Regulation and isoform function of the V-ATPases

Abstract

The vacuolar (H(+))-ATPases are ATP-dependent proton pumps that acidify intracellular compartments and, in some cases, transport protons across the plasma membrane of eukaryotic cells. Intracellular V-ATPases play an important role in normal physiological processes such as receptor-mediated endocytosis, intracellular membrane trafficking, pro-hormone processing, protein degradation, and the coupled uptake of small molecules, such as neurotransmitters. They also function in the entry of various pathogenic agents, including many envelope viruses, like influenza virus, and toxins, like anthrax toxin. Plasma membrane V-ATPases function in renal pH homeostasis, bone resorption and sperm maturation, and various disease processes, including renal tubular acidosis, osteopetrosis, and tumor metastasis. V-ATPases are composed of a peripheral V(1) domain containing eight different subunits that is responsible for ATP hydrolysis and an integral V(0) domain containing six different subunits that translocates protons. In mammalian cells, most of the V-ATPase subunits exist in multiple isoforms which are often expressed in a tissue specific manner. Isoforms of one of the V(0) subunits (subunit a) have been shown to possess information that targets the V-ATPase to distinct cellular destinations. Mutations in isoforms of subunit a lead to the human diseases osteopetrosis and renal tubular acidosis. A number of mechanisms are employed to regulate V-ATPase activity in vivo, including reversible dissociation of the V(1) and V(0) domains, control of the tightness of coupling of proton transport and ATP hydrolysis, and selective targeting of V-ATPases to distinct cellular membranes. Isoforms of subunit a are involved in regulation both via the control of coupling and via selective targeting. This review will begin with a brief introduction to the function, structure, and mechanism of the V-ATPases followed by a discussion of the role of V-ATPase subunit isoforms and the mechanisms involved in regulation of V-ATPase activity.

Fig. 1. Structure and mechanism of the V-ATPase

The V-ATPase is composed of two domains, V₁ and V₀. The peripheral V₁ domain is composed of eight different subunits (A-H, shown in yellow and orange) and is responsible for ATP hydrolysis, whereas the integral V₀ domain is composed of six subunits (in yeast these are subunits a, c, c′, c″, d and e, shown in blue and grey), and is involved in proton translocation across the membrane. ATP hydrolysis drives the rotation of a central rotor, which is composed of the D, F, d and proteolipid (c, c′, c″) subunits. Subunit a possesses two hemi-channels and a crucial arginine residue (shown in red), which are required for proton translocation. The hemi-channels allow protons to reach buried glutamic acid residues (shown in green) on the proteolipid ring from the cytoplasmic side of the membrane and to leave from these sites to the luminal side of the membrane following interaction of the glutamate residues with the a subunit arginine residue. The V₁ and V₀ domain are connected by a central stalk, which is composed of subunits D, F and d, and three peripheral stalks, which are composed of subunits C, E, G and the N-terminal cytoplasmic domain of subunit a. The peripheral stalks hold the A₃B₃ hexamer stationary with respect to subunit a. The non-homologous region of subunit A, which is absent from the F₁F_O ATP synthases, is involved in reversible dissociation (see text).

Fig. 2. Regulation of V-ATPase activity by reversible dissociation

In yeast, glucose depletion initiates the reversible dissociation of the V-ATPase into a V₁ complex minus C, free subunit C and V₀. Dissociation requires an intact microtubule network whereas reassembly of V₁ back onto V₀ is mediated by the RAVE complex (composed of Skp1, Rav1 and Rav2) and aldolase. Dissociation may also be stimulated by altered interactions of the non-homologous region of subunit A with the V₀ domain. Glucose-dependent assembly of the V-ATPase is driven by activation of the Ras/cAMP/protein kinase A (PKA) pathway. The free forms of V₁ and V₀ are silenced with respect to ATP hydrolysis and passive proton translocation.