Direct Observation of Stepping Rotation of V-ATPase Reveals Rigid Coupling between V _o and V ₁ Motors

V-ATPases are rotary motor proteins which convert chemical energy of ATP into electrochemical potential of ions across the cell membrane. V-ATPases consist of two rotary motors, V _o and V ₁ , and Enterococcus hirae V-ATPase (EhV _o V ₁ ) actively transports Na ⁺ in V _o (EhV _o ) by using torque generated by ATP hydrolysis in V ₁ (EhV ₁ ). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhV _o V ₁ wild-type, aE634A, and BR350K mutants under the 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 EhV _o is the rate-limiting, wild-type and aE634A exhibited 10-pausing positions reflecting 10-fold symmetry of the EhV _o rotor and almost no backward steps. Duration time before 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 EhV _o and EhV ₁ , respectively. Distribution of duration time before forward step was well fitted by a sum of two exponential decay functions with distinct time constants. Furthermore, frequent backward steps smaller than 36° were observed. Small backward steps were also observed during long, three ATP cleavage pauses of BR350K. These results indicate that EhV _o and EhV ₁ do not share pausing positions and Na ⁺ and ATP bindings occur at different angles, and the coupling between EhV _o and EhV ₁ is not elastic but rigid.