Direct observation of stepping rotation of V-ATPase reveals rigid component in coupling between Vo and V1 motors.

ABSTRACT

V-ATPases are rotary motor proteins that convert the chemical energy of ATP into the electrochemical potential of ions across cell membranes. V-ATPases consist of two rotary motors, Vo and V1, and Enterococcus hirae V-ATPase (EhVoV1) actively transports Na+ in Vo (EhVo) by using torque generated by ATP hydrolysis in V1 (EhV1). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhVoV1 wild-type, aE634A, and BR350K mutants under various Na+ and ATP concentrations ([Na+] and [ATP], respectively) by using a 40-nm gold nanoparticle as a low-load probe. When [Na+] was low and [ATP] was high, under the condition that only Na+ binding to EhVo is rate limiting, wild-type and aE634A exhibited 10 pausing positions reflecting 10-fold symmetry of the EhVo rotor and almost no backward steps. Duration time before the forward steps was inversely proportional to [Na+], confirming that Na+ binding triggers the steps. When both [ATP] and [Na+] were low, under the condition that both Na+ and ATP bindings are rate limiting, aE634A exhibited 13 pausing positions reflecting 10- and 3-fold symmetries of EhVo and EhV1, respectively. The distribution of duration time before the forward step was fitted well by the sum of two exponential decay functions with distinct time constants. Furthermore, occasional backward steps smaller than 36° were observed. Small backward steps were also observed during three long ATP cleavage pauses of BR350K. These results indicate that EhVo and EhV1 do not share pausing positions, Na+ and ATP bindings occur at different angles, and the coupling between EhVo and EhV1 has a rigid component.