Recent Insights into the Structure, Regulation, and Function of the V-ATPases

Abstract

The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and are also present at the plasma membrane. They function in such processes as membrane traffic, protein degradation, virus and toxin entry, bone resorption, pH homeostasis, and tumor cell invasion. V-ATPases are large multisubunit complexes, composed of an ATP-hydrolytic domain (V1) and a proton translocation domain (V0), and operate by a rotary mechanism. This review focuses on recent insights into their structure and mechanism, the mechanisms that regulate V-ATPase activity (particularly regulated assembly and trafficking), and the role of V-ATPases in processes such as cell signaling and cancer. These developments have highlighted the potential of V-ATPases as a therapeutic target in a variety of human diseases.

Keywords: V-ATPase; acidification; cancer; cell signaling; membrane traffic; proton transport.

Figure 1. Structural model of the V-ATPase

Electron cryomicroscopy structure of the yeast V-ATPase with known crystal structures of individual subunits from S.cerevisiae and T. thermophilus fitted into the map. The V₁ cytosolic domain, made up of subunits A-H, is involved in ATP hydrolysis, while the integral V₀ domain, made up of subunits a, d, e, c, and c″, conducts protons across the membrane. ATP hydrolysis in the A3B3 complex drives central stalk movement (subunits D, F, and d), which in turn rotates the proteolipid ring to allow proton transport across the membrane through hemichannels located in subunit a. Subunits E, G, C and H and the N-terminal domain of subunit a make up the peripheral stalks and serve to tether V₁ to V₀. Note that subunit e is absent from the preparation used for this cryo-EM structure due to its loss during detergent extraction [7]. Figure adapted from [7].

Figure 2. Regulation of V-ATPase assembly in yeast and mammalian cells

Regulated assembly represents an important mode of regulating V-ATPase activity in cells. The reversible dissociation of the V₁ and V₀ domains occurs in response to glucose depletion in both yeast and mammalian cells and in response to molting in insect cells, likely as a means to conserve cellular stores of ATP. Assembly of V₁ and V₀ occurs during maturation of dendritic cells to aid in antigen processing and in response to EGF (epidermal growth factor). Assembly in yeast is promoted by activation of PKA (protein kinase A), interaction with aldolase and the heterotrimeric complex RAVE (Regulator of the ATPase of Vacuolar and Endosomal memranes), whereas dissociation requires catalytic activity and intact microtubules. Assembly in mammalian cells is promoted by activation of PI3K and, in dendritic cells, activation of mTORC1 (mechanistic target of rapamycin complex 1).

Figure 3. V-ATPase dependent cell signaling

A, Signaling pathways that rely on the low pH generated by the V-ATPase. Wnt and Notch signaling rely on the V-ATPase to maintain a proper pH environment in vesicles of the endocytic pathway for activation and subsequent signaling and control of gene transcription. B, Signaling pathways that rely on the V-ATPase, independent of its role in proton pumping. The V-ATPase-Ragulator complex is necessary for activation of mTORC1 and AMPK (AMP-activated protein kinase) downstream of cellular amino acids and ATP levels, respectively.

Figure 4. Functions of V-ATPase in cancer

A, The V-ATPase aids cancer cell survival, likely by its role in regulating cytoplasmic pH. Cancer cells experience an increased acid load due to enhanced glycolysis that can induce apoptosis. By transporting protons out of the cytosol, the V-ATPase helps prevent apoptosis due to this increased acid production. B, The V-ATPase may contribute to the development of drug resistance, either by promoting the transport of drugs into intracellular compartments or into the extracellular space or by preventing drug entry. C, The V-ATPase is involved in cancer cell migration, possibly through direct interaction with cytoskelet al proteins, such as actin, or by promoting the trafficking of proinvasive factors, such as EGF receptor, to the leading edge. D, V-ATPases contribute to cancer cell invasion. Upregulation of the a3 isoform occurs in several invasive cancer cell lines and is believed to localize V-ATPases to the plasma membrane, where they contribute to an acidic extracellular microenvironment that promotes the activation and activity of pH-dependent proteases, such as the cathepsins, that allow cancer cells to invade through extracellular matrix. Intracellular V-ATPases may also contribute to the activation of proteases involved in invasion.