Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the F(O) sector of Escherichia coli ATP synthase.
Subunit rotation within the F(1) catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded F(O) sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. F(O)F(1) was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607--6612]. Membranes were treated with N,N'-dicyclohexyl-[(14)C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a--c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little (14)C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a-c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/P(i). Furthermore, preincubation with MgADP and azide to inhibit F(1) or with high concentrations of N,N'-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.
Hutcheon
M L
ML
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.
Duncan
T M
TM
Ngai
H
H
Cross
R L
RL
D000255:Adenosine Triphosphate / Q000378:metabolism; D003432:Cross-Linking Reagents; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D004735:Energy Transfer; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases