Figure 3.

V₁H_chim and C associate with V_oND to form coupled V₁V_o-ATPase. A, V_oND was immobilized on streptavidin-coated BLI sensors via biotinylated MSP (step 1). Sensors were then dipped into 0.4 μm of V₁ mutants in presence of 1 μm C (association; step 2) followed by buffer (dissociation; step 3). Association with V_oND was most efficient with V₁H_chim (red trace). Sensors were then dipped in PreScission protease to verify that the BLI signal was not due to nonspecific binding (step 4). Inset shows an enlarged view of the association and dissociation steps. B, equimolar amounts of V₁H_chim and V_oND, and a 2-fold molar excess of C subunit were incubated at 22 °C, and the ConA-sensitive MgATPase activity was measured as a function of time. Each point represents the mean ± S.E. of two separate reconstitutions from two individual purifications. Inset, specific MgATPase activities of reconstituted V₁H_chimV_oND and V₁H_wtV_oND (± S.E. from two independent purifications) compared with purified V₁V_oND (29). C, following association of the V₁H_chimV_oND complex, sensors were dipped in wells containing buffer (green) or buffer + 1 mm MgATP (blue) for dissociation rate measurement. The dissociation phase of WT V₁V_oND in buffer (red) and buffer +1 mm MgATP (orange) is included for comparison (data from Ref. 29).