V-ATPase

Vacuolar-type 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.
The Archaea-type ATPase is a related group of ATPases found in archaea that often work as an ATP synthase. It forms a clade V/A-ATPase with V-ATPase. Most members of either group shuttle protons, but a few members have evolved to use sodium ions instead.

Roles played by V-ATPases

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 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, 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. In addition, other variety of biological processes, such as toxin delivery, viral entry, membrane targeting, apoptosis, regulation of cytoplasmic pH, proteolytic process, and acidification of intracellular systems, are important roles of V-ATPases.
V-ATPases also play a significant role in cell morphogenesis development. Disruption of the gene vma-1 gene which encodes for the catalytic subunit of the enzyme severely impairs the rate of growth, differentiation, and the capacity to produce viable spores in fungus Neurospora crassa.

Structure

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 V_o domain or the V₁ domain.
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 V_o 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 V-ATPase. The mammalian V_o 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 V_o 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 V_o 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 V_o 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 to aid its assembly and maturation. Vma21p coordinates assembly of the V_o subunits as well as escorting the V_o domain into vesicles for transport to the Golgi.

V₁

The V₁ domain of the V-ATPase is the site of ATP hydrolysis. Unlike V_o, the V₁ domain is hydrophilic. 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.

Subunit	Human Gene	Note
A, B	ATP6V1A, ATP6V1B1, ATP6V1B2	Catalytic hexamer.
C	ATP6V1C1, ATP6V1C2
D	ATP6V1D	Central rotor stalk, responsible for ion specificity.
E, G	ATP6V1E1, ATP6V1E2, ATP6V1G1, ATP6V1G2, ATP6V1G3
F	ATP6V1F
H	ATP6V1H

Subunit C

V-ATPase C represents the C terminal subunit that is part of the V1 complex, and is localised to the interface between the V1 and Vo complexes.

Subunit C function

The C subunit plays an essential role in controlling the assembly of V-ATPase, acting as a flexible stator that holds together the catalytic and membrane sectors of the enzyme. The release of subunit C from the ATPase complex results in the dissociation of the V1 and Vo subcomplexes, which is an important mechanism in controlling V-ATPase activity in cells. Essentially, by creating a high electrochemical gradient and low pH, this powers the enzyme to create more ATP.

Subunits E, G

These related subunits make up the stalk of A/V-ATPase. They are important in assembly, and may function as pushrods in activity. E has a cap to connect to A/B, while G does not. They likely evolved from a single protein by gene duplication.

Subunit H

, is only involved in activity and not in assembly. This subunit also acts as an inhibitor of free V1 subunits; it stops ATP hydrolysis when V1 and Vo are dissociated.

V_o

The V_o domain is responsible for proton translocation. Unlike the F-type ATP synthase, the V_o domain generally transports protons against their own concentration gradient. Rotation of the V_o domain transports the protons in movement coordinated with the V₁ domain, which is responsible for ATP hydrolysis. The V_o domain is hydrophobic and composed of several dissociable subunits. These subunits are present in the V_o domain to make this a functional proton translocase; they are described below.

Subunit	Human Gene	Note
a/I	ATP6V0A1, ATP6V0A2, ATP6V0A4
c	ATP6V0B, ATP6V0C	Ring of varied size.
d/C	ATP6V0D1, ATP6V0D2
e	ATP6V0E1, ATP6V0E2	9 kDa hydrophobic assembly protein.
AC45/S1	ATP6AP1	Accessory subunit
S2	ATP6AP2	Accessory subunit

Subunit a/I

The 116kDa subunit and subunit I are found in the Vo or Ao complex of V- or A-ATPases, respectively. The 116kDa subunit is a transmembrane glycoprotein required for the assembly and proton transport activity of the ATPase complex. Several isoforms of the 116kDa subunit exist, providing a potential role in the differential targeting and regulation of the V-ATPase for specific organelles.
The function of the 116-kDa subunit is not defined, but its predicted structure consists of 6–8 transmembranous sectors, suggesting that it may function similar to subunit a of FO.

Subunit d/C

Subunit d in V-ATPases, called subunit C in A-ATPases, is a part of the Vo complex. They fit onto the middle of the c ring, so are thought to function as a rotor. There are two versions of this subunit in eukaryotes, d/d1 and d2.
In mammals, d1 is the ubiquitously expressed version and d2 is expressed in specific cell types only.