds directly to NADH dehydrogenase ubiquinone flavoprotein 2 (Ndufv2), a core component of complex I, stabilizing the holoenzyme. Loss of Ft leads to loss of complex I activity, increases in reactive oxygen species, and a switch to aerobic glycolysis. Defects in mitochondrial activity in ft mutants are independent of Hippo and PCP signaling and are reminiscent of the Warburg effect.

Experimental relocation of the mitochondrial ATP9 gene to the nucleus reveals forces underlying mitochondrial genome evolution.

Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms.

Two nuclear life cycle-regulated genes encode interchangeable subunits c of mitochondrial ATP synthase in Podospora anserina.

An F(1)F(O) ATP synthase in the inner mitochondrial membrane catalyzes the late steps of ATP production via the process of oxidative phosphorylation. A small protein subunit (subunit c or ATP9) of this enzyme shows a substantial genetic diversity, and its gene can be found in both the mitochondrion and/or nucleus. In a representative set of 26 species of fungi for which the genomes have been entirely sequenced, we found five Atp9 gene repartitions. The phylogenetic distribution of nuclear and mitochondrial Atp9 genes suggests that their evolution has included two independent transfers to the nucleus followed by several independent episodes of the loss of the mitochondrial and/or nuclear gene. Interestingly, we found that in Podospora anserina, subunit c is exclusively produced from two nuclear genes (PaAtp9-5 and PaAtp9-7), which display different expression profiles through the life cycle of the fungus. The PaAtp9-5 gene is specifically and strongly expressed in germinating ascospores, whereas PaAtp9-7 is mostly transcribed during sexual reproduction. Consistent with these observations, deletion of PaAtp9-5 is lethal, whereas PaAtp9-7 deletion strongly impairs ascospore production. The P. anserina PaAtp9-5 and PaAtp9-7 genes are therefore nonredundant. By swapping the 5' and 3' flanking regions between genes we demonstrated, however, that the PaAtp9 coding sequences are functionally interchangeable. These findings show that after transfer to the nucleus, the subunit c gene in Podospora became a key target for the modulation of cellular energy metabolism according to the requirements of the life cycle.

Mitochondrial ATP synthase disorders: molecular mechanisms and the quest for curative therapeutic approaches.

In mammals, the majority of cellular ATP is produced by the mitochondrial F1F(O)-ATP synthase through an elaborate catalytic mechanism. While most subunits of this enzymatic complex are encoded by the nuclear genome, a few essential components are encoded in the mitochondrial genome. The biogenesis of this multi-subunit enzyme is a sophisticated multi-step process that is regulated on levels of transcription, translation and assembly. Defects that result in diminished abundance or functional impairment of the F1F(O)-ATP synthase can cause a variety of severe neuromuscular disorders. Underlying mutations have been identified in both the nuclear and the mitochondrial DNA. The pathogenic mechanisms are only partially understood. Currently, the therapeutic options are extremely limited. Alternative methods of treatment have however been proposed, but still encounter several technical difficulties. The application of novel scientific approaches promises to deepen our understanding of the molecular mechanisms of the ATP synthase, unravel novel therapeutic pathways and improve the unfortunate situation of the patients suffering from such diseases.

A yeast model of the neurogenic ataxia retinitis pigmentosa (NARP) T8993G mutation in the mitochondrial ATP synthase-6 gene.

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.