F1F0-ATP Synthase Complex Interactions In Vivo Can Occur in the Absence of the Dimer Specific Subunit e

Abstract

Evidence suggests membrane bound F(1)F(0)-ATPase complexes form stable associations such that dimers can be retrieved from detergent lysates of mitochondria isolated from a range of sources including algae, higher plants, yeast and bovine heart, and plant chloroplasts. The physiological relevance of these interactions is not clear but may be connected with the formation and structure of mitochondrial cristae. We sought to demonstrate, in vivo, the association of F(1)F(0)-ATPases in yeast cells co-expressing two b subunits each fused at its C-terminus to a GFP variant appropriate for fluorescence resonance energy transfer (FRET; BFP as the donor and GFP as the acceptor fluorophore). Both subunit b-GFP and b-BFP fusions were assembled into functional complexes. FRET was observed from enzyme complexes in molecular proximity in respiring cells providing the first demonstration of the association, in vivo, of F(1)F(0)-ATPase complexes. Moreover, FRET was observed within cells lacking the dimer specific subunit e, indicating structured associations can occur within the inner membrane in the absence of subunit e.

Structure of the yeast F 1 F o -ATP synthase dimer and its role in shaping the mitochondrial cristae

“…The assembly of ATP synthase dimers into rows has been implied to result from two different dimer geometries, one with an angle of approximately 35°and the other with an angle of approximately 90°(32) between monomers. It has been postulated that these two different dimers are formed by interaction of either subunits e and g or of subunits 4, i, a, and h (18)(19)(20)(21)(22). We show here that subunits e, g, and 4 are each essential for dimer formation.…”

“…The formation of ATP synthase dimer rows has been assumed to be protein-driven and considerable effort has been invested into finding the proteins responsible (16, 18-21, 26, 27). Subunits e and g have been widely assumed to provide the protein contacts between dimers (19)(20)(21)(22). We demonstrate here that mutants lacking subunits e or g do not have ATP synthase dimers in situ, and that subunits e, g, and su4TM1 are all essential for dimer formation in the membrane.…”

“…However, in subsequent work small amounts of ATP synthase dimers were reported in digitonin extracts of these mutants (18). Biochemical cross-linking (19)(20)(21) and FRET (fluorescence resonance energy transfer) studies (22) likewise suggested dimers of the mitochondrial ATP synthase without subunits e and g, casting further doubt on the role of these subunits in dimer formation.…”

Characterization of Domain Interfaces in Monomeric and Dimeric ATP Synthase

“…This does not mean that subunits e and g were essential for the formation of dimers in the membrane (24,25). It just means that dimeric/ oligomeric ATP synthase is stabilized by subunits e and g so that dimeric/oligomeric ATP synthase can be isolated under the conditions of BN-PAGE.…”

“…This term is potentially misleading because low amounts of subunit g and/or subunit e can be isolated with monomeric yeast ATP synthase following solubilization by digitonin and separation by BN-PAGE as described under "Results". The presence of subunits e and g favors dimerization but is not essential for dimerization, suggesting that other F 0 -proteins are also involved (24,25). Cross-linking evidence for the involvement of subunits h, i, and b in supporting the dimerization interface has recently been presented (25)(26)(27).…”