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

Abstract

The molecular pathogenic mechanism of the human mitochondrial diseases neurogenic ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome was determined in cultured human cells harboring homoplasmic T8993G/T8993C point mutations in the mitochondrial ATP6 gene, which encodes subunit 6 of the F 1 F 0 -ATP synthase. Immunoprecipitation and blue native electrophoresis showed that F 1 F 0 -ATP synthase assembles correctly in homoplasmic mutant mitochondria. The mutants exhibited a tendency to have an increased sensitivity to subsaturating amounts of oligomycin; this provided further evidence for complete assembly and tight coupling between the F 1 and F 0 sectors. Furthermore, human ATP synthase dimers and higher homo-oligomers were observed for the first time, and it was demonstrated that the mutant enzymes retain enough structural integrity to oligomerize. A reproducible increase in the proportion of oligomeric-to-monomeric enzyme was found for the T8993G mutant suggesting that F 1 F 0 oligomerization is regulated in vivo and that it can be modified in pathological conditions. Despite correct assembly, the T8993G mutation produced a 60% inhibition in ATP synthesis turnover. In vitro denaturing conditions showed F 1 F 0 instability conferred by the mutations, although this instability did not produce enzyme disassembly in the conditions used for determination of ATP synthesis. Taken together, the data show that the primary molecular pathogenic mechanism of these deleterious human mitochondrial mutations is functional inhibition in a correctly assembled ATP synthase. Structural instability may play a role in the progression of the disease under potentially denaturing conditions, as discussed.

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

“…A recent study of human (homoplasmic) NARP cells where the assembly of the ATP synthase has been thoroughly investigated led to the conclusion that this mutation has no significant effect on the assembly of the ATP synthase (45). In addition, the authors of this study provide evidence that the ATP synthase supramolecular structure is unaffected in NARP cells.…”

“…In human NARP cells the assembly of the ATP synthase was reported in several studies to be unaffected by T8993G indicating that the impaired ATP production in these cells would be caused by a local disruption of the enzyme proton channel (19,45). However, others concluded that T8993G destabilizes the ATP synthase or slows down its assembly as indicated by an increased accumulation of ATP synthase subcomplexes in the mutant cells/tissues analyzed (13,21,44).…”

“…E. coli ATP synthase was found to be very sensitive to the leucine to arginine patho-genic change induced by T8993G (at position 207 of the homologous subunit a) as evidenced by a complete loss of both ATP synthesis and ATP-driven proton pumping activities (22). However, whereas the assembly of L156R-Atp6p in human cells containing high levels, up to 100%, of T8993G was found to be unaffected (19,45), the mutated L207R-subunit a failed to insert into the bacterial ATP synthase complex (23). The more sophisticated structure of mitochondrial ATP synthase, with at least 10 subunits not present in the bacterial enzyme (24), together with important differences in subunit a and Atp6p structures (10), might be responsible for the different sensitivities of the bacterial and human ATP synthases to the leucine to arginine pathogenic change.…”

Selectivity of TMC207 towards Mycobacterial ATP Synthase Compared with That towards the Eukaryotic Homologue

“…The human ovarian cancer cell line OVCAR3 was grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 100 units/ml penicillin-streptomycin. Mitochondria and submitochondrial particles (SMPs) were isolated and prepared according to methods outlined previously (6,9,22). ATP synthesis activity by human mitochondria and mycobacterial membrane vesicles was measured as described previously (6, 9, 11).…”

Mechanisms of Mitochondrial Dysfunction in Autism

“…BNG and CNG also assess supercomplex formation. This type of analysis has been performed with muscle, platelets and fibroblasts from patients with suspected mitochondrial diseases [13][14][15][16][17][18]. For BNG, multisubunit OXPHOS enzymes bind a charged dye (Coomassie brilliant blue) which allows electrophoretic separation in the first dimension by the size of the complex.…”