Structure, Functioning, and Assembly of the ATP Synthase in Cells from Patients with the T8993G Mitochondrial DNA Mutation: COMPARISON WITH THE ENZYME IN Rho0 CELLS COMPLETELY LACKING mtDNA

Brian H Robinson,José J Garcı́A,Isla Ogilvie,Roderick A Capaldi

doi:10.1074/jbc.275.15.11075

Brian H Robinson, José J Garcı́A + Show 2 more

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https://doi.org/10.1074/jbc.275.15.11075

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Abstract

The structure and functioning of the ATP synthase of human fibroblast cell lines with 91 and 100%, respectively, of the T8993G mutation have been studied, with MRC5 human fibroblasts and Rho(0) cells derived from this cell line as controls. ATP hydrolysis was normal but ATP synthesis was reduced by 60% in the 100% mutants. Both activities were highly oligomycin-sensitive. The levels of F(1)F(0) were close to normal, and the enzyme was stable. It is concluded that the loss of ATP synthesis is because of disruption of the proton translocation step within the F(0) part. This is supported by membrane potential measurements using the dye JC-1. Cells with a 91% mutation load grew well and showed only a 25% loss in ATP synthesis. This much reduced effect for only a 9% difference in mutation load mirrors the reduced pathogenicity in patients. F(1)F(0) has been purified for the first time from human cell lines. A partial complex was obtained from Rho(0) cells containing the F(1) subunits associated with several stalk, as well as F(0) subunits, including oligomycin sensitivity conferring protein, b, and c subunits. This partial complex no longer binds inhibitor protein.

Highlights

The structure and functioning of the ATP synthase of human fibroblast cell lines with 91 and 100%, respectively, of the T8993G mutation have been studied, with MRC5 human fibroblasts and Rho0 cells derived from this cell line as controls
ATP hydrolysis was normal but ATP synthesis was reduced by 60% in the 100% mutants
Functional Parameters of Fibroblasts with the T8993G Mutation—Two different fibroblast cell lines containing the T8993G mutation were examined in this study, one with 91% of mutant mtDNA and the second with 100%, i.e. homoplasmic for the mutation

Summary

Introduction

The structure and functioning of the ATP synthase of human fibroblast cell lines with 91 and 100%, respectively, of the T8993G mutation have been studied, with MRC5 human fibroblasts and Rho0 cells derived from this cell line as controls. ATP hydrolysis was normal but ATP synthesis was reduced by 60% in the 100% mutants It is concluded that the loss of ATP synthesis is because of disruption of the proton translocation step within the F0 part. Cells with a 91% mutation load grew well and showed only a 25% loss in ATP synthesis. A partial complex was obtained from Rho0 cells containing the F1 subunits associated with several stalk, as well as F0 subunits, including oligomycin sensitivity conferring protein, b, and c subunits. This partial complex no longer binds inhibitor protein. Depending on the percentage of mtDNA molecules with this mutation, patients present with neurogenic muscle weakness, atraxia, and retinitis pigmentosa or Leigh’s Syndrome (above 95% mutation) [20]

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