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The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V1 domain is an ATPase, normally coupled to membrane-bound proton pump Vo via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V1 from the membrane and curtail ATP hydrolysis. To investigate the molecular basis for these processes, we have carried out cryo-electron microscopy three-dimensional reconstruction of deactivated V1 from Manduca sexta. In the resulting model, three peripheral stalks that are parts of the mechanical stator of the V-ATPase are clearly resolved as unsupported filaments in the same conformations as in the holoenzyme. They are likely therefore to have inherent stiffness consistent with a role as flexible rods in buffering elastic power transmission between the domains of the V-ATPase. Inactivated V1 adopted a homogeneous resting state with one open active site adjacent to the stator filament normally linked to the H subunit. Although present at 1:1 stoichiometry with V1, both recombinant subunit C reconstituted with V1 and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V1 that could indicate positional variability. If the position of H can vary, existing mechanistic models of deactivation in which it binds to and locks the axle of the V-ATPase rotary motor would need to be re-evaluated., Graphical abstract, Highlights • Dissociation of vacuolar H+-ATPase domains deactivates its V1 motor. • V1 has one “open” catalytic site linked to the stator filament bound by subunit H. • Movement of subunit H to prevent rotary catalysis is possible. • Three stator filaments project from deactivated V1, indicating inherent stiffness. • This work gives new insight into energetic coupling and control in V-ATPases.

Publisher Summary This chapter focuses on the isolation of goblet cell apical membrane from tobacco hornworm midgut and purification of its vacuolar-type ATPase. The only known method for isolation of the goblet cell apical membrane from larval lepidopteran midgut is described in this chapter. This chapter also described is the purification of the ATPase associated with this membrane as well as procedures for measuring both membrane-bound and purified solubilized ATPase activity. The chapter also discusses a detailed description of reliable and sensitive colorimetric methods for measurement of inorganic phosphate and protein is included. This chapter presents a discussion on the isolation of the goblet cell apical membrane. The isolated posterior midgut segment is placed lumen side down and the Malpighian tubules are removed.this chapter concludes with the discussion on Purification of the goblet cell apical membrane ATPase.