V-ATPase

Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved evolutionarily ancient enzyme with remarkably diverse functions in eukaryotic organisms. V-ATPases acidify a wide array of intracellular organelles and pump protons across the plasma membranes of numerous cell types. V-ATPases couple the energy of ATP hydrolysis to proton transport across intracellular and plasma membranes of eukaryotic cells. It is generally seen as the polar opposite of ATP synthase because ATP synthase is a proton channel that uses the energy from a proton gradient to produce ATP. V-ATPase however, is a proton pump that uses the energy from ATP hydrolysis to produce a proton gradient. Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved evolutionarily ancient enzyme with remarkably diverse functions in eukaryotic organisms. V-ATPases acidify a wide array of intracellular organelles and pump protons across the plasma membranes of numerous cell types. V-ATPases couple the energy of ATP hydrolysis to proton transport across intracellular and plasma membranes of eukaryotic cells. It is generally seen as the polar opposite of ATP synthase because ATP synthase is a proton channel that uses the energy from a proton gradient to produce ATP. V-ATPase however, is a proton pump that uses the energy from ATP hydrolysis to produce a proton gradient. V-ATPases are found within the membranes of many organelles, such as endosomes, lysosomes, and secretory vesicles, where they play a variety of roles crucial for the function of these organelles. For example, the proton gradient across the yeast vacuolar membrane generated by V-ATPases drives calcium uptake into the vacuole through an H+/Ca2+ antiporter system. In synaptic transmission in neuronal cells, V-ATPase acidifies synaptic vesicles. Norepinephrine enters vesicles by V-ATPase. V-ATPases are also found in the plasma membranes of a wide variety of cells such as intercalated cells of the kidney, osteoclasts (bone resorbing cells), macrophages, neutrophils, sperm, midgut cells of insects, and certain tumor cells. Plasma membrane V-ATPases are involved in processes such as pH homeostasis, coupled transport, and tumor metastasis. V-ATPases in the acrosomal membrane of sperm acidify the acrosome. This acidification activates proteases required to drill through the plasma membrane of the egg. V-ATPases in the osteoclast plasma membrane pump protons onto the bone surface, which is necessary for bone resorption. In the intercalated cells of the kidney, V-ATPases pump protons into the urine, allowing for bicarbonate reabsorption into the blood. The yeast V-ATPase is the best characterized. There are at least thirteen subunits identified to form a functional V-ATPase complex, which consists of two domains. The subunits belong to either the Vo domain (membrane associated subunits, lowercase letters on the figure) or the V1 domain (peripherally associated subunits, uppercase letters on the figure). The V1 includes eight subunits, A-H, with three copies of the catalytic A and B subunits, three copies of the stator subunits E and G, and one copy of the regulatory C and H subunits. In addition, the V1 domain also contains the subunits D and F, which form a central rotor axle. The V1 domain contains tissue-specific subunit isoforms including B, C, E, and G. Mutations to the B1 isoform result in the human disease distal renal tubular acidosis and sensorineural deafness. The Vo domain contains six different subunits, a, d, c, c', c', and e, with the stoichiometry of the c ring still a matter of debate with a decamer being postulated for the tobacco hornworm (Manduca sexta) V-ATPase. The mammalian Vo domain contains tissue-specific isoforms for subunits a and d, while yeast V-ATPase contains two organelle-specific subunit isoforms of a, Vph1p, and Stv1p. Mutations to the a3 isoform result in the human disease infantile malignant osteopetrosis, and mutations to the a4 isoform result in distal renal tubular acidosis, in some cases with sensorineural deafness. The V1 domain is responsible for ATP hydrolysis, whereas the Vo domain is responsible for proton translocation. ATP hydrolysis at the catalytic nucleotide binding sites on subunit A drives rotation of a central stalk composed of subunits D and F, which in turn drives rotation of a barrel of c subunits relative to the a subunit. The complex structure of the V-ATPase has been revealed through the structure of the M. Sexta and Yeast complexes that were solved by single-particle cryo-EM and negative staining, respectively. These structures have revealed that the V-ATPase has a 3-stator network, linked by a collar of density formed by the C, H, and a subunits, which, while dividing the V1 and V0 domains, make no interactions with the central rotor axle formed by the F, D, and d subunits. Rotation of this central rotor axle caused by the hydrolysis of ATP within the catalytic AB domains results in the movement of the barrel of c subunits past the a subunit, which drives proton transport across the membrane. A stoichiometry of two protons translocated for each ATP hydrolyzed has been proposed by Johnson. In addition to the structural subunits of yeast V-ATPase, associated proteins that are necessary for assembly have been identified. These associated proteins are essential for Vo domain assembly and are termed Vma12p, Vma21p, and Vma22p. Two of the three proteins, Vma12p and Vma22p, form a complex that binds transiently to Vph1p (subunit a) to aid its assembly and maturation. Vma21p coordinates assembly of the Vo subunits as well as escorting the Vo domain into vesicles for transport to the Golgi. The V1 domain of the V-ATPase is the site of ATP hydrolysis. This soluble domain consists of a hexamer of alternating A and B subunits, a central rotor D, peripheral stators G and E, and regulatory subunits C and H. Hydrolysis of ATP drives a conformational change in the six A|B interfaces and with it rotation of the central rotor D. Unlike with the ATP synthase, the V1 domain is not an active ATPase when dissociated.