Impaired ATP Synthase Assembly Associated with a Mutation in the Human ATP Synthase Subunit 6 Gene

Leo G.J Nijtmans,Nadine S Henderson,Giuseppe Attardi,Ian J Holt

doi:10.1074/jbc.m008114200

Abstract

Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome. To enable analysis of this mutation in control nuclear backgrounds, two different cell lines were transformed with mitochondria carrying NARP mutant mitochondrial DNA. Transformant cell lines had decreased ATP synthesis capacity, and many also had abnormally high levels of two ATP synthase sub-complexes, one of which was F(1)-ATPase. A combination of metabolic labeling and immunoblotting experiments indicated that assembly of ATP synthase was slowed and that the assembled holoenzyme was unstable in cells carrying NARP mutant mitochondrial DNA compared with control cells. These findings indicate that altered assembly and stability of ATP synthase are underlying molecular defects associated with the NARP mutation in subunit 6 of ATP synthase, yet intrinsic enzyme activity is also compromised.

Highlights

IntroductionATP synthase (or complex V) is the enzyme of aerobic ATP production. It is located in the inner mitochondrial membrane of eukaryotic cells together with four respiratory chain enzymes that generate the proton motive force, which in turn drives ATP synthesis
ATP synthase is the enzyme of aerobic ATP production
MtDNA can be isolated from its host nuclear DNA, and any mitochondrial dysfunction in the transformant cells can be ascribed to mtDNA, where appropriate controls are in place

Summary

Introduction

ATP synthase (or complex V) is the enzyme of aerobic ATP production. It is located in the inner mitochondrial membrane of eukaryotic cells together with four respiratory chain enzymes that generate the proton motive force, which in turn drives ATP synthesis. One of the earliest disease-associated point mutations of mtDNA1 to be described was localized to ATP synthase subunit 6 gene, hereafter called A6 (3). Wallace and coworkers (8) use 143B osteosarcoma cells that lack mtDNA (␳o cells) as recipients of mitochondria carrying T8993G mtDNA They found that mitochondria with T8993G mtDNA, isolated from this control nuclear background, had reduced state III respiration, indicative of decreased ATP synthase activity. Muscle mitochondria harboring T8993G mtDNA were shown to contain sub-complexes of ATP synthase, raising the possibility that the underlying defect in this disease was holoenzyme assembly or stability (9). We demonstrate for the first time that mutant subunit A6 of ATP synthase is linked to impaired assembly of complex V in human cells

Methods

Results

Conclusion