Structure of V-ATPase from citrus fruit

We used the Legionella pneumophila effector SidK to affinity purify the endogenous vacuolar-type ATPases (V-ATPases) from lemon fruit. The preparation was sufficient for cryoelectron microscopy, allowing structure determination of the enzyme in two rotational states. The structure defines the ATP:H+ ratio of the enzyme, demonstrating that it can establish a maximum DpH of -3, which is insufficient to maintain the low pH observed in the vacuoles of juice sac cells in lemons and other citrus fruit. Compared with yeast and mamma-lian enzymes, the membrane region of the plant V-ATPase lacks subunit f and possesses an unusual config-uration of transmembrane a helices. Subunit H, which inhibits ATP hydrolysis in the isolated catalytic region of V-ATPase, adopts two different conformations in the intact complex, hinting at a role in modulating activity in the intact enzyme.