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

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.

ATP6 Homoplasmic Mutations Inhibit and Destabilize the Human F1F0-ATP Synthase without Preventing Enzyme Assembly and Oligomerization

“…The F 1 F 0 disassembly intermediate marked with an asterisk (Fig. 8, lower panel) is compatible with those described previously for T8993G mitochondria (25)(26)(27), which mainly contain F 1 and c subunits. The detergent present in the samples (3 g of dodecyl maltoside/g of protein) probably accounts for the short times that are sufficient to observe enzyme disassembly in this experiment.…”

“…Therefore, the L156R/L156P mutations per se do not impair the assembly route of the human F 1 F 0 -ATP synthase. Our results are in apparent contrast with the large amounts of sub-assembled F 1 F 0 complexes found previously with BN-PAGE of T8993G mutant samples (25)(26)(27). However, most of those studies did not address whether ATP6 mutations directly impair enzyme assembly, because they used postmortem (26) or biopsy muscular tissues (25), which have sustained metabolic stress because of disease and tissue extraction.…”

“…In this scenario soluble F 1 and incomplete F 1 F 0 complexes would be found in mitochondria, as reported for mtDNA-less Rho 0 cells lacking ATP6 and ATP8 (9,25). Incomplete F 1 F 0 complexes have been found in postmortem heart tissue from MILS patients (26), in NARP/ MILS cybrids (27), and in skeletal muscle biopsies from these patients (25). In contrast, a normal content of F 1 F 0 subunits, including subunit 6, was found in the enzyme isolated from patient cultured fibroblasts (9).…”

“…To distinguish between them, concluding evidence on the effect of NARP/MILS mutations on F 1 F 0 assembly was limited because the reported human F 1 F 0 structural studies have been done mostly with heteroplasmic cells and provided apparently opposing results (9,(25)(26)(27)). Here we demonstrate the proper assembly of the F 1 F 0 complex in two T8993G homoplasmic human cell cultures, namely fibroblasts and cybrids, by immunoprecipitation (Fig.…”

“…Among the possible consequences of ATP6 point mutations, impaired F 1 F 0 assembly (26,27), enzyme uncoupling (11,40), and proton conduction blockade through F 0 (6, 7, 9) have all been proposed. To distinguish between them, concluding evidence on the effect of NARP/MILS mutations on F 1 F 0 assembly was limited because the reported human F 1 F 0 structural studies have been done mostly with heteroplasmic cells and provided apparently opposing results (9,(25)(26)(27)).…”