ATP HYDROLYSIS BY MEMBRANE-BOUND ESCHERICHIA-COLI F0F1 CAUSES ROTATION OF THE GAMMA-SUBUNIT RELATIVE TO THE BETA-SUBUNITS

We recently demonstrated that the gamma subunit in soluble F-1-ATPase from Escherichia coli rotates relative to surrounding beta subunits du ring catalytic turnover (Duncan et al. (1995) Proc. Natl. Acad. Sci. U SA 92, 10964-10968). Here, we extend our studies to the more physiolog ically relevant membrane-bound F0F1 complex. It is shown that beta D38 0C-F-1, containing a beta-gamma intersubunit disulfide bond, can bind to F-1-depleted membranes and can restore coupled membrane activities upon reduction of the disulfide. Using a dissociation/reconstitution a pproach with crosslinked PD380C-F-1, beta subunits containing an N-ter minal Flag epitope (beta(flag)) were incorporated into the two non-cro sslinked beta positions and the hybrid F-1 was reconstituted with memb rane-bound F-0. Following reduction and ATP hydrolysis, reoxidation re sulted in a significant amount of crosslinking of beta(flag) to the ga mma subunit. This demonstrates that gamma rotates within F-1 during ca talytic turnover by membrane-bound F0F1. Furthermore, the rotation of gamma is functionally coupled to F-0, since preincubation with DCCD to modify F-0 blocked rotation.