FIGURE 5.

Activation of His₆-VPS4A by liposomes containing Ni²⁺-NTA-DOGS. A, effect of the indicated reagent or manipulation on ATP hydrolysis by 0.2 μm VPS4A: from left to right, liposomes containing 5% Ni²⁺-NTA-conjugated to DOGS (with molar excess of Ni²⁺-NTA compared with His₆ tag on VPS4A), liposomes containing DOPC only, Ni²⁺-NTA-Sepharose, 0.5 μm NiSO₄, VPS4A with its His₆ tag removed together with Ni²⁺-NTA containing liposomes, and VPS4A alone. B, specific activity of ATP hydrolysis by VPS4A when increasing amounts of enzyme are added to a fixed amount of liposomes containing 5% Ni²⁺-NTA-conjugated DOGS. C, effect of changing the density of Ni²⁺-NTA-DOGS present in liposomes (from 2 to 15 mol %, adjusted with varying amounts of DOPC) on VPS4A activity. D, effects of liposomes containing 5% Ni²⁺-NTA-DOGS on AAA⁺ ATPases including VPS4A, VPS4B, spastin, and NSF. Basal activity of each enzyme is shown with black bars and activity in the presence of Ni²⁺-NTA containing liposomes with gray bars. Error bars indicate S.D. from one experiment conducted in duplicate or triplicate; all experiments were repeated three times unless otherwise noted.