Rotation of the proteolipid ring in the V-ATPase

V0V1-ATPase is a proton-translocating ATPase responsible for acidification of eukaryotic intracellular compartments and for ATP synthesis in archaea and some eubacteria. We demonstrated recently the rotation of the central stalk subunits in V-1, a catalytic sector of V0V1-ATPase (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315), but the rotation of the proteolipid ring, a predicted counterpart rotor in the membrane V-0 sector, has remained to be proven. V0V1-ATPase that retained sensitivity to N',N'-dicyclohexylcarbodiimide was isolated from Thermus thermophilus, immobilized onto a glass surface through the N termini of the A subunits of V-1, and decorated with a bead attached to a proteolipid subunit of V-0. Rotation of beads was observed in the presence of ATP, and direction of rotation was always counterclock-wise viewed from the membrane side. The rotation proceeded at similar to3.0 rev/s in average at 4 mM ATP and was abolished by N',N'-dicyclohexylcarbodiimide treatment. Thus, the rotation of the central stalk in V-1 accompanies rotation of a proteolipid ring of V-0 in the functioning V0V1-ATPase.