Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L

Abstract

Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.

The champions' mitochondria: is it genetically determined? A review on mitochondrial DNA and elite athletic performance

“…The function of subunit A6L is still unclear (36). Recent work by Wittig et al (82) suggests that both proteins ATPase6 and A6L are important for the stabilization of ATP synthase dimer/oligomers.…”

Assessing Actual Contribution of IF1, Inhibitor of Mitochondrial FoF1, to ATP Homeostasis, Cell Growth, Mitochondrial Morphology, and Cell Viability

“…When cells are deprived of oxygen and glucose, IF1-suppressed cells lose cellular ATP and die more rapidly than control cells, suggesting a role of IF1 in maintaining ATP concentration ([ATP]) in energy crises (9). Several groups reported that IF1 facilitates dimer formation of F o F 1 in the inner mitochondrial membranes (10 -12) and contributes to cristae formation (9,(13)(14)(15)(16)(17)(18). This profound effect of IF1 on mitochondrial morphology remains controversial (19,20).…”

Time-Delayed In Vivo Assembly of Subunit a into Preformed Escherichia coli FoF1 ATP Synthase

“…The successful time-delayed assembly of subunit a into preformed F O F 1 -a complexes in vivo supports the view that subunit a is the last subunit being integrated into the enzyme complex (23)(24)(25), thereby finally generating a functional ATP synthase. From an energetic point of view, it appears favorable that the formation of the proton-conducting unit by interaction of subunit a with the c 10 ring is the last step during assembly, ensuring that partially assembled subcomplexes do not result in proton leakage, which might be lethal for the cell.…”

“…The minimal catalytic unit stably present in the cytoplasm is composed of ␣ 3 ␤ 3 ␥ (20, 21), and complex formation of subunit ␣ with other F 1 subunits is a prerequisite for the binding of subunit ␦ to the N-terminal region of subunit ␣ (22). In the case of both thermophilic Bacillus PS3 and human 0 cells, a stable F O F 1 subcomplex lacking subunit a can be purified (23)(24)(25), clearly indicating that the F 1 subunits are associated with the subunit c ring and the peripheral stalk prior to attachment of subunit a. The analysis of partially assembled subcomplexes is a prerequisite for defining the sequence of events in the assembly process of the ATP synthase.…”