Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

Thomas Krebstakies,Boris Zimmermann,Peter Gräber,Karlheinz Altendorf,Michael Börsch,Jörg-Christian Greie

doi:10.1074/jbc.m506251200

Thomas Krebstakies, Boris Zimmermann + Show 4 more

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https://doi.org/10.1074/jbc.m506251200

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Abstract

In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at gammaCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9-10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly.

Highlights

Least of the two copies of subunit b [9, 10], which are supposed to undergo transient elastic deformation to compensate for the torque, which is built up by the propelling rotor [5, 11, 12]
Rates of ATP synthesis and hydrolysis catalyzed by labeled F1F0 and F1, respectively
Binding affinities have been determined in solution for subunits of the peripheral stator [20, 22, 46]

Summary

Introduction

Least of the two copies of subunit b [9, 10], which are supposed to undergo transient elastic deformation to compensate for the torque, which is built up by the propelling rotor [5, 11, 12]. In the case of the interaction of subunit b with F1, binding affinities have so far been determined only in solution by several techniques, including fluorometric tryptophan quenching [1, 22, 24] and fluorescence resonance energy transfer (FRET)6 [20] In these assays, only truncated forms of subunit b lacking the membrane part were used, thereby confusing the interpretation of the corresponding results with a rather weak dissociation constant for dimerization [20, 22]. It has previously been shown that, in the case of reconstituted F0 and its subcomplexes, all three subunits a, b, and c are necessary for the functional binding of F1 [11, 23, 27] Both rotor (subunit c) and stator (subunit b) components of F0 contribute to F1 binding in vivo. Binding of F1 to F0 and Its Subcomplexes in ATP Synthase that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme

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