title: ATP hydrolysis by membrane-bound Escherichia coli F0F1 causes rotation of the gamma subunit relative to the beta subunits.
abstract: We recently demonstrated that the gamma subunit in soluble F1-ATPase from Escherichia coli rotates relative to surrounding beta subunits during catalytic turnover (Duncan et al. (1995) Proc. Natl. Acad. Sci. USA 92, 10964-10968). Here, we extend our studies to the more physiologically relevant membrane-bound F0F1 complex. It is shown that beta D380C-F1, containing a beta-gamma intersubunit disulfide bond, can bind to F1-depleted membranes and can restore coupled membrane activities upon reduction of the disulfide. Using a dissociation/reconstitution approach with crosslinked beta D380C-F1, beta subunits containing an N-terminal Flag epitope (beta flag) were incorporated into the two non-crosslinked beta positions and the hybrid F1 was reconstituted with membrane-bound F0. Following reduction and ATP hydrolysis, reoxidation resulted in a significant amount of crosslinking of beta flag to the gamma subunit. This demonstrates that gamma rotates within F1 during catalytic turnover by membrane-bound F0-F1. Furthermore, the rotation of gamma is functionally coupled to F0, since preincubation with DCCD to modify F0 blocked rotation.
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse 13210, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhou
authors.forename: T M
authors.initials: TM
authors.lastname: Duncan
authors.forename: V V
authors.initials: VV
authors.lastname: Bulygin
authors.forename: M L
authors.initials: ML
authors.lastname: Hutcheon
authors.forename: R L
authors.initials: RL
authors.lastname: Cross
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D006868:Hydrolysis; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D012399:Rotation
chemical_list: D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases