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 a, b, 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). Mechanisms of coupling H+ movement to ATP synthesis, based on structural data for the a and c subunits, have been proposed (Allegretti et al. 2015). The asymmetric features of the a subunit make it the master regulator determining which of the two functions, ATP production or ATP dissipation, will be performed (Nesci et al. 2015). The structure of the a subunit (5ARA) reveals an unusual two transmembrane α-helices at a 70° angle to the plane of the membrane (Tsirigos et al. 2017). The 3.6 Å structure of the yeast Fo domain has been solved by cryoEM, revealing how the protons travel through the complex, how the complex dimerizes, and how the dimers bend the membrane to produce cristae (Guo et al. 2017).
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. Several intact F-type ATPase complexes have been purified from different fungal species and analyzed for their properties and subunit comopositions after solubilization with various detergents (Liu et al. 2015).
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, but rings having anywhere from 8 protomers to 15 protomers have been reported (Watt et al., 2010). 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, pro-hormone processing, protein degradation, etc (Kane, 2006). 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 (Toei et al., 2010). 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 crystal structure of the central axis DF complex of a prokaryotic V-ATPase has been solved (Saijo et al., 2011).
By altering the pH of intracellular compartments, V-ATPases dictate enzyme activities, govern the dissociation of ligands from receptors and promote the coupled transport of substrates across membranes. In tissues where V-ATPases are expressed at the plasma membrane, they can serve to acidify the extracellular microenvironment. Additional roles that seem independent of H+ translocation include fusogenicity, cytoskeletal tethering and metabolic sensing (Maxson and Grinstein 2014). CryoEM studies have revealed conformational heterogeneity and characteristic TMSs that are highly tilted with respect to the membrane (Mazhab-Jafari and Rubinstein 2016).
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. The Vesicular proton pump (V-ATPase) in nerve terminals following exocytosis continues to pump, promoting neurotransmitter uptake via endocytosis (Tabares and Betz, 2010). 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 (Toei et al., 2010).
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). Evolution of the hexameric transmembrane ring of the eukaryotic V-ATPase proton pump has been investigated (Finnigan et al., 2012).
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). Surprisingly, Uchihashi et al. (2011), using high speed atomic force microscopy, have shown that the F1 ATPase exhibits rotary catalysis in the absence of the rotor. Post-translational modifications modulate both the F0 and F1 proteins, affecting the ATPase activity in response to different stimuli (Nesci et al. 2017).
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 halotolerant cyanobacterium, Aphanothece halophytica, contains an Na+-dependent F1F0-ATP synthase with a potential role in salt-stress tolerance (Soontharapirakkul et al., 2011).
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.
Subunit a of the vacuolar H+-ATPases is an integral membrane 100kDa subunit, thought to contribute to and leave buried carboxyl groups on the proteolipid subunits (c, c' and c'') during proton translocation. Subunit a contains a large N-terminal cytoplasmic domain followed by a C-terminal domain containing eight transmembrane (TM) helices. TM7 contains a buried arginine residue (Arg-735) that is essential for proton transport and is located on a helical face that interacts with the proteolipid ring (Toei et al., 2011). Residues important for proton transport are located on the cytoplasmic half of TM7 and TM8 and the luminal half of TM3, TM4 and TM7. The cytoplasmic hemi-channel is located at the interface of TM7 and TM8 of subunit a and the proteolipid ring, whereas the luminal hemi-channel is located within subunit a at the interface of TM3, TM4 and TM7.
Rotary ATPases/synthases from Thermus thermophilus and Enterococcus hirae are maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover, subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently, specific lipid and nucleotide binding can be linked with distinct regulatory roles (Zhou et al., 2011).
Rotations of F1-ATPase occurs reversibly in discrete 120° steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis. Toyabe et al. (2011) found that the maximal work performed by F1-ATPase per 120° step is nearly equal to the thermodynamic maximal work that can be extracted from a single ATP hydrolysis event under a broad range of conditions. Their results support a nearly 100% free-energy transduction efficiency and a tight mechanochemical coupling.
The ability of c-rings to rotate within the F(o) complex derives from the interplay between the ion-binding sites and their nonhomogenous microenvironment (Pogoryelov et al., 2010). Pogoryelov et al. (2010) demonstrated how the thermodynamic stability of the so-called proton-locked state is maximized by the lipid membrane. By contrast, a hydrophilic environment at the a-subunit-c-ring interface appears to unlock the binding-site conformation and promotes proton exchange with the surrounding solution. Rotation thus occurs as c-subunits stochastically alternate between these environments, directionally biased by the electrochemical transmembrane gradient. Intrinsic features of the annular arrangement of c subunits, the c-ring, provides the core of the membrane-embedded FO domain. The c-ring constitution is linked to the number of ions, H+ or Na+, channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the 'molecular currency unit' of energy transfer (Nesci et al. 2016).
Allegretti et al. 2015 reported the structure of a native, active mitochondrial ATP synthase dimer, determined by single- particle electron cryomicroscopy at 6.2 Å resolution. The structure showed four long, horizontal membrane-intrinsic α-helices in the a-subunit, arranged in two hairpins at an angle of approximately 70 degrees relative to the c-ring helices. It had been proposed that a strictly conserved membrane-embedded arginine in the a-subunit couples proton translocation to c-ring rotation. A fit of the conserved carboxy-terminal a-subunit sequence places the conserved arginine next to a proton-binding c-subunit glutamate. The map shows a slanting solvent-accessible channel that extends from the mitochondrial matrix to the conserved arginine. Another hydrophilic cavity on the lumenal membrane surface defines a direct route for the protons to an essential histidine-glutamate pair. The results explain how ATP production is coupled to proton translocation (Allegretti et al. 2015).
The molecular structure of the transmembrane domain of ATP synthases is responsible for inner mitochondrial membrane bending. According to a hypothesized mechanism, ATP synthase dissociation from dimers to monomers, triggered by Ca2+ binding to F1, allows mitochondrial permeability transition pore formation at the interface between the detached monomers (Nesci 2018).
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 + Pi