Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and ATP synthases. The V₁ complex consumes ATP to drive rotation of a central rotor that pumps protons across membranes via the V_o complex. Eukaryotic V-ATPases are regulated by reversible disassembly of subunit C, V₁ without C, and V_O. ATP hydrolysis is thought to generate an unknown rotary state that initiates regulated disassembly. Dissociated V₁ is inhibited by subunit H that traps it in a specific rotational position. Here, we report the first single-molecule studies with high resolution of time and rotational position of Saccharomyces cerevisiae V₁-ATPase lacking subunits H and C (V₁ΔHC), which resolves previously elusive dwells and angular velocity changes. Rotation occurred in 120° power strokes separated by dwells comparable to catalytic dwells observed in other rotary ATPases. However, unique V₁ΔHC rotational features included: 1) faltering power stroke rotation during the first 60°; 2) a dwell often occurring ∼45° after the catalytic dwell, which did not increase in duration at limiting MgATP; 3) a second dwell, ∼2-fold longer occurring 112° that increased in duration and occurrence at limiting MgATP; 4) limiting MgATP-dependent decreases in power stroke angular velocity where dwells were not observed. The results presented here are consistent with MgATP binding to the empty catalytic site at 112° and MgADP released at ∼45°, and provide important new insight concerning the molecular basis for the differences in rotary positions of substrate binding and product release between V-type and F-type ATPases.

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Yanagisawa S, Bukhari ZA, Parra KJ, Frasch WD

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