Molecular Basis of the Pathogenic Mechanism Induced by the m.9191T>C Mutation in Mitochondrial ATP6 Gene.

Xin Su,Alain Dautant,François Godard,Jean-Paul di Rago,Marine Bouhier,Déborah Tribouillard-Tanvier,Roza Kucharczyk,Teresa Zoladek

doi:10.3390/ijms21145083

Abstract

Probing the pathogenicity and functional consequences of mitochondrial DNA (mtDNA) mutations from patient’s cells and tissues is difficult due to genetic heteroplasmy (co-existence of wild type and mutated mtDNA in cells), occurrence of numerous mtDNA polymorphisms, and absence of methods for genetically transforming human mitochondria. Owing to its good fermenting capacity that enables survival to loss-of-function mtDNA mutations, its amenability to mitochondrial genome manipulation, and lack of heteroplasmy, Saccharomyces cerevisiae is an excellent model for studying and resolving the molecular bases of human diseases linked to mtDNA in a controlled genetic background. Using this model, we previously showed that a pathogenic mutation in mitochondrial ATP6 gene (m.9191T>C), that converts a highly conserved leucine residue into proline in human ATP synthase subunit a (aL222P), severely compromises the assembly of yeast ATP synthase and reduces by 90% the rate of mitochondrial ATP synthesis. Herein, we report the isolation of intragenic suppressors of this mutation. In light of recently described high resolution structures of ATP synthase, the results indicate that the m.9191T>C mutation disrupts a four α-helix bundle in subunit a and that the leucine residue it targets indirectly optimizes proton conduction through the membrane domain of ATP synthase.

Highlights

Mitochondria provide aerobic eukaryotes with ATP though the process of oxidative phosphorylation (OXPHOS) [1]
The leucine residue at position 222 of human subunit a that is changed into proline by the m.9191T>C mutation corresponds to aL242 in the mature yeast protein (252 in unprocessed yeast subunit a) (Table 1)
Arabidopsis thaliana (At) the time we found that an equivalent of the m.9191T>C mutation of the mitochondrial ATP6 gene strongly compromises yeast ATP synthase assembly [18], complete structures of this enzyme were not yet available, and it was difficult to understand the molecular basis of this effect

Summary

Introduction

Mitochondria provide aerobic eukaryotes with ATP though the process of oxidative phosphorylation (OXPHOS) [1]. Devastating human neuromuscular disorders like neuropathy ataxia retinitis pigmentosa (NARP) and maternally inherited Leigh syndrome (MILS) have been associated to specific mutations in the mitochondrial ATP6 gene, which encodes the subunit a [10,11,12] In most cases these mutations co-exist with wild type mitochondrial DNA molecules (heteroplasmy), which makes it difficult to know precisely how they impact ATP synthase from patient’s cells and tissues. Its mitochondrial genome can be modified [21], and owing to the instability of heteroplasmy in this organism [22], it is possible to obtain homoplasmic clones where all the mtDNA molecules carry the same mutation We found in this way that an equivalent of the m.9191T>C mutation identified in patients presenting with MILS [23] severely compromises the assembly/stability of yeast ATP synthase and reduces by 90% the rate of mitochondrial ATP synthesis [18]. Due to the high propensity of proline residues to break α-helices, this can explain the detrimental consequences of the leucine-to-proline change on the stability/assembly of subunit a (see below)

Methods

Results

Conclusion