CryoEM of V-ATPases: Assembly, disassembly, and inhibition

In eukaryotic cells, the internal pH of membrane-bounded compartments is tightly regulated. This control is crucial for the function of proteins within organelles and, by establishing a transmembrane proton motive force, facilitates the transport of small molecules across the membrane [1]. To control pH, protons are pumped from the cytosol into the lumen of endosomes, lysosomes, the trans-Golgi, and secretory vesicles by the enzyme complexes known as vacuolar-type adenosine triphosphatases (V-ATPases). V-ATPase activity, combined with the buffering capacity and the activities of other transporters and ion channels, determines the ultimate pH of each intracellular compartment [2]. In some specialized cells, V-ATPases also localize to the plasma membrane where they pump protons out of the cell to acidify the extracellular environment for cell-specific functions. These functions include facilitating bone resorption by osteoclasts [3,4], controlling blood pH by renal α-intercalated cells [5], allowing sperm maturation in the epididymis and vas deferens [6], and enabling tumor invasion and metastasis by some cancer cells [7]. Owing to their many physiological roles, the malfunction of V-ATPases is implicated in various diseases [8]. As reviewed recently [9], advances in electron cryomicroscopy (cryoEM) of V-ATPases, primarily with the enzyme from the yeast Saccharomyces cerevisiae, revealed fundamental principles of how these macromolecular machines hydrolyze ATP to pump protons (Figure 1A). Briefly, ATP hydrolysis in the soluble catalytic V1 region of the enzyme (subunits A3B3CDE3FG3H) induces a rotor subcomplex to rotate (subunits D, F, c8, c′, c″, d, and Voa1p). Rotation of the rotor against the a subunit within the membrane-embedded VO region (subunits a, c8, c′, c″, d, e, f, and Voa1p) drives transmembrane proton translocation (Figure 1A and B). In addition to revealing the overall structures of V-ATPases, cryoEM showed the enzyme in three rotational states, which have become known as “State 1,” “State 2,” and “State 3” [10,11]. Interpolation between these conformations of the enzyme illustrates the conformational changes in the V1 and VO regions of the enzyme that are needed for rotary catalysis. In the past few years, continued advances have begun to allow examination of the structures of V-ATPases from animals and plants, the structural bases for assembly and regulation of activity, and how inhibitors of V-ATPases bind to the enzyme to block its activity.

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