3.A.2 The H⁺- or Na⁺-translocating F-type, V-type and A-type ATPase (F-ATPase) Superfamily

F-type ATPases are found in eukaryotic mitochondria and chloroplasts as well as in bacteria. V-type ATPases are found in vacuoles of eukaryotes and in bacteria. A-type ATPases are found in archaea. All such systems are multisubunit complexes with at least 3 dissimilar subunits embedded as a complex in the membrane (F₀, a:b:c = 1:2:~12) and (usually) at least 5 dissimilar subunits attached to F₀ (F₁, α:β:γ:δ:ε = 3:3:1:1:1 for F-type ATPases). The eukaryotic proteins are more complicated than the bacterial enzyme complexes. The α, β, δ and F₁ hexamer (α₃β₃) comprise the stator. The rotor (which consists of the c, ε and γ subunits) is believed to rotate relative to the stator in response to either ATP hydrolysis by F₁ or proton transport through F₀. H⁺ transport and ATP synthesis may therefore be coupled mechanically. The F₁ portion of the bovine mitochondrial F-type ATPase has been solved to 2.8 Å resolution. Cross-linking studies suggest that the H⁺ channel consists of a and c subunits where the external aqueous access channel is located to the inside of the a-subunit 4-helix bundle (Schwem and Fillingame, 2006). Minimal requirements for proton translocation by the ATP synthase include a positive charge in subunit a and a weak interface between subunit a and oligomeric subunit c (Ishmukhametov et al., 2007). These enzymes can pump H⁺ or Na⁺. A Na⁺ A-type ATPase from Pyrococcus furiosus has been described (Pisa et al., 2007b). ATP binding to the ε-subunit plays a regulatory role; ATP binding may stabilize the ATPase-active form by fixing the ε-subunit into the folded conformation (Kato et al., 2007).

All eukaryotic F-type ATPases pump 3-4 H⁺ out of mitochondria, or into thylakoids of chloroplasts, per ATP hydrolyzed. Bacterial F-type ATPases pump 3-4 H⁺ and/or Na⁺ (depending on the system) out of the cell per ATP hydrolyzed. These enzymes also operate in the opposite direction, synthesizing ATP when protons or Na⁺ flow through the 'ATP synthase' down the proton electrochemical gradient (the 'proton motive force' or pmf) (Ferguson et al., 2006). V-type ATPases may pump 2-3 H⁺ per ATP hydrolyzed, and these enzymes cannot catalyze pmf-driven ATP synthesis. It has been proposed that this difference between F-type and V-type ATPases is due to a 'proton slip' that results from an altered structure in the membrane sector of V-type ATPases (Perzov et al., 2001). This probably results from duplication (intragenic and/or intergenic) of the proteolipid (c) subunit.

The structure of the membrane integral rotor ring of the proton translocating F(1)F(0)-ATPase synthase from spinach chloroplasts was determined to 3.8 A resolution by X-ray crystallography (Vollmar et al., 2009). The rotor ring consists of 14 identical protomers which are symmetrically arranged around a central pore. Comparisons to the c(11)-rotor ring of the sodium translocating ATPase from Ilyobacter tartaricus show that the conserved carboxylates involved in proton or sodium transport, respectively, are 10.6-10.8 A apart in both c-ring rotors. This finding suggests that both ATPases have the same gear distance despite their different stoichiometries. The putative proton binding site at the conserved carboxylate E61 in the chloroplast ATP synthase differs from the sodium binding site in Ilyobacter. Residues adjacent to the conserved carboxylate show increased hydrophobicity and reduced hydrogen bonding. The crystal structure reflects the protonated form of the chloroplast c-ring rotor. Vollmar et al., 2009 proposed that upon deprotonation, the conformation of E61 is changed to another rotamer and becomes fully exposed to the periphery of the ring. Reprotonation of E61 by a conserved arginine in the adjacent a subunit returns the carboxylate to its initial conformation.

Phylogenetic clustering of the integral membrane constituents of F-type ATPases generally corresponds to the phylogenies of the organisms of origin, and consequently the systems in different organisms are probably orthologues. The a subunit of F₀ (one copy per complex) spans the membrane five or six times. The b subunits (2 copies per complex; heterodimeric in plant chloroplasts and blue green bacteria) span the membrane once; and the c subunits (called DCCD-binding lipoproteins; reportedly 10, 11, 12, or 14 copies per complex depending on the system) span the membrane two times. Some F-type ATPases such as the Na⁺-translocating ATPase of Acetobacterium woodii probably contains 3 dissimilar but homologous c-subunit proteolipids of 8 and 18 kDa. The V-type ATPase of S. cerevisiae also has 3 dissimilar c-subunits as mentioned in the next paragraph. While c-subunits in the E. coli F-ATPase have 2 TMSs (one acive site asp per subunit and 12 copies per complex), V-type ATPases have 4 TMSs (one active site glu per subunit and 6 copies per complex), and an archaeal A-ATPase has 6 TMSs (2 active site glus per subunit and 4 subunits per complex).

The α, β and c-subunits of F-type ATPases are homologues to the B, A and c- (or K-) subunits of V-type and A-type ATPases, respectively. Other subunits in these protein complexes are probably homologous to each other, but this fact cannot always be demonstrated by statistical analyses of the sequences. Thus, for the A-type ATPase of Methanosarcina mazei, the V-type ATPase of yeast, and the F-type ATPase of E. coli, respectively, the following subunit equivalences have been suggested: A = Vma1 (A) = β; B = Vma2 (B) = α; C = Vma6 (d) = no E. coli F-type ATPase equivalent; Vma8 (D) = γ; Vma4 (E) = δ; F = Vma7 (F) = ε; I = Vphl/stvl = a+b ?, and K = Vma3 (c) = c. Additionally, the yeast v-type ATPase has 3 dissimilar c-subunits: Vma3(c), Vmal1(c') and Vma6(c'), and three subunits, Vma13(H), Vma5(c) and Vma10(G) which are not found in either the A- or F-type ATPases. All of the yeast vacuolar ATPase subunits have an equivalent subunit in the V-type ATPases of clathrin-coated vesicles of higher eukaryotes.

Eukaryotic V-type ATPases acidify Golgi-derived vesicles, clathrin-coated vesicles, synaptic vesicles, liposomes, and plant vacuoles and function in protein trafficking, receptor-mediated endocytosis, neurotransmitter release, pH regulation, waste management, etc (Kane, 2006). There are 13 subunits, 8 (A-H) in V₁ and 5 (a, c, c', c'', d and e). The c-subunits are arranged in a ring with the a-subunit on the outside of the ring. The proton channel may be at the a/c interface, and c rotates relative to a when ATP is hydrolyzed and H⁺ is translocated. Rotation of V-type ATPases has been demonstrated (Imamura et al., 2003). Two important mechanisms of regulating V-ATPase activity in vivo are reversible dissociation of the V₁ and V₀ domains and changes in coupling efficiency of proton transport and ATP hydrolysis (Cipriano et al., 2008). V-type ATPase proteolipids can form symmetrical 6-membered rings as is true for gap junction sheets from Nephrops norvegicus which are formed by a protein identical to the 4 TMS V-ATPase c-subunit. A 14th subunit, Ac45, associated with V₀, is found in some mammalian tissues.

The V-ATPase ring contains three different subunits, c, c' and c'' and is therefore probably asymmetric if two or all three are present. In the yeast vacuolar ATPase, C (VMA3p) and C' (VMA11p) are 4 TMS proteins with N- and C-termini in the vacuolar lumen, but C'' (VMA16p) is a 5 TMS protein with N-terminus in the cytosol and the C-terminus in the lumen (Flannery et al., 2004). Althought TMHMM predicts 7TMSs for the a subunit (P32563) of the yeast (S. cerevisiae) V-type ATPase, Wang et al (2008) provide evidence for an eight transmembrane helix model in which the C-terminus is located on the cytoplasmic side of the membrane. By angular reconstitution from electron microscopic images, a 21 Å resolution structure shows an asymmetric protein ring with two small openings on the lumenal side and one large opening on the cytoplasmic side. The central hole on the lumenal side is covered by a globular protein while the cytoplasmic opening is covered by two elongated proteins (Wilkens and Forgac, 2001).

In the reversible coupled rotatory mechanism for ATP-hydrolysis driven H⁺ transport, the coupling scheme is now basically complete. During rotation of single molecules of F₁, phosphate release drives the last 40° of the 120° step, and the 40° rotation accompanies reduction of the affinity for phosphate. Moreover, the affinity for ADP decreases with rotation, and thus, ADP release contributes part of energy for rotation (Adachi et al., 2007; Senior, 2007).

The rotary proton- and sodium-translocating ATPases are reversible molecular machines present in all cellular life forms that couple ion movement across membranes with ATP hydrolysis or synthesis. Sequence and structural comparisons of F- and V-type ATPases have revealed homology between their catalytic and membrane subunits, but not between the subunits of the central stalk that connects the catalytic and membrane components. It has been proposed that these ATPases originated from membrane protein translocases which evolved from RNA translocases (Mulkidjanian et al., 2007). The Na⁺-pumping ATP synthase of Acetobacterium woodii has an unusual feature: its membrane-embedded rotor is a hybrid made of F and V-like subunits in a stoichiometry of 9:1, apparently not variable with the growth conditions (Schmidt et al., 2009).

Bacterial operons for F(1)F(0)-ATP synthase typically include an uncI gene encodes a small hydrophobic protein. When Suzuki et al. (2007) expressed a hybrid F(1)F(0) (F(1) from thermophilic Bacillus PS3 and Na⁺-translocating F(0) from Propionigenium modestum) in E. coli cells, they found that uncI derived from P. modestum was indispensable to produce active enzyme; without uncI, c-subunits in F(1)F(0) existed as monomers but not as a functional c(11)-ring. A plasmid containing only uncI and the c-subunit gene produced c(11)-ring, but a plasmid containing only the c-subunit gene did not. Direct interaction of the UncI protein with c-subunits was demonstrated. Na⁺ induced dissociation of His-tagged UncI protein from the c(11)-ring but not from c-monomers. Thus, UncI is a chaperone-like protein that assists the c(11)-ring assembly from the c-monomers in the membrane (Suzuki et al., 2007). Ozaki et al., (2008) have confirmed these observations.

The prokaryotic V-ATPase of Enterococcus hirae, closely related to the eukaryotic enzymes, provides a unique opportunity to study ion translocation by V-ATPases because it transports Na⁺ ions, which are easier to detect by x-ray crystallography and radioisotope experiments (Murata et al., 2008). The purified rotor ring (K-ring) of the E. hirae V-ATPase binds one Na⁺ ion per K-monomer with high affinity, which is competitively inhibited by Li⁺ or H⁺. The K-ring structure was determined at 2.8 A in the presence of Li⁺. Association and dissociation rates of the Na⁺ to and from the purified K-ring were extremely slow compared with the Na⁺ translocation rate estimated from the enzymatic activity, suggesting that interaction with the stator subunit (I-subunit) is essential for Na⁺ binding to and release from the K-ring.

The generalized transport reaction for F-type, V-type and A-type ATPases is:

nH⁺ (in) [or nNa⁺ (in)] + ATP nH⁺ (out) [or nNa⁺ (out)] + ADP + P_i.

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