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

Malgorzata Rak,Emmanuel Tetaud,Stéphane Duvezin-Caubet,Nahia Ezkurdia,Maïlis Bietenhader,Joanna Rytka,Jean-Paul Di Rago

doi:10.1074/jbc.m703053200

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.

Highlights

NARP5 and MILS are mitochondrial disorders associated with point mutations of the mitochondrial DNA in the ATP6 gene encoding the Atp6p subunit of the ATP synthase
Converting Atp6p Leucine Residue 183 into Arginine Severely Compromises the Yeast Respiratory Growth—Leucine residue 156 of human Atp6p changed into arginine by the T8993G mutation corresponds to leucine residue 183 of yeast Atp6p
The MR14 respiratory growth deficiency was rescued by crossing with a synthetic ␳Ϫ strain (SDC30) containing in its mitochondria the wild type ATP6 gene only, which proved that no other genetic alteration than atp6-L183R, in nuclear or mitochondrial DNA, was involved in the expression of MR14 respiratory growth-deficiency phenotype

Summary

Introduction

In the F0, the core of the proton channel consists in a ring of 10 –12 Atp9p subunits and one Atp6p subunit (referred to as subunits c and a in Escherichia coli, respectively). Proton movements through this channel coincide with rotation of the Atp9p-ring [5,6,7,8,9], which results in conformational changes favoring ATP synthesis in the F1 [1]. The most common and studied NARP/MILS mutation is T8993G converting a highly conserved leucine residue into arginine, at Atp6p amino acid position 156 [11]. MATa ade his trp leu112 ura CAN1 arg8::HIS3 MAT␣ leu2⌬ ura ade101 arg8::URA3 kar MAT␣ leu2⌬ ura ade101 arg8::URA3 kar

Methods

Results

Conclusion