Abstract
We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before. Specific removal of subunits alpha, beta, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the gamma-alpha(3)beta(3) rotor-stator interface. Loss of two F(1)-parts from dimeric ATP synthase led to the isolation of a dimeric subcomplex containing membrane and peripheral stalk proteins thus identifying the membrane/peripheral stalk sectors immediately as the dimerizing parts of ATP synthase. Almost all subunit a was found associated with a ring of 10 c-subunits in two-dimensional blue native/SDS gels. We therefore postulate that c10a1-complex is a stable structure in resting ATP synthase until the entry of protons induces a breaking of interactions and stepwise rotation of the c-ring relative to the a-subunit in the catalytic mechanism. Dimeric subunit a was identified in SDS gels in association with two c10-rings suggesting that a c10a2c10-complex may constitute an important part of the monomer-monomer interface in dimeric ATP synthase that seems to be further tightened by subunits b, i, e, g, and h. In contrast to the monomer-monomer interface, the interface between dimers in higher oligomeric structures remains largely unknown. However, we could show that the natural inhibitor protein Inh1 is not required for oligomerization.
Highlights
We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before
Dimeric ATP synthase was solubilized from mitochondrial membranes using low Triton X-100/protein ratios (0.5– 0.6 g/g as described in Ref. 13), separated by preparative blue native (BN) gels, and extracted by native electroelution [29] for Tricine-SDS-PAGE [30] and doubled SDS-PAGE analysis [31]
Selective Dissociation of Stator Subunits ␣, , h, and oligomycin sensitivity conferring protein (OSCP) from ATP Synthase—To identify the most labile subunits and domains of yeast ATP synthase, the multiprotein complex was isolated as a dimer by 1-D BN-PAGE (dimeric complex V (VD); Fig. 1A) and subsequently disassembled under very mild conditions namely by 2-D BN-PAGE using the common cathode buffer for BN-PAGE but supplemented with 0.02% dodecyl -D-maltoside (DDM)
Summary
We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before. Mitochondrial F1F0-ATP synthase from yeast and mammals is commonly isolated as a catalytically functional monomeric complex (9 –12), but several lines of evidence suggested that this complex is dimeric in the membrane [13, 14] or even oligomeric [15,16,17]. The term “dimer-specific subunits” originated historically from the presence of subunits e and g in dimeric yeast ATP synthase and a complete lack of these subunits in the monomeric ATP synthase isolated by blue native (BN)1-.
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