3.A.3 The P-type ATPase (P-ATPase) Superfamily
Nearly all of the members of this superfamily, found in bacteria, archaea and eukaryotes, catalyze cation uptake and/or efflux driven by ATP hydrolysis. Clustering on the phylogenetic tree is usually in accordance with specificity for the transported ion(s). Many of these protein complexes are multisubunit with a large subunit serving the primary ATPase and ion translocation functions. In eukaryotes, they are present in the plasma membranes or endoplasmic reticular membranes. In prokaryotes, they are localized to the cytoplasmic membranes. Gastric H+-translocating ATPases (ouabain-insensitive) comprise a subgroup of the larger and more diverse Na+/K+ ATPase family (ouabain-sensitive) (Family 1). The ouabain-binding pocket can be functionally reconstituted in the gastric H+,K+ ATPase by substitution of only seven residues (Qiu et al., 2005). Ca2+ ATPases of prokaryotes and eukaryotes comprise a very diverse family (Family 2) including in eukaryotes plasma membrane, golgi, and sarcoplasmic reticular types. Sarcoplasmic reticular Ca2+ ATPases (SERCA) in brown adipose tissue can uncouple ATP hydrolysis from Ca2+ transport and be thermogenic (de Meis, 2003). H+-translocating P-type ATPases of plants and fungi comprise their own family (Family 3). Plant P-ATPases have been reviewed by Wdowikowska and Klobus, (2011). Distinct bacterial enzymes specific for K+ (Family 7; only in prokaryotes) or Mg2+ (Family 4, mostly in prokaryotes; uptake), Cu2+, Ag+, Zn2+, Co2+, Pb2+, Ni2+, and/or Cd2+ (Family 6; efflux) and Cu2+ or Cu+ (Family 5; uptake or efflux, depending on the system) have been characterized, and each of these types of enzymes comprises a distinct family. Cu2+ or Cu+-translocating ATPases from bacteria, archaea and animals cluster together, and at least some of these also transport Ag+. The Cu+/Ag+ (Family 5 and heavy metal (Family 6)) ATPases have an 8 TMS topology (Mandal et al., 2002). A cys-pro-cys motif in CopA of E. coli (TC #3.A.3.5.5) is essential for Cu+/Ag+ efflux and phosphoenzyme formation (Fan and Rosen, 2002).
P-type ATPases play essential roles in numerous processes, which in humans include nerve impulse propagation, relaxation of muscle fibers, secretion and absorption in the kidney, acidification of the stomach and nutrient absorption in the intestine. Published evidence suggests that uncharacterized families of P-type ATPases with novel specificities exist. Thever & Saier (2009) analyzed the fully sequenced genomes of 26 eukaryotes including animals, plants, fungi and unicellular eukaryotes for P-type ATPases. They reported the organismal distributions, phylogenetic relationships, probable topologies and conserved motifs of nine functionally characterized families and 13 uncharacterized families of these enzyme transporters. A similar but more extensive study on Prokaryotic P-type ATPase was presented by Chan et al. 2010 (see below). Family 9 Na+- or K+-ATPases can be found in fungi, plants bryophytes and protozoa (Rodríguez-Navarro and Benito, 2010). The Na+/K+-ATPase acts as a receptor for cardiotonic steroids such as ouabain. Cardiotonic steroids inhibit this enzyme's activity, resulting in modulation of Ca2+ levels, which are essential for homeostasis in neurons. Two points regarding the interplay between the Na+/K+-ATPase and Ca2+ signalling in the brain are (1) Na+/K+-ATPase impairment causes illness and neuronal death due to deficient Ca2+ signalling and (2) benefits to the brain result from modulating Na+/K+-ATPase activity. These interactions play an essential role in neuronal cell fate determination (Kinoshita et al. 2022).
Chan et al. (2010) analyzed P-type ATPases in all major prokaryotic phyla for which complete genome sequence data were available and compared the results with those for eukaryotic P-type ATPases. Topological type I (heavy metal) P-type ATPases predominate in prokaryotes (approx. tenfold) while type II ATPases (specific for Na+,K+, H+ Ca2+, Mg2+ and phospholipids) predominate in eukaryotes (approx. twofold). Many P-type ATPase families are found exclusively in prokaryotes (e.g. Kdp-type K+ uptake ATPases (type III) and all ten prokaryotic functionally uncharacterized P-type ATPase (FUPA) familes), while others are restricted to eukaryotes (e.g. phospholipid flippases and all 13 eukaryotic FUPA families) (Thever and Saier, 2009). Horizontal gene transfer has occurred frequently among bacteria and archaea, which have similar distributions of these enzymes, but rarely between most eukaryotic kingdoms, and even more rarely between eukaryotes and prokaryotes. In some bacterial phyla (e.g. Bacteroidetes, Flavobacteria and Fusobacteria), ATPase gene gain and loss as well as horizontal transfer occurred seldom in contrast to most other bacterial phyla. Some families (i.e., Kdp-type ATPases) underwent far less horizontal gene transfer than other prokaryotic families, possibly due to their multisubunit characteristics. Functional motifs are better conserved across family lines than across organismal lines, and these motifs can be family specific, facilitating functional predictions. In some cases, gene fusion events created P-type ATPases covalently linked to regulatory catalytic enzymes. In one family (FUPA Family 24), a type I ATPase gene (N-terminal) is fused to a type II ATPase gene (C-terminal) with retention of function only for the latter. Several pseudogene-encoded nonfunctional ATPases were identified. Genome minimalization led to preferential loss of P-type ATPase genes. Chan et al. (2010) suggested that in prokaryotes and some unicellular eukaryotes, the primary function of P-type ATPases is protection from extreme environmental stress conditions. The classification of P-type ATPases of unknown function into phylogenetic families provides guides for future molecular biological studies (Chan et al., 2010).
Many eukaryotic P-type ATPases are monomeric or homodimeric enzymes of the catalytic subunit that hydrolyzes ATP. They contain the aspartyl phosphorylation site and catalyzes ion transport. The Na+,K+-ATPases, the Ca2+-ATPases and the (fungal) H+-ATPases of higher organisms exhibit 10 transmembrane α helical spanners (TMSs), some of them highly tilted. Additional subunits that appear to lack catalytic activity may be present in the ATPase complex. For example, the 10 TMS catalytic α-subunit of the Na+,K+-ATPase of animals is tightly complexed to the 1 TMS β-subunit and the tissue-specific, regulatory, 1 TMS γ-subunit. The β-subunit, which may influence the activity of the α-subunit, probably functions to facilitate proper insertion of the α-subunit into the membrane, to allow proper targeting to a subcellular membrane site in post-translational processing, and to stabilize the catalytic subunit. The β-subunit can therefore be considered to be an auxiliary protein of the Na+,K+-ATPase catalytic subunit. The γ-subunit of the Na+,K+-ATPase has been reported to influence kinetic parameters and is homologous to a family of pore-forming peptides, the peptides of the phospholemman family (TC #1.A.27), and the C-subunits of V-type ATPases (TC #3.A.2). This γ-subunit is induced under stress conditions and modulates Na+,K+-ATPase activity and cell growth (Wetzel et al., 2004). The Na+, K+-ATPase can serve as a steroid hormone receptor (Schoner, 2002). Several other P-type ATPases also depend on small proteolipids, the functions of which are uncertain.
The annular lipid-protein stoichiometry in a native pig kidney Na+/K+ -ATPase preparation has been studied by [125I]TID-PC/16 labeling, giving results that indicated that the transmembrane domain of the Na+/K+ -ATPase in the E1 state is less exposed to the lipids than in the E2 state, i.e., the conformational transitions are accompanied by changes in the numbers of annular lipids but not in the affinity of these lipids for the protein (Mangialavori et al. 2011). The lipid-protein stoichiometry was 23 ± 2 (α subunit) and 5.0 ± 0.4 (β subunit) in the E1 conformation and 32 ± 2 (α subunit) and 7 ± 1 (β subunit) in the E2 conformation.
The stoichiometries of transport are sometimes known and complex. In the case of the Na+,K+-ATPases, 3 Na+ are exchanged for 2 K+ per ATP molecule hydrolyzed. The gastric H+-translocating ATPases replace H+ for K+ but with an H+/K+ stoichiometry of 2:2. Thus, although these two enzymes are ~65% identical, the Na+,K+-ATPases are electrogenic while the H+,K+-ATPases are electroneutral. Gastric H+, K+-ATPase transports 2 moles of H+ together with two H2O (two H3O+) per mole of ATP hydrolyzed in isolated hog gastric vesicles. Protons are charge-transferred from the cytosolic side to H2O in sites 2 and 1, the H2O coming from the cytosol, and H3O+ in these sites are transported into the lumen during the conformational transition from E1P to E2P (Morii et al., 2008). Ca2+ ATPases may catalyze Ca2+/K+ or Ca2+/H+ antiport. A single organism often possesses multiple isoforms of these enzymes. Caloxin-derived peptides in the micromolar range are inhibitors of plasma membrane calcium ATPases (Boutin et al. 2022).
Considerable evidence is available showing that animals have Cl- translocating, Cl- stimulating P-type ATPases. Although extensive biochemical data are available, the protein sequence of any one such Cl- ATPase has not yet been determined (Gerencser, 1993; Inagaki et al., 1996; Zeng et al., 1999). Evidence for mammalian iron-inducible, iron-transporting ATPases is also available (Baranano et al., 2000). Finally bacterial Na+-transporting P-type ATPases probably exist (Ueno et al., 2000). Evidence for a Na+-P-type ATPase has been obtained for the halotolerant cyanobacterium, Aphanothece halophytica (Wiangon et al., 2007). Thus the breadth of substrates transported by P-type ATPases is likely to be much greater than currently recognized.
The Na+,K+-ATPase acts as a signal transducer and transcription activator, modulating cell growth, apoptosis, and cell motility. A prominent binding motif linking the Na+,K+-ATPase to intracellular signaling effectors is the N-terminal tail of the Na+,K+-ATPase catalytic α-subunit which binds directly to the N-terminus of the inositol 1,4,5-trisphosphate receptor (Zhang et al., 2006). Three amino acyl residues, LKK, conserved in most species and most α-isoforms, are essential for binding. In wild-type cells, low concentrations of ouabain trigger low frequency calcium oscillations that activate NF-κB and protect from apoptosis. Thus, the LKK motif binds the inositol 1,4,5-trisphosphate receptor and triggers an anti-apoptotic calcium signal. However, the N-terminal hydrophilic region in front of the first TMS does not interact with the transported cation (Na+, K+, or Ca2+) although this first TMS does (Einholm et al., 2007). Of the P-type ATPases, only Na+, K+-ATPases are receptors that respond to endogenous cardiotonic steroids such as ouabain and marinobufagenin (steroid 'hormones') which regulate Na+ excretion and blood pressure (Liu and Xie, 2010).
P-type ATPases provide a polar transmembrane pathway, to which access is strictly controlled by coupled gates that are constrained to open alternately, thereby enabling thermodynamically uphill ion transport. Reyes and Gadsby (2006) have examined the ion pathway through the N+, K+-ATPase, a representative P-type pump, after uncoupling its extra- and intracellular gates with the marine toxin palytoxin. They found a wide outer vestibule penetrating deep into the Na+, K+-ATPase, where the pathway narrows and leads to a charge-selectivity filter. Acidic residues in this region, which are conserved to coordinate pumped ions, allow the approach of cations but exclude anions. Reversing the charge at just one of those positions converts the pathway from cation selective to anion selective. Cysteine scans from TM1 to TM6 in the Na+, K+-ATPase revealed a single unbroken cation pathway that traverses palytoxin-bound Na+,K+-pump-channels from one side of the membrane to the other (Takeuchi et al. 2008). This pathway comprises residues from TM1, TM2, TM4 and TM6, passes through ion-binding site II, and is probably conserved in structurally and evolutionarily related P-type pumps. Close structural homology among the catalytic subunits of Ca2+-, Na+, K+- and H+, K+-ATPases argues that their extracytosolic cation exchange pathways all share these physical characteristics (Reyes and Gadsby, 2006). The mechanistic details of type II ATPases, notably those for which 3-d structures are available (Na, K+-, gastri H+, K+-, Ca2+ and H+, K+-ATPase); TC Families 1,2 and 3, respectively, as well as prepared cation translocation pathways, have been discussed by Bublitz et al. (2010). P-type ATPase in several Rosaceae species including the pear have been identified (Zhang et al. 2020).
The X-ray crystal structure at 3.5 Å resolution of the pig renal Na+,K+-ATPase has been determined with two rubidium ions bound in an occluded state in the transmembrane part of the α-subunit (Morth et al., 2007). Several of the residues forming the cavity for rubidium/potassium occlusion in the Na+,K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of the sarco(endo)plasmic reticulum. The β- and γ-subunits specific to the Na+,K+-ATPase are associated with transmembrane helices αM7/αM10 and αM9, respectively. The γ-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the α-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.
The Na+,K+-ATPase can be transformed into an ion channel using pharmacological agents. Palytoxin (PTX), produced by soft coral of the genus Palythoa, binds to the ATPase with a Kd of 20 pM and creates a monovalent cation-selective channel with a single channel conductance of 10 pS (Rossini and Bigiani, 2011). The presence of external Na+ seems to be essential for channel activation (Wu et al., 2003). When the N-terminal 35 residues are removed from the ATPase, the toxin-activated channel does not exhibit a time-dependent inactivation gating at positive potentials as is characteristic of the wild-type protein. The truncated pump exhibits no electrogenic current, and the ion stoichiometry for active transport is altered. Addition of the synthetic peptide restores activity towards wild type. The N-terminal peptide therefore appears to act as an inactivation gate (similar to Shaker B channels of the VIC family (TC #1.A.1)). It may also play a critical role in determining the ion stoichiometry (Wu et al., 2003). Fluorometric studies indicate that under normal conditions, α- and β-subunits move towards each other during the E2 to E1 transition (Dempski et al., 2006).
The structures of the sarcoplasmic reticular Ca2+-ATPase have been solved at 2.6 Å resolution for the complex to which 2 Ca2+ are bound, and at 3.1 Å resolution for the complex lacking Ca2+ (Toyoshima et al., 2000; Toyoshima and Nomura, 2002). A total of eight different states of the Ca2+ -ATPase, representing the many steps in the reaction cycle, have been visualized by high resolution x-ray crystallography (Toyoshima et al., 2007; Toyoshima, 2008). The two Ca2+ are located side by side, surrounded by 4 transmembrane helices, two of which are unwound for efficient coordination geometry. There are 3 cytoplasmic domains, one, the central catalytic domain, bearing the phosphorylation site, a second bearing the adenosine nucleotide binding site, and a third of unknown function. The central domain has the same fold as haloacid dehydrogenases (Aravind et al., 1998; Stokes and Green, 2000). The Ca2+-free form shows large conformational differences from the Ca2+-bound form with the three cytoplasmic domains tightly associated to form a single headpiece and six of the ten TMSs largely rearranged. These latter rearrangements guarantee the release of external Ca2+ and create a pathway for entry of Ca2+ from the cytoplasm. ATPase activity and Ca2+ binding are cooperatively interdependent, but the two processes can be separated by mutations (Zhang et al., 2002).
Structures are available for both the E1 and E2 states of the Ca2+ ATPase showing that Ca2+ binding induces major changes in all three cytoplasmic domains relative to each other (Xu et al., 2002). Xu et al. proposed how Ca2+ binding induces conformational changes in TMS4 and 5 in the membrane domain (M) that in turn induce rotation of the phosphorylation domain (P). The nucleotide binding (N) and β-sheet (β) domains are highly mobile, with N flexibly linked to P, and β flexibly linked to M. Modeling of the fungal H+ ATPase, based on the structures of the Ca2+ pump, suggested a comparable 70º rotation of N relative to P to deliver ATP to the phosphorylation site (Kühlbrandt et al., 2002). One report suggests that this S.R. Ca2+ ATPase is homodimeric (Ushimaru and Fukushima, 2008).
Crystal structures (Gadsby, 2007) have shown that the conserved TGES loop of the Ca2+-ATPase is isolated in the Ca2E1 state but becomes inserted in the catalytic site in E2 states. Anthonisen et al. (2006) characterized the kinetics of the partial reaction steps of the transport cycle and the binding of the phosphoryl analogs BeF, AlF, MgF, and vanadate in mutants with alterations to the TGES residues. The data provide functional evidence supporting a role of Glu183 in activating the water molecule involved in the E2P → E2 dephosphorylation and suggest a direct participation of the side chains of the TGES loop in the control and facilitation of the insertion of the loop in the catalytic site. The interactions of the TGES loop furthermore seem to facilitate its disengagement from the catalytic site during the E2 → Ca2E1 transition.
Olesen et al. (2007) have described functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase. These represent the phosphoenzyme intermediates associated with (1) Ca2+ binding, (2) Ca2+ translocation and (3) dephosphorylation. They are based on complexes with a functional ATP analogue, beryllium fluoride or aluminium fluoride. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers a conformational change that leads to opening of a luminal exit pathway defined by the transmembrane segments M1 through M6. M1-M6 represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells (Olesen et al., 2007). Moller et al. (2010) reveiwed structural studies of various conformers of the Ca2+ ATPase, (SERCA1a), present in skeletal muscle. The structures corresponding to the various intermediary states. They have been obtained after stabilization with structural analogues of ATP or metal fluorides as mimicks of inorganic phosphate. It is possible to provide a detailed structural description of both ATP hydrolysis and Ca2+ transport through the membrane.
The structure of a P-type proton pump was determined by X-ray crystallography by Pederson et al., (2007). Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane. The structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement. The plasma membrane H+-ATPase, SmPHA4, negatively regulates the biosynthesis of tanshinones in Salvia miltiorrhiza.(Li et al. 2021).
Detailed high resolution x-ray structures of heavy metal P1B-type ATPases were not available prior to 2011 when Gourdon et al. (2011) reported the structure of CopA, a Cu+-ATPase from Legionella pneumophile at 3.2 Å resolution. The results provided the first in depth description of a heavy metal-translocatin P1B-type ATPase. A three-stage copper transport pathway involves several well conserved residues. A P1B-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative copper entry point at the intracellular interface. An ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site is probable (Gourdon et al. 2011).
As in other P-type ATPases, metal binding to transmembrane metal-binding sites (TM-MBS) in Cu+-ATPases is required for enzyme phosphorylation and subsequent transport. However, Cu+ does not access Cu+-ATPases in a free (hydrated) form but is bound to a chaperone protein. The delivery of Cu+ by Archaeoglobus fulgidus Cu+ chaperone CopZ to the corresponding Cu+-ATPase, CopA, has been studied (González-Guerrero and Argüello, 2008). CopZ interacted with and delivered the metal to the N-terminal metal binding domain(s) of CopA (MBDs). Cu+-loaded MBDs, acting as metal donors, were unable to activate CopA or a truncated CopA lacking MBDs. Conversely, Cu+-loaded CopZ activated the CopA ATPase and CopA constructs in which MBDs were rendered unable to bind Cu+. Furthermore, under nonturnover conditions, CopZ transferred Cu+ to the TM-MBS of a CopA lacking MBDs altogether. Thus, MBDs may serve a regulatory function without participating directly in metal transport, and the chaperone delivers Cu+ directly to transmembrane transport sites of Cu+-ATPases (González-Guerrero and Argüello, 2008). Wu et al (2008) have determined structures of two constructs of the Cu (CopA) pump from Archaeoglobus fulgidus by cryoelectron microscopy of tubular crystals, which revealed the overall architecture and domain organization of the molecule. They localized its N-terminal MBD within the cytoplasmic domains that use ATP hydrolysis to drive the transport cycle and built a pseudoatomic model by fitting existing crystallographic structures into the cryoelectron microscopy maps for CopA. The results also similiarly suggested a Cu-dependent regulatory role for the MBD.
In the Archaeoglobus fulgidus CopA (TC# 3.A.3.5.7), invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites (TM-MBSs). These bind Cu+ with high affinity in a trigonal planar geometry. The cytoplasmic Cu+ chaperone CopZ transfers the metal directly to the TM-MBSs; however, loading both of the TM-MBSs requires binding of nucleotides to the enzyme. In agreement with the classical transport mechanism of P-type ATPases, occupancy of both transmembrane sites by cytoplasmic Cu+ is a requirement for enzyme phosphorylation and subsequent transport into the periplasmic or extracellular milieu. Transport studies have shown that most Cu+-ATPases drive cytoplasmic Cu+ efflux, albeit with quite different transport rates in tune with their various physiological roles. Archetypical Cu+-efflux pumps responsible for Cu+ tolerance, like the Escherichia coli CopA, have turnover rates ten times higher than those involved in cuproprotein assembly (or alternative functions). This explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high copper environments. Structural and mechanistic details of copper-transporting P-type ATPase functionhave been described (Meng et al. 2015).
Chintalapati et al. (2008) have characterized two copper-transporting ATPases, CtrA2 and CtrA3 from Aquifex aeolicus. CtrA2 has a CPC metal-binding sequence in TM6 and a CxxC metal-binding N-terminal domain, while CtrA3 has a CPH metal-binding motif in TM6 and a histidine-rich N-terminal metal-binding domain. CtrA2 is activated by Ag+ and Cu+ and presumably transports reduced Cu+, while CtrA3 is activated by, and presumably transports, the oxidized copper ion. Both CtrA2 and CtrA3 are thermophilic proteins with an activity maximum at 75 degrees C. Electron cryomicroscopy of two-dimensional crystals of CtrA3 yielded a projection map at approximately 7 A resolution with density peaks indicating eight membrane-spanning alpha-helices per monomer. A fit of the Ca-ATPase structure to the projection map indicates that the arrangement of the six central helices surrounding the ion-binding site in the membrane is conserved, and suggests the position of the two additional N-terminal transmembrane helices that are characteristic of the heavy metal, eight-helix P(1B)-type ATPases (Chintalapati et al., 2008).
Transmembrane helices contain a cation-binding cysteine-proline-cysteine/histidine/serine (CPx) motif for catalytic activation and cation translocation. In addition, most Cu-ATPases possess the N-terminal Cu-binding CxxC motif required for regulation of enzyme activity. In cells, the Cu- ATPases receive copper from soluble chaperones and maintain intracellular copper homeostasis by efflux of copper from the cell or transport of the metal into the intracellular compartments (Migocka 2015).
The 8TMS CadA of Listeria monocytogenes (Family 6) confirs resistance to cadmium. Residues in TMS6 (Cys354 and Cys356), TMS8 (Asp692) and TMS3 (Met149) may bind Cd2+ (Wu et al., 2006). However, the two cysteine residues in the CPC motif act at different steps: Cys354 is involved in Cd2+ binding while Cys356 is involved in Cd2+ occlusion. The two equivalent cysteines in the yeast Cu2+ ATPase may also act at different steps. The conserved Glu164 may be required for Cd2+ release. Possibly two Cd2+ are involved in the reaction cycle of CadA (Wu et al., 2006). A hemerythrin-like two iron-binding domain in a P1B-type transport ATPase from Acidothermus cellulolyticus has been identified (Traverso et al., 2010).
The phospholipid translocating P-type ATPases (Family 8; TC #3.A.3.8) are found only in eukaryotes. They appear to function with β-subunits of about 400 aas with 2 TMSs. These have been functionally characterized from yeast and protozoans (TC #8.A.27; Perez-Victoria et al., 2006). These enzymes catalyze the ATP-dependent flipping of phospholipids and lysophospholipids from the outer leaflet of the cytoplasmic membrane to the inner leaflet. Residues defining phospholipid flippase substrate specificity have been identified (Baldridge and Graham, 2012). In the yeast Saccharomyces cerevisiae, Family 3.A.3.8 lipid flipping ATPases play a pivotal role in the biogenesis of intracellular transport vesicles, polarized protein transport and protein maturation. However, in mammals, these ATPases act in concert with members of the CDC50 protein family, putative beta-subunits for these ATPases, and many function as part of the vesicle-generating machinery (Paulusma and Oude Elferink, 2010). Family 8 ATPases may exert their cellular functions by combining enzymatic phospholipid translocation activity with an enzyme-independent action. The latter can involve the timely recruitment of proteins involved in cellular signalling, vesicle coat assembly and cytoskeleton regulation (van der Velden et al., 2010). The beta-subunit, CDC50A, allows the stable expression, assembly, subcellular localization, and lipid transport activity of the P4-ATPase ATP8A2 (Coleman and Molday, 2011). Residues in phospholipid specificity have been identified (Baldridge and Graham 2012).
Asymmetric phosopholipid distribution in the plasma membranes of animals is disrupted during apoptosis, exposing phosphatidylserine (PtdSer) on the cell surface. ATP11C (adenosine triphosphatase type 11C) and CDC50A (cell division cycle protein 50A) are required for aminophospholipid translocation from the outer to the inner plasma membrane leaflet due to their flippase activity. ATP11C contain caspase recognition sites, and mutations at these sites generate caspase-resistant ATP11C without affecting its flippase activity. Cells expressing caspase-resistant ATP11C do not expose PtdSer during apoptosis and are not engulfed by macrophages, suggesting that inactivation of the flippase activity is required for apoptotic PtdSer exposure. CDC50A-deficient cells displayed PtdSer on their surface and were engulfed by macrophages, indicating that PtdSer is sufficient as an 'eat me' signal. CDC50A serves as a chaparone protein for most phospholipid-flipping ATPases, targetting them (including ATP11C) to the plasma membrane (Segawa et al. 2014).
P4-ATPases mediate the translocation of phospholipids from the outer to the inner leaflet and maintain lipid asymmetry, which is critical for membrane trafficking and signaling pathways. Hiraizumi et al. 2019 reported cryo-EM structures of six distinct intermediates of the human ATP8A1-CDC50a heterocomplex at resolutions of 2.6 to 3.3 angstroms, elucidating the lipid translocation cycle of this P4-ATPase. ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site in the phosphorylation domain, accompanied by lateral shifts of the first and second transmembrane helices, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment (Hiraizumi et al. 2019).
The generalized reactions for P-type ATPases are:
nMe1 (out) + mMe2 (in) + ATP → nMe1 (in) + mMe2 (out) + ADP + Pi.
Phospholipid (outer leaflet of the membrane) + ATP → Phospholipid (inner leaflet) + ADP + Pi
Na+-, K+-ATPase (Na+ efflux; K+ uptake). Kinetic alterations due to a missense mutation in the alpha2 subunit cause familial hemiplegic migraine type 2 (Segall et al. 2004). Mutation in the γ-subunit causes renal hypomagnesemia, associated with hypocalciurea (Cairo et al., 2008). The Na/K-ATPase is an important signal transducer that not only interacts and regulates protein kinases, but also functions as a scaffold (Li and Xie, 2009). Capsazepine, a synthetic vanilloid, converts the Na, K-ATPase to a Na-ATPase (Mahmmoud, 2008a). There are alternative α- and β-subunits, α1, α2,... β1, β2,... in muscle which form α1β1, α1β2, α2β1 and α2β2, heterodimers, each with differing Na+ affinities (4-13mM) (Kristensen and Juel, 2010). α3 and β3 isoforms have also been identified. The γ-subunit is the same as TC# 1.A.27.2.1. Poulsen et al. (2010) have proposed a second ion conduction pathway in the C-terminal part of the ATPase. The two C-terminal tyrosines stabilize the occluded Na/K pump conformations containing Na or K ions (Vedovato and Gadsby, 2010). Na+, K+-ATPase mutations causing familial hemiplegic migraines type 2 (FHM2) inhibit phosphorylation (Schack et al., 2012). Salt, the vascular Na+/K+ ATPase and the endogenous glycosides, ouabain and marinobufagenin, play roles in systemic hypertension (Hauck and Frishman, 2012). Protein kinase A (PKA) phosphorylation of Ser936 (in the intracellular loop between transmembrane segments M8 and M9) opens an intracellular C-terminal water pathway leading to the third Na+-binding site (Poulsen et al., 2012). PKA-mediated phosphorylation regulates activity in vivo. Ser-938 is located (Einholm et al. 2016). E960 on the Na+-K+-ATPase and F28 on phospholemman (PLM) are critical for phospholemman (PLM) inhibition, but there is at least one additional site that is important for tethering PLM to the ATPase. Mutations in the Na+/K+-ATPase α3 subunit gene (ATP1A3) cause rapid-onset dystonia-parkinsonism, a rare movement disorder characterized by sudden onset of dystonic spasms and slow movements (Doğanli et al. 2013). The 3-d strcuture of the Na+-bound Na+,K+-ATPase at 4.3 Å resolution reveals the positions of the three Na+ ions (Nyblom et al. 2013). Mutations cause adrenal hypertension (Kopec et al. 2014) as well as alternating hemiplegia of childhood (AHC) and rapid-onset dystonia- parkinsonism (RDP) (Rosewich et al. 2014). Differences in the structures of the ouabain-, digonxin- and bufalin-bound enzyme have been reported (Laursen et al. 2015). ATPase inhibitors have been shown to be effective anti-cancer agents (Alevizopoulos et al. 2014). Cys45 in the β-subunit can be glutathionylated, regulating the activity of the enzyme (Garcia et al. 2015). ATP1A2 mutations play a role in migraine headaches (Friedrich et al. 2016). The beta2 subunit is essential for motor physiology in mammals, and in contrast to beta1 and beta3, beta2 stabilizes the Na+-occluded E1P state relative to the outward-open E2P state (Hilbers et al. 2016). Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli as well as epigenetic signals modulate the transcription of Na,K-ATPase subunits (Li and Langhans 2015). Čechová et al. 2016 have identified two cytoplasmic pathways along the pairs of TMSs, TMS3/TMS7 or TM6S/TMS9 that allow hydration of the cation binding sites or transport of cations from/to the bulk medium. Dissipation of the transmembrane gradient of K+ and Na+ due to ouabain inhibition increases Ptgs2 and Nr4a1 transcription by increasing Ca2+ influx through L-type Ca2+ channels that, in turn, leads to CaMKII-mediated phosphorylation of CREB and calcineurin-mediated dephosphorylation of NFAT, respectively (Smolyaninova et al. 2017). ZMay play a role in the development of gastric adenocarcinomas (Wang et al. 2017). Mutations F785L and T618M give rise to familial rapid onset dystonia parkonsonism by distinct mechanisms (Rodacker et al. 2006). Reacts with methylglyoxal to inhibit its activity (Svrckova et al. 2017). Accumulation of beta-amyloid (Abeta) at the early stages of Alzheimer's disease is accompanied by reduction of Na,K-ATPase functional activity. Petrushanko et al. 2016 showed that monomeric Abeta(1-42) forms a tight (Kd of 3 mμM), enthalpy-driven equimolar complex with alpha1beta1 Na,K-ATPase. Complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Abeta(1-42) is localized in the "gap" between the α- and β-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Abeta(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activities and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increases in the Abeta(1-42) level (Petrushanko et al. 2016). Two distinct phospholipids bind to two distinct sites on the ATPase, affecting activity and stability (Habeck et al. 2017). Five cysteinyl residues (C452, C456, C457, C577, and C656) serve as the cisplatin binding sites on the cytoplasmic loop connecting transmembrane helices 4 and 5 (Šeflová et al. 2018). Mutations can cause F/SHM with moderate penitrance (Prontera et al. 2018). Arginine substitution of a cysteine in transmembrane helix M8 converts the Na+,K+-ATPase to an electroneutral pump similar to the gastric H+,K+-ATPase (Holm et al. 2017). Early onset life-threatening epilepsy can be associated with ATP1A3 gene variants (Ishihara et al. 2019), and loss of Na/K pump function is the common feature of mutants that induce hyperaldosteronism (Meyer et al. 2019). Three Na+ sites are defined in the Na+-bound crystal structure of the Na+, K+-ATPase. Sites I and II overlap with two K+ sites in the K+-bound structure, whereas site III is unique and Na+ specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na+ at site III, but does not contribute to K+ coordination at sites I and II. Nielsen et al. 2019 addressed the functional role of Q925 in the various conformational states of this-ATPase by examining the mutants Q925A/G/E/N/L/I/Y both enzymatically and electrophysiologically, thereby revealing their Na+ and K+ binding properties. Q925 substitutions had minor effects on Na+ binding from the intracellular side of the membrane, but mutations Q925A and Q925G increased the apparent Na+ affinity, but caused dramatic reductions of the binding of K+ as well as Na+ from the extracellular side of the membrane. Thus, an interaction between sites III and I and a possible gating function of Q925 in the release of Na+ at the extracellular side are supported (Nielsen et al. 2019). The alpha2 Na+/K+-ATPase isoform mediates LPS-induced neuroinflammation (Leite et al. 2020). Apical periodontitis induces changes on oxidative stress parameters and increases Na+/K+-ATPase activity in adult rats (Barcelos et al. 2020). Familial hemiplegic migraine type 2 can be due to a missense mutation (L425H) in ATP1A2 (Antonaci et al. 2021). Mutations in ATP1A3 and FXYD genes (TC# 1.A.27) can cause childhood-onset schizophrenia (Chaumette et al. 2020). The ATPase plays a role in cell adhesion, motility, and migration of cancer cells (Silva et al. 2021). TMS2 moves outward as Na+ is deoccluded from the E1 conformation (Young and Artigas 2021). Kinetic properties and crystal structures of the Na+,K+-ATPase in complex with cardiotonic steroids (CTS) has revealed differences between CTS subfamilies. Ladefoged et al. 2021 found beneficial effects of K+ on bufadienolide binding in contrast to the well-known antagonism between K+ and cardenolides. Bufadienolide binding is affected by (i) electrostatic attraction of the lactone ring by a cation and (ii) the ability of a cation to stabilize and "shape" the site constituted by transmembrane helices of the alpha-subunit (αM1-6). The latter effect was due to varying coordination patterns involving amino acid residues from helix bundles αM1-4 and αM5-10. Substituents on the steroid core of a bufadienolide add to and modify the cation effects (Ladefoged et al. 2021). The Na+-K+-ATPase functions in the developing hippocampus (Shao et al. 2021). CryoEM analyses of the Na+,K+-ATPase in the two E2P states with and without cardiotonic steroids has revealed mechanistic details (Kanai et al. 2022). A conserved ion-binding site tyrosine plays a role in ion selectivity of the Na+/K+ pump (Spontarelli et al. 2022). Essential roles of the Na+K+-ATPase in ischemic pathology provide a platform for the improvement in clinical research on ischemic stroke (Zhu et al. 2022). In intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex. alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance the NKA turnover rate (Seflova et al. 2022).
3 component systems:
Na+-, K+-ATPase from α, β, γ heterotrimer of Homo sapiens
α1 (ATP1A1) (P05023)
α2 (ATP1A2) (P50993)
α3 (ATP1A3) (P13637)
β1 (ATP1B1) (P05026)
β2 (ATP1B2) (Q58I19)
β3 (ATP1B3) (P54709)
γ1 (ATP1G1) (P54710)
Putative archaeal Na+, K+ ATPase, Mac8 (encoded with methylcobalamin: coenzyme M methyltransferase; methanol-specific, a metal chaparone protein and an electron transfer protein) (Chan et al., 2010).
Putative Na+/K+ ATPase of Methanosarcina acetivorans (Q8THY0)
Na+,K+-ATPase α2 subunit, ATP1a2a or ATPA2A. Deficiency causes brain ventricle dilation and embryonic motility in zebra fish. Is essential for skeletal and heart muscle function (Doganli et al. 2012).
ATPA2 of Danio rerio (Q90X34)
Na+,K+-ATPase subunits α (837 aas) and β (302 aas) of the blood fluke ().
Na+,K+-ATPase subunits α and β of Schistosoma mansoni
H+/K+-ATPase, ATP12A or ATP1AL1 of 1039 aas. Plays a role in myocardial relaxation (Knez et al. 2014), but also functions in airway surface liquid acidification which impaires airway host defenses in cells lacking or compromised for CFTR (3.A.1.202.1) (Shah et al. 2016). This non-gastric H+/K+ ATPase (ATP12A) is expressed in mammalian spermatozoa (Favia et al. 2022).
ATP12A of Homo sapiens
Na+/K+-ATPase of 1227 aas and 10 TMSs. Involved in cell signaling, volume regulation, and maintenance of electrochemical gradients (Morrill et al. 2016).
ATPase of Paramecium tetraurelia
Silkworm nerve Na+,K+-ATPase, α-subunit of 1009 aas and 10 TMSs (77% identical to the human ortholog). and the β-subunit of 326 aas and 1 TMS (30% identical to the human homolog). This ATPase, in contrast to mamalian ATPases, has high affinity for K+, but low affinity for Na+, suggesting that the β-subunit is responsible for the difference in Na+ affinity (Homareda et al. 2017).
Na+,K+-ATPase of Bombyx mori (domestic silkworm)
ATPase, Na+/K+ transporting subunit alpha 4, ATP1A4, of 1029 aas and 10 TMSs. Alternatively spliced transcript variants, encoding different isoforms, have been identified. ATP1A4 is exclusively expressed in germ cells and sperm and is essential for male fertility as it interacts with signaling molecules in both raft and non-raft fractions of the sperm plasma membrane to regulate capacitation-associated signaling, hyperactivation, sperm-oocyte interactions, and activation. ATP1A4 activity and expression increase during capacitation, challenging the widely accepted dogma of sperm translational quiescence (Tiwari et al. 2022).
ATP1A4 of Homo sapiens
H+-, K+-ATPase (gastric; H+ efflux; K+ uptake). Two H3O+ may be transported per ATP hydrolyzed. Howeve, a cryo-electron microscope structure suggests that 1 H+ and 1 K+ are transporter per ATP hydrolyzed, providing the energy needed to generate the one million fold H+ concentration gradient effected by this enzyme (Abe et al. 2012). The detailed mechanism has been discussed, and the roles of essential residues have been proposed (Shin et al. 2011). A number of inhibitors of acid secretion have been identified, and these are of pharmacological importance (Shin et al. 2011). The catalytic alpha subunit has ten transmembrane segments with a cluster of intramembranal carboxylic amino acids located in the middle of TMSs 4, 5, 6 and 8. The beta subunit has one TMS with the N terminus in the cytoplasm. The extracellular domain of the beta subunit contains six or seven N-linked glycosylation sites. N-glycosylation is important for enzyme assembly, maturation and sorting (Shin et al. 2009). The cryo-EM structure with bound BYK99, a high-affinity member of K+-competitive, imidazo[1,2-a]pyridine inhibitors, has been solved (Abe et al. 2017).
Gastric H+-, K+-ATPase from Homo sapiens
Non-gastric H+-, K+- or NH4+-ATPase (Swarts et al., 2005; Worrell et al., 2008)
H+-, K+ or NH4+-ATPase of Rattus norvegicus (P54708)
Spiny dogfish Na+,K+-ATPase (3-d structure solved at 2.4 Å resolution, Shinoda et al., 2009). The α-subunit is 88% identical to the human Na+,K+ ATPase (TC# 3.A.3.1.1).
Na+,K+-ATPase α, β, and γ subunits of Squalus acanthias
α (1028aas; Q4H132)
β (305aas; C4IX13)
γ (94aas; Q70Q12)
H+/K+-ATPase α-subunit (1534aas) (Ramos et al., 2011)
H+/K+ ATPase of Aspergillus oryzae (Q2U3D2)
Putative Na+/K+-ATPase, Mhun_0636 (encoded in an operon with two half sized TrkA homologues, Mhun_0637 and Mhun_0638, that together may regulate the ATPase)
Mhun_0636-8 of Methanospirillum hungatei
Mhun_0636 (Q2FLJ9) Mhun_0637 (Q2FLJ8) Mhun_0638 (Q2FLJ6)
Ouabain-insensitive K+-independent Na+-ATPase ɑ-subunit, AtnA; very similar to the human ɑ-1 chain of the Na+,K+-ATPase (3.A.3.1.1) (Rocafull et al., 2011).
AtnA of Cavia porcellus (B3SI05)
P-type ATPase 13a1 of 1193 aas
ATPase 13a1 of Ricinus communis (Castor bean)
Putative Mn2+-exporting P-type ATPase of 1343 aas.
ATPase of Albugo laibachii
This protein was reviously designated the functionally uncharacterized P-type ATPase 16 (FUPA16) (Thever and Saier 2009). Probable manganese exporter by similarity.
Putative Mn2+ ATPase of Tetrahymena thermophila (Q23QW3)
P-type ATPase with N-terminal MACPF domain (TC# 1.C.39) of 1982 aas
MACPF-Mn2+ P-type ATPase of Tetrahymena thermophila
This protein was previously designated the functionally uncharacterized P-type ATPase 17 (FUPA17) (Thever and Saier 2009), but it has been shown to be a Ca2+/Mn2+-exporting ATPase designated Cation-transporting ATPase 5 (Cta5 or ATP13A2) (Furune et al. 2008).
ATPase of Schizosaccharomyces pombe (O14022)
This protein was previously designated the functionally uncharacterized P-type ATPase 18 (FUPA18 of 1491 aas) (Thever and Saier 2009). It may be a Mn2+-ATPase (by similarity).
FUPA18a of Cryptosporidium parvum (Q5CW06)
This protein was previously designated the functionally uncharacterized P-type ATPase 19 (FUPA19 of 1807 aas) (Thever and Saier 2009). The unusually large size and number of TMSs is unique to this protein. Whether this is a consequence of an artifact of sequencing is not known. It may be a Mn2+-ATPase (by similarity).
ATPase of Tetrahymena thermophilus
This protein was previously designated the functionally uncharacterized P-type ATPase 20 (FUPA20) (Thever and Saier 2009). It may be a Mn2+-exporting ATPase (by similarity).
ATPase of Tetrahymena thermophila (Q22V52)
This protein was previously designated the functionally uncharacterized P-type ATPase 21 (FUPA21 of 1372 aas) (Thever and Saier 2009). It may be a Mn2+-ATPase (by similarity).
ATPase of Thalassiosira pseudonana
This protein was previously designated the functionally uncharacterized P-type ATPase 22 (FUPA22 of 1212-2393 aas) (Thever and Saier 2009). It may be a Mn2+-exporting ATPase (by similarity).
ATPase of Cryptosporidium parvum (Q5CTJ9)
Mn2+-exporting ATPase, ATP13A1 of 1204 aas. Defects cause Mn2+-dependent neurological disorders. Orthologous to the yeast Mn2+-ATPase, Spf1 (Cohen et al. 2013). It is present in the endoplasmic reticulum while the other P5 ATPases, A2 - A5, are in overlapping compartments of the endosomal system (Sørensen et al. 2018). It complements the yeast ER ATPase, SPF1 (TC#3.A.3.10.3) although ATP13A2 - 5 do not, and unlike these latter proteins, it seems to have 12 (rather than 10) TMSs, with the two extra ones in an N-terminal domain (Sørensen et al. 2018). ATP13A1 (Spf1 in yeast) directly interacts with the TMSs of mitochondrial tail-anchored proteins (McKenna et al. 2020). P5A-ATPase mediates the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo-electron microscopy structures of Saccharomyces cerevisiae Spf1 (TC# 3.A.3.10.3) revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an alpha-helical TMS. Thus, the P5A-ATPase can dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TMS dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis (McKenna et al. 2020). It has been designated as a transmembrane islocase (Dederer and Lemberg 2021).
ATP13A1 of Homo sapiens
Zebrafish ATP13A2 (Parkinson''s disease protein) is essential for embryonic survival (Lopes da Fonseca et al. 2013). A missense variant in Australian Cattle Dogs give rise to late onset neuronal ceroid lipofuscinosis (Schmutz et al. 2019).
ATP13A2 of Danio rerio (Q7SXR0)
Probable divalent cation transporting ATPase 13A4, ATP13A4, of 1196 aas and 10 TMSs. This protein had been suggested to be a Mg2+ transporter, but the evidence is equivocal (Schäffers et al. 2018). It may be a Mn2+/Ca2+ exporter. This protein as well as ATP13A2 has been implicated in Parkinson's disease and autism spectrum disorder (Sørensen et al. 2018). ATPA2 - 5 are all in compartments of the endosomal system and all have 10 TMSs with overlapping functions, often in different amounts in different tissues (Sørensen et al. 2018).
ATP13A4 of Homo sapiens
Divalent cation transporting ATPase of 1207 aas and 9 putative TMSs, Catp-6. C. elegans has three paralogues, Catp5, Catp6 and Catp7, with overlapping tissue expression patterns and functions (Zielich et al. 2018).
Catp-5 of Caenorhabditis elegans
Manganese transporter of 1179 aas and 12 probable TMSs (Ticconi et al. 2004). Mediates manganese transport into the endoplasmic reticulum. The ATPase activity is required for cellular manganese homeostasis. Plays an important role in pollen and root development through its impact on protein secretion and transport processes (Jakobsen et al. 2005). Functions together with LPR1 and LPR2 in a common pathway that adjusts root meristem activity to phosphate availability (Ticconi et al. 2009).
PDR2 of Arabidopsis thaliana (Mouse-ear cress)
The endoplasmic reticular ATPase, Spf1 or Cod1. Plays a role in ER Mn2+ homeostasis by pumping Mn2+ into the ER lumen (Cronin et al., 2002; Cohen et al. 2013). Deletion of the gene results in ER stress and lowered Mn2+ in the ER lumen (Cohen et al. 2013). This SPF1-ATPase is a transmembrane helix dislocase, used to remove some mislocated TM proteins from the outer membranes of mitochondria (McKenna et al. 2020). Spf1p exhibits unique structures at its N-terminus, including two putative additional transmembrane domains, and a large insertion connecting the P domain with transmembrane segment M5 (Petrovich et al. 2021). The Spf1p P5A-ATPase "arm-like" domain is not essential for ATP hydrolysis but its deletion impairs autophosphorylation (Grenon et al. 2021).
Spf1 of Saccharomyces cerevisiae (P39986)
P-type ATPase of 1308 aas
ATPase of Babesia equi
P-type ATPase of 1291 aas
ATPase of Cryptosporidium parvum
Putative Mn2+-exporting P-type ATPase of 1146 aas.
APase of Encephalitozoon cuniculi (Q8SRH4)
This protein, ATP13A2, was orginally designated the functionally uncharacterized P-type ATPase, FUPA13 (Thever and Saier 2009). It is the Parkinson''s disease (PD) gene product, PARK9 (ATP13A2), and its defect gives rise to multiple abnormalities (Dehay et al. 2012). It is similar to the probable manganese exporter in yeast, Ypk1 (TC# 3.A.3.10.8), and may have the same function, but in lysosomes, it is a polyamine (spermine/spermidine) exporter (van Veen et al. 2020). Toxic levels of manganese or abnormal levels of polyamines may cause a syndrome simiilar to PD (Chesi et al. 2012). Manganese homeostasis in the nervous system has been reviewed (Chen et al. 2015). The progression of PD may involve the lysosome and different autophagy pathways (Gan-Or et al. 2015). It exhibits an activity-independent scaffolding role in trafficking/export of intracellular cargo in response to proteotoxic stress (Demirsoy et al. 2017). Mutations cause rare early onset Parkinson's disease (Suleiman et al. 2018). ATP13A2 modulates astrocyte-mediated neuroinflammation via NLRP3 inflammasome activation, thus bringing to light a direct link between astrocyte lysosomes and neuroinflammation in the pathological model of PD (Qiao et al. 2016). ATP13A2 and its close homologs, collectively known as P5B-ATPases, are polyamine transporters in endo-/lysosomes. Cryo-EM structures of human ATP13A2 in five distinct conformational intermediates, which together, represent a near-complete transport cycle of ATP13A2, have been determined. The structural basis of the polyamine specificity was revealed by an endogenous polyamine molecule bound to a narrow, elongated cavity within the transmembrane domain. The structures show an atypical transport path for a water-soluble substrate, in which polyamines may exit within the cytosolic leaflet of the membrane (Sim et al. 2021). Spermine is exported from the lysosome. The transmembrane domain serves as a substrate binding site, and the C-terminal domain is essential for protein stability and may play a regulatory role (Chen et al. 2021). The carcinogenic effects of ATP13A2 in different tumors has been studied (Zheng and Li 2021). High-resolution cryo-EM structures of human ATP13A2 in five distinct conformational intermediates have been determined, which together, represent a near-complete transport cycle of ATP13A2. The structural basis of the polyamine specificity was revealed by an endogenous polyamine molecule bound to a narrow, elongated cavity within the transmembrane domain. The structures show an atypical transport path for a water-soluble substrate, in which polyamines may exit within the cytosolic leaflet of the membrane (Sim et al. 2021). Mutations in ATP13A2 aare associated with mixed neurological presentations and iron toxicity due to nonsense-mediated decay (Kırımtay et al. 2021). The importance of the protein in regulating neuronal integrity has been established, and the structural dynamics and catalytic mechanism have been proposed (Mateeva et al. 2021).
PARK9 of Homo sapiens
This protein, P5B-ATPase, was originally designated the functionally uncharacterized P-type ATPase 14 (FUPA14) (Thever and Saier 2009), but it has been shown to be a vacuolar ATPase, Ypk1, that functions in manganese detoxification and homeostasis (Chesi et al. 2012). It is therefore likely to catalyze export of manganese ions from the cytoplasm into the vacuole. However, it also transporter polyamines such as spermine . The 3-D structure has been determined to 3.4 Å, and it revealed three separate transport cycle intermediates, including spermine-bound conformations (Li et al. 2021). In the absence of cargo, Ypk9 rests in a phosphorylated conformation auto-inhibited by the N-terminus. Spermine uptake into vesicles is accomplished through an electronegative cleft lined by transmembrane segments 2, 4 and 6 (Li et al. 2021).
Ypk1 of Saccharomyces cerevisiae (gi6324865)
This protein was previously designated the functionally uncharacterized P-type ATPase (FUPA15) (Thever and Saier 2009). Probable manganese exporter by similarity (see 3.A.3.10.7 and 3.A.3.10.8).
Putative Mn2+-ATPase of Dictyostelium discoideum
Plasma membrane Ca2+-ATPase (efflux), PMCA4 (Giacomello et al. 2013). The CD147 immunosupression protein interacts via its immunomodulatory domains with PMCA4 to bypass T-cell receptor proximal signaling and inhibit interleukin-2 (IL-2) expression (Supper et al. 2016). Deletion of residues 300 - 349, corresponding the the residues deleted in a natural splice variant (de Tezanos Pinto and Adamo 2006). The plasma membrane Ca2+ pump PMCA4z Is more active than splicing variant PMCA4x (Corradi et al. 2021).
Plasma membrane Ca2+-translocating ATPase, PMCA4, of Homo sapiens (P23634)
Endomembrane plasma membrane-type Ca2+ ATPase, ACA2 (Arabidopsis Ca2+ ATPase isoform 2) (lacks a C-terminal calmodulin domain, but activity is stimulated 5x by calmodulin which binds to an N-terminal inhibitory domain (Harper et al., 1998; Kamrul Huda et al. 2013).
ACA2 of Arabidopsis thaliana
Endoplasmic reticular (ER)-type Ca2+/Mn2+ ATPase, ECA1; 80% identical to and orthologous to the Medicago truncatula MCA8 protein of 1081 aas (F9W2W4). 42 P-type II Ca2+ ATPase genes have been found in Triticum aestivum. which may play roles in plant growth, development and signalling during abiotic and biotic stresses (Taneja and Upadhyay 2018).
ECA1 of Arabidopsis thaliana
Autoinhibited Ca2+ ATPase (ACA9) (expressed in pollen plasma membrane and required for male fertility), calmodulin-binding (Schiøtt et al., 2004).
ACA9 of Arabidopsis thaliana
Endomembrane (Golgi) Ca2+/Mn2+-ATPase, ECA3 (one of 4 close paralogues in A. thaliana (Mills et al., 2008; Kamrul Huda et al. 2013)
ECA3 of Arabidopsis thaliana (Q0WP80)
Ca2+-ATPase, Pmc1 (uptake into vacuoles) (Espeso 2016).
Vacuolar membrane Ca2+-translocating ATPase from Saccharomyces cerevisiae Pmc1
Cac1 of Clostridium acetobutylicum (Q97JK5)
Pmo1 of Petrotoga mobilis (A9BJX0)
Sth1 of Streptococcus thermophilus (Q5M0A4)
Putative Ca2+ ATPase most similar to Golgi Ca2+ ATPases of eukaryotes
Putative Ca2+ ATPase of Methanococcus vannielii (A6URW9)
Putative Ca2+-ATPase (48% identical to 3.A.3.2.23) (like Golgi Ca2+-ATPases of eukaryotes)
Putative Ca2+-ATPase of Aguifex aeolicus (O66938)
Plasma membrane Ca2+-ATPase, isoform 1a (PMCA1) (78% identical to PMCA4 (TC# 3.A.3.2.1)). Maitotoxin converts it into a Ca2+-permeable nonselective cation channel (Sinkins et al., 2009). The C-terminal tail contains most of the regulatory sites including that for calmodulin. The pump is also regulated by acidic phospholipids, kinases, a dimerization process, and numerous protein interactors. In mammals, four genes code for the four basic isoforms. Isoform complexity is increased by alternative splicing of primary transcripts. Pumps 2 and 3 are expressed preferentially in the nervous system (Calì et al. 2017). This enzyme has two essential auxillary subunits, basigin and neuroplastin (NPTN), and the 3-d structure of the complex of PMCA1 with NPTN has been solved at 3.9 Å resolution (Gong et al. 2018). Methylene blue activates PMCA activity and cross-interacts with amyloid beta-peptide, blocking Abeta-mediated PMCA inhibition (Berrocal et al. 2018).
PMCA1 of Homo sapiens (P20020)
The M535L virus Ca2+/Mn2+ efflux pump (transcribed during viral infection) (Bonza et al., 2010)
M535L Ca2+ pump of Paramecium bursaria chlorella virus, MT325 (A7IUR5)
Plasma Membrane Ca2+-type ATPase, NCA-2 (most like 3.A.3.2.2) (Bowman et al., 2011).
NCA-2 of Neurospora crassa (Q9UUY2)
The probable Mg2+/Ca2+ ATPase antiporter (catalyzes Mg2+ uptake and Ca2+ efflux in a single coupled step; Neef et al. 2011)
Antiporter of Streptococcus pneumoniae (Q04JJ5)
The putative Ca+ ATPase with an extra C-terminal TMS followed by a lysin (LysM) domain of ~210aas. LysM domains are often found in cell wall degradative enzymes and have peptidoglycan binding sites. Found in Nitrosococcus oceani as well as Nitrosococcus halophilus. The ATPase domain is 46% identical to 3.A.3.2.4.
Putative Ca2+ ATPase of Nitrosococcus halophilus (D5C355)
Ca2+-ATPase, Pmr1 (efflux) (also transport Mn2+ and Cd2+) (Lauer et al., 2008)
Golgi Ca2+-ATPase Pmr1 of Saccharomyces cerevisiae
Pleasma membrane Ca2+-ATPase of parenchymal tissue of the liver fluke, PMCA. Interacts with a calmodulin-like protein, FhCaM1 in a calcium ion dependent fashion (Moore et al. 2012).
PMCA of Fasciola helpatica
Sarcoplasmic reticulum Ca2+ ATPase, Atp6. The inhibitors, artemisinin and its anti-malarial derivatives, artesunate and artemether, bind to a hydrophobic pocket in a transmembrane region near the membrane surface (Naik et al. 2011; Meier et al. 2018). Other inhibitors include arterolane and thapsigargin (Meier et al. 2018).
Atp6 of Plasmodium falciparum
Lobster intracellular SERCA Ca2+ ATPase of 1020 aas. In related species, expression of the gene is increased under hypersaline conditions, and the enzyme is ivolved in salinity stress adaptation (Wang et al. 2013).
ATPase of Palinurus argus
Crustacian plasma membrane calcium ATPase of 1170 aas (Chen et al. 2013).
Calcium ATPase of Callinectes sapidus (blue crab)
Ca2+/Mn2+-exporting ATPase, Pmr1 of 899 aas (Furune et al. 2008). It plays a role in the control of cell division involving Mn2+ sensitivity and the Cwh43 protein (see TC# 9.B.131.1.9 for details) (Nakazawa et al. 2019).
Pmr1 of Schizosaccharomyces pombe
Calcium-exporting ATPase, Pmc1 of 1096 aas (Furune et al. 2008)..
Pmc1 of Schizosaccharomyces pombe
SERCA Ca2+-ATPase of 1093 aas (Docampo et al. 2013).
SERCA ATPase of Toxoplasma gondii
SERCA P-type ATPase of 1036 aas.
SERCA ATPase of Paramecium tetraurelia
Plasma membrane Ca2+ ATPase (PMCA) of 1146 aas (Plattner 2014).
PMCA of Paramecium tetraurelia
Plasma membrae Ca2+ ATPase (PMCA) of 1064 aas (Lescasse et al. 2005).
PMCA of Oxytricha trifallax (Sterkiella histriomuscorum)
Ca2+-ATPase of 905 aas and 10 TMSs, Pma1
Putative Ca2+-ATPase of Synechocystis sp. pMA1
Plasma membrane Ca2+ ATPase, isoform 2, of 1243 aas, ATP2b2. The mouse orthologue, of 1198 aas (P9R0I7), when mutated (I1023S in TMS 10 and R561S in the catailytic core) gives rise to semi-dominant hearing loss (Carpinelli et al. 2013). Neuroplastin (TC# 8.A.23.1.8) expression is essential for hearing and hair cell PMCA expression (Lin et al. 2021).
ATP2b2 of Homo sapiens
P-type Na+-ATPase of 889 aas (Takemura et al. 2009).
Na+-ATPase of Exiguobacterium aurantiacum
Plasma membrane Ca2+-ATPase of 1033 aas, ACA12. Can replace ACA9 which is normally required for male fertility. ACA12 is not stimulated by calmodulin (Limonta et al. 2014).
ACA12 of Arabidopsis thaliana
SERCA1 of 1001 aas. Several 3-D structures are known (e.g., 3W5B). One has an ATP analogue, a Mg2+ and two Ca2+ ions in the respective binding sites (Toyoshima and Mizutani 2004). In this state, the ATP reorganizes the three cytoplasmic domains (A, N and P), which are widely separated without nucleotide, by directly bridging the N and P domains. The structure of the P-domain itself is altered by the binding of the ATP analogue and Mg2+. As a result, the A-domain is tilted so that one of the TMSs moves to lock the cytoplasmic gate of the transmembrane Ca2+-binding sites. This appears to be the mechanism for occluding the bound Ca2+ ions, before releasing them into the lumen of the sarcoplasmic reticulum (Toyoshima and Mizutani 2004). Molecular dynamics simulations provided evidence for the role of the Mg2+ and K+ bound states in the transport mechanism (Espinoza-Fonseca et al. 2014). Animal SERCAs are inhibited by three short single TMS membrane proteins, phospholamban (TC# 1.A.50.1), sarcolipin (1.A.50.2) and myoregulin (1.A.50.3), and the inhibitory actions of these peptides on SERCA are counteracted by a peptide called DWORF (Dwarf ORF) (Nelson et al. 2016; Anderson et al. 2015). Norimatsu et al. 2017 have resolved the first layer of phospholipids surrounding the transmembrane helices. Phospholipids follow the movements of associated residues, causing local distortions and changes in thickness of the bilayer. The entire protein tilts during the reaction cycle, governed primarily by a belt of Trp residues, to minimize energy costs accompanying the large perpendicular movements of the transmembrane helices. A class of Arg residues extend their side chains through the cytoplasm to exploit phospholipids as anchors for conformational switching (Norimatsu et al. 2017). The human ortholog (O14983) is 96% identical to this rabbit enzyme. These enzymes are inhibited by the fungal mycotoxins, cyclopiazonic acid and thapsigargin (Darby et al. 2016; Houdou et al. 2019). The human ortholog is 97% identical and is of the same length. The SERCA residue Glu340 mediates interdomain communication that guides Ca2+ transport (Geurts et al. 2020). The SERCA Ca2+-ATPase may serve as a calcium-sensitive membrane-endoskeleton sensor in the sarcoplasmic reticulum (Nakamura et al. 2021). The C-terminal proton release pathway is a functional element of SERCA which provides a mechanistic model for its operation in the catalytic cycle of the pump (Espinoza-Fonseca 2021).
SERCA of Oryctolagus cuniculus (rabbit)
Crayfish basolateral plasma membrane Ca2+-ATPase, PMCA, of 1190 aas (Wheatly et al. 2007). 80% identical to the human orthologue.
PMCA of Procambarus clarkii (Red swamp crayfish)
The calmodulin-sensitive plasma membrane Ca2+-ATPase (PMCA) of 1080 aas and 10 TMSs. It has a non-canonical calmodulin (CaM) binding domain that contains a C-terminal 1-18 motif (Pérez-Gordones et al. 2017).
PMCA of Trypanosoma equiperdum
Ca2+-ATPase of 880 aas and 10 TMSs, Ca1. Key intermediates have been identified; Ca2+ efflux is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+ (Dyla et al. 2017).
Ca1 of Listeria monocytogenes
Putative Ca2+ P-type ATPase, TMEM94, of 1356 aas and 10 TMSs in the usual 2 + 2 + 6 TMS arrangement. This protein is very distantly related to all other members of the 3.A.3.2 family within the P-type ATPase superfamily, and therefore may have a different or unique function (Zhang et al. 2018).
TMEM94 of Homo sapiens
Sarco/endoplasmic reticulum Ca2+ ATPase of 1018 aas and 10 TMSs (Roegner et al. 2018).
SARCA of Callinectes sapidus
Sarcoplasmic reticular Calcium-ATPase of 993 aas and 10 TMSs. Elongation and contraction of scallop sarcoplasmic reticulum (SR): ATP Stabilizes Ca2+-ATPase crystalline array elongation of SR vesicles (Nakamura et al. 2022).
Ca2+-ATPase of Mizuhopecten yessoensis (Yesso scallop)
The Golgi Ca2+, Mn2+-ATPase, hSPCA1, ATP2C1 or Hussy-28 (efflux) (the Hailey-Hailey disease protein). Involved in responses to Golgi stress, apoptosis and midgestational death (Okunade et al., 2007). SPCA1 transports Mn2+ from the cytosol into the Golgi. Increasing Golgi Mn2+ transport increased cell viability upon Mn2+ exposure, supporting a role in the management of Mn2+ -induced neurotoxicity (Mukhopadhyay and Linstedt, 2011). SPCA1 governs the stability of TMEM165 (TC# 2.A.106.2.2) in Hailey-Hailey disease (Roy et al. 2020).
hSPCA1 of Homo sapiens
Plasma membrane calcium-transporting ATPase, ATP2B3, of 1220 aas and 10 TMSs. This 3ATP-driven Ca2+ ion pump is involved in the maintaining basal intracellular Ca2+ levels at the presynaptic terminals (Calì et al. 2015; Tauber et al. 2016). It uses ATP as an energy source to transport cytosolic Ca2+ ions across the plasma membrane to the extracellular compartment, and
may counter-transport protons.
ATP2B3 of Homo sapiens
The sarco/endoplasmic reticulum Ca2+ -ATPase, SERCA2b or ATP2A2, is encoded by the ATP2A2 gene. Mutatioins give rise to Darier''s disease; the spectrum of mutations have been related to patients' phenotypes (Ahn et al., 2003; Godic et al. 2010). SERCA1 functions as a heat generator in mitochondria of brown adipose tissue (de Meis et al., 2006). It normally functions as a Ca2+:H+ antiporter (Karjalainen et al., 2007). Capsaicin converts SERCA to a Ca2+ non-transporting ATPase that generates heat, and is thus a natural drug that augments uncoupled SERCA, resulting in thermogenesis (Mahmmoud, 2008b). Oligomeric interactions of the N-terminus of sarcolipin with the Ca-ATPase have been documented (Autry et al., 2011), and these interactions also uncouple ATP hydrolysis from Ca2+ transport (Sahoo et al. 2015) resulting in thermogenesis. TMS 11, absent in SERCA1a and SERCA2a, functions in regulation (Gorski et al. 2012). The bovine SERCA has also been crystallized (2.9 Å resolution; Sacchetto et al., 2012). These enzymes are regulated differentially by phospholamban (PLN; 1.A.50.1.1) and sarcolipin (SLN; 1.A.50.2.1) as noted above (Gorski et al. 2013). SERCA2 is regulated by TMEM64 (9.B.27.5.1), a 380 aa 6 TMS membrane protein of the DedA family (TC# 9.B.27) which regulates Ca2+ oscillations by direct interaction with CIRCA2, modulating its activity and influencing osteoblast differentiation (Kim et al. 2013). Animal SERCAs are inhibited by three short single (C-terminal) TMS membrane proteins, phospholamban (TC# 1.A.50.1), sarcolipin (1.A.50.2) and myoregulin (1.A.50.3), and the inhibitory actions of these peptides on SERCA are counteracted by a peptide called DWORF (Dwarf ORF) (Nelson et al. 2016; Anderson et al. 2015). Small ankyrin 1 (sAnk1; TC#8.A.28.1.2) and sarcolipin (TC# 1.A.50.2.1) interact in their transmembrane domains to regulate SERCA (Desmond et al. 2017). Reduced SERCA function preferentially affects Wnt signaling by retaining E-cadherin in the endoplasmic reticulum and promotes apoptosis (Suisse and Treisman 2019). There is strong coupling between the chronological order of deprotonation, the entry of water molecules into the TM region, and the opening of the cytoplasmic gate. Deprotonation of E309 and E771 is sequential with E309 being the first to lose the proton. Deprotonation promotes the opening of the cytoplasmic gate but leads to a productive gating transition only if it occurs after the transmembrane domain has reached an intermediate conformation (Rui et al. 2018). Coordination at cation binding sites I and II is optimized for Ca2+ and to a lesser extent for Mg2+ and K+ (Sun et al. 2019). Methyglyoxal reacts with and inhibits SERCA (Zizkova et al. 2018). The phospholamban pentamer alters the function of SERCA (Glaves et al. 2019). TMS11 followed by the luminal tail is inhibitory. Inoue et al. 2019 determined the crystal structures of SERCA2b and its C-terminal splicing variant SERCA2a, both in the E1-2Ca2+-adenylylmethylenediphosphonate (AMPPCP) state. TMS11 is located adjacent to TMS10 and interacts weakly with a part of the L8/9 loop as well as the N-terminal end of TMS10, thereby inhibiting the SERCA2b catalytic cycle (Inoue et al. 2019). Accordingly, mutational disruption of the interactions between TMS11 and its neighboring residues caused SERCA2b to display SERCA2a-like ATPase activity. The authors proposed that TMS11 serves as a key modulator of SERCA2b activity by fine-tuning the intramolecular interactions with other transmembrane regions. Kabashima et al. 2020 revealed what ATP binding does to the Ca2+ pump and how nonproductive phosphoryl transfer is prevented in the absence of Ca2+. They reported that the A-domain takes an E1 position, and the N-domain occupies exactly the same position as that in the E1.ATP.2Ca2+ state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without filling the two transmembrane Ca2+-binding sites. This explains what ATP binding does to SERCA, and how nonproductive phosphorylation is prevented in E2 (Kabashima et al. 2020). Nonannular lipid binding is not necessary for the stability of the E2 state but may become functionally significant during the E2-to-E1 transition (Espinoza-Fonseca 2019). Structural changes induced by the binding of rutin arachidonate to SERCA1a may shift proton balance near the titrable residues Glu771 and Glu309 into neutral species, hence preventing the binding of calcium ions to the transmembrane binding sites and thus affecting calcium homeostasis (Rodríguez and Májeková 2020). SERCA2a is a key protein in the Ca2+ cycle during heart failure (Zhihao et al. 2020). Covalent conjugates of fullerene derivatives with xanthene dyes inhibit SERCA (Tatyanenko et al. 2020). Autophosphorylation of the pump with two bound Ca2+ ions triggers a large conformational change that opens a gate on the luminal side of the membrane allowing the release of the ions. In response to phosphorylation, the cytoplasmic domains are partially reconfigured into an intermediate state on the path toward the E2 state with a closed luminal gate (Thirman et al. 2021). In preB cells, loss of SERCA2 leads to reduced V(D)J recombination kinetics due to diminished RAG-mediated DNA cleavage (Chen et al. 2021). A series of structural changes may accompany the ordered dissociation of ADP, the A-domain rotation, and the rearrangement of the transmembrane (TM) helices. The luminal gate then opens to release Ca2+ toward the SR lumen. Intermediate structures on the pathway are stabilized by transient sidechain interactions between the A- and P-domains. Lipid molecules between TM helices play a key role in the stabilization (Kobayashi et al. 2021). Structural bases for the conformational and functional regulation of human SERCA2b have been reported (Zhang and Inaba 2022).
SERCA2b of Homo sapiens (P16615)
Ca2+-ATPase (efflux) with broad Ca2+ dependence (3.2-320 μm). Probably inhibited by cipargamin and SJ1733 (Meier et al. 2018).
PfATPase4 of Plasmodium falciparum
Functionally uncharacterized P-type ATPase family 23 (FUPA23) (8 proteins from Actinomycetes; 650-802 aas) (Chan et al. 2010).
FUPA23a of Streptomyces coelicolor (Q9KXM5)
FUPA23c of Trichodesmium erythraeum (Q10YH7)
Functionally uncharacterized P-type ATPase family 24 (FUPA24) (6 proteins of Actinomycetes; 760-1625 aas) (Chan et al. 2010).
FUPA24a of Mycobacterium bovis (Q7U2U7)
Functionally uncharacterized P-type ATPase family 24 (FUPA24) (1607aas); The first half is most like type I (Copper) ATPases, while the second half is most like type II ATPases (Ca2+).
FUPA24b of Thermomicrobium roseum (B9L3W5)
Functionally uncharacterized P-type ATPase family 24 (FUPA24) (1430aas)
FUPA24c of Haliangium ochraceum (D0LKA4)
Functionally uncharacterized P-type ATPase family 24 (FUPA24) (1446aas)
FUPA24d of Hahella chejuensis (ABC27339)
Functionally uncharacterized P-type ATPase family 25 (FUPA25.1) (4 proteins from Actinomycetes; 645-776 aas) (Chan et al. 2010).
FUPA25a of Streptomyces coelicolor (Q9RJ01)
P-type ATPase with a C-terminal hemeerythrin (Hr) domain (Traverso et al., 2010). The Hr domain binds two iron ions per monomer (a diiron center) and may provide a regulatory or more direct function in iron transport (Traverso et al., 2010).
P1B-5- ATPase of Acidothermus cellulolyticus (A0LQU2)
Functionally uncharacterized P-type ATPase family 27 (FUPA27) (multiple proteins from α-, β- and γ- proteobacteria; 817-851aas) (Chan et al. 2010).
FUPA27a of Neisseria meningitidis (Q9JZI0)
Functionally uncharacterized P-type ATPase family 27 (FUPA27), Lbi2 (
FUPA27b of Leptospira biflexa (B0STR2)
Functionally uncharacterized ε-proteobacteria P-type ATPase
FUPA27c of Nitratiruptor sp. SB155-2 (A6Q500)
The Cu2+ - ATPase, CtpA. Required for assembly of periplasmic and membrane embedded copper-dependent oxidases, but not for copper tolerance (Hassani, et al. 2010). Possibly CtpA delivers Cu2+ directly to the enzymes in the membrane rather than catalyzing transmembrane transport: similar to (3.A.3.27.1).
CtpA of Rubrivivax gelatinosus (Q5GCB0)
Cu+ export ATPase, CopA2; provides copper for cytochrome oxidase assembly (González-Guerrero et al. 2010; Raimunda et al. 2013).
CopA2 of Pseudomonas aeruginosa
Functionally uncharacterized P-type ATPase family 29 (FUPA29) (1 protein from a δ-proteobacterium, 798 aas) (Chan et al. 2010).
FUPA29a of Bdellovibrio bacteriovorus (Q6MK07)
Functionally uncharacterized P-type ATPase family 29 (FUPA29) (2 proteins from flavobacteria; 792-795)
FUPA29b of Flavobacterium johnsoniae (A5FGV9)
Plamsa membrane proton-pumping ATPase, Pma1, of 1003 aas and 10 putative TMSs in a 2 + 2 + 6 TMS arrangement. Leptosphaeria maculans, lacking this enzyme, displays a total loss of pathogenicity towards its host plant (Brassica napus). The mutant is unable to germinate on the host leaf surface and is thus blocked at the pre-penetration stage. Reduction in Pma1 activity may disturb the electrochemical transmembrane gradient, thus leading to conidia defective in turgor pressure generation on the leaf surface. L. maculans possesses a second plasma membrane H+-ATPase-encoding gene, termed pma2 (Remy et al. 2008).
Pma1 of Leptosphaeria maculans
Probable H+ pumping P-type ATPase of 1068 aas and 10 TMSs, PMA1 (Shan et al. 2006). PnPMA1 is differentially expressed during pathogen asexual development with a more than 10-fold increase in expression in germinated cysts, the stage at which plant infection is initiated, compared to vegetative or sporulating hyphae or motile zoospores. PnPMA1 contains all the catalytic domains characteristic of H+-ATPases but also has a distinct domain of approximately 155 amino acids that forms a putative cytoplasmic loop between transmembrane domains 8 and 9 (Shan et al. 2006).
PMA1 of Phytophthora nicotianae
ATPase-7, AHA7, of 961 aas and 10 TMSs. 73% identical to AHA2 with which it shares function. AHA7 is autoinhibited by a sequence present in the extracellular loop between transmembrane segments 7 and 8. Autoinhibition of pump activity is regulated by extracellular pH, suggesting negative feedback regulation of AHA7 during establishment of an H+ gradient. Restriction of root hair elongation is dependent on both AHA2 and AHA7, with each having different roles in this process (Hoffmann et al. 2018).
AHA7 of Arabidopsis thaliana (Mouse-ear cress)
MntA of Lactobacillus plantarum
Plasma membrane H+-ATPase, TbHA1 (912 aas) (3 isoforms are present in T. brucei) (Luo et al., 2006). This and another H+-ATPase, (UniProt acc # Q388Z3; 97% identical to TbHA1) have been found to be essential for bloodstream-form Trypanosoma brucei through a genome-wide RNAi screen (Schmidt et al. 2018).
TbHA1 of Trypanosoma brucei (AAP30857)
Plamsa membrane H+-ATPase, Pma1 (pumps protons out of the cell to generate a membrane potential and regulate cytosolic pH) (Liu et al., 2006; Petrov, 2009). TMSs 4,5,6 and 8 comprise the H+ pathway where essential and important residues have been identified (Miranda et al., 2010). Residues in the loop between TMSs 5 and 6 play roles in protein stability, function, and insertion (Petrov 2015). Pma1 interacts with the plamsa membrane Cch1/Mid1 (1.A.1.11.10) to regulate its activity by influencing the membrane potential (Cho et al. 2016). Asp739 and Arg811 are important residues for the biogenesis and function of the enzyme as H+ transport determinants (Petrov 2017). Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Zhao et al. 2021 reported a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. The Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 A downward shift and a 40 degrees rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes enabled the authors to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane (Zhao et al. 2021).
H+-ATPase of Saccharomyces cerevisiae (P05030)
Plasma membrane H+ ATPase, AHA1 Three isoforms, AHA1, 2 & 3, exhibit different kinetic properties (Palmgren and Christensen, 1994). Both the N- and C-termini are directly involved in controlling the pump activity (Ekberg et al., 2010). Methyl jasmonate elicits stomatal closure in many plant species including A. thaliana, and stomatal closure is accompanied by large ion fluxes across the plasma membrane. These events appear to be mediated by AHA1 (Yan et al. 2015). It is involved in root nutrient uptake, epidermal stomatal opening, phloem sucrose loading and unloading, and hypocotyl cell elongation (Ding et al. 2021). Auxin activates two distinct, antagonistically acting signalling pathways that converge on rapid regulation of apoplastic pH, a causative determinant of growth. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H+-ATPases for apoplast acidification, while intracellular canonical auxin signalling promotes net cellular H+ influx, causing apoplast alkalinization (Li et al. 2021).
AHA1 of Arabidopsis thaliana
Plasma membrane H+ ATPase, AHA6 (binds 14-3-3 proteins induced by phosphorylation of Thr948, causing activation; preferentially expressed in pollen; Bock et al., 2006) (82% identical to 3.A.3.3.7).
AHA6 of Arabidopsis thaliana (Q9SH76)
Proton pumping ATPase, AHA2. 94% identical to AHA1 (3.A.3.3.7); generates the plasma membrane pmf. Cation-binding pockets have been identified (Ekberg et al. 2010). The pump has been reconstituted into "nanodiscs" in a functionally monomeric form (Justesen et al. 2013). Regulated at the post-translation level by cis-acting auto-inhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species such as lysophospholipids (Wielandt et al. 2015). Pumping is stochastically interrupted by long-lived (~100 seconds) inactive or leaky states. Allosteric regulation by pH gradients modulates the switch between these states but not the pumping or leakage rates (Veshaguri et al. 2016). They dynamics of the pump have been examined (Guerra and Bondar 2015). AHA2 drives root cell expansion (Hoffmann et al. 2018). This protein is 81% identical to the barley (Hordeum vulgare) HA1 of 956 aas and 10 TMSs. Plasma membrane H+-ATPase (HA1 and HA2) activity and/or expression is important for regulating the activity of K+ transporters and channels under drought stress conditions (Cai et al. 2019). Herbivore exposure enhances A. nanus tolerance to salt stress by activating the jasmonic acid-signalling pathway, increasing plasma membrane H+-ATPase activity, promoting cytosolic Ca2+ accumulation, and then restricting K+ leakage and reducing Na+ accumulation in the cytosol (Chen et al. 2020).
Proton pumping ATPase of Arabidopsis thaliana
Functionally uncharacterized P-type ATPase family 30 (FUPA30) (4 proteins from α-, β- and δ-proteobacteria; 825-896 aas) (Chan et al. 2010).
FUPA30a of Bdellovibrio bacteriovorus (Q6MPD9)
FUPA30d of Anabaena variabilis (Q3M5P5)
Functionally uncharacterized P-type ATPase family 31 (FUPA31) (3 proteins from γ-proteobacteria; 673-1068) (most closely related to FUPA32 homologues) (probably an active enzyme) (Chan et al. 2010).
FUPA31a of Methylococcus capsulatus (Q606V3)
Functionally uncharacterized P-type ATPase family 31 (FUPA31b) (probably a pseudogene). Bears a C-terminal domain of the EcsC family (see 3.A.1.143.1) not found in other P-type ATPases.
FUPA31b of Methylococcus capsulatus (Q606U9)
Functionally uncharacterized P-type ATPase family 32 (FUPA32) (multiple proteins from α-, β-, γ-, δ- and ε-proteobacteria (690-720 aas) (Chan et al. 2010).
FUPA32a of Azoarcus sp. EbN1 (Q5P8C0)
FUPA32c of Clostridium bartiettii (A6NST6)
FUPA32e of Treponema denticola (Q73QH0)
FUPA32f of Methanobrevibacter smithii (A5UJX0)
FUPA32g of Akkermansia muciniphila (B2UR24)
FUPA32h of Thermosynechococcus elongatus (Q8DL41)
Mg2+ ATPase (1182 aas; 18-20 TMSs) with an N-terminal (residues 1-325) transmembrane domain of 8-10 TMSs; homologous to residues 493-791 in O53781 of Mycobacterium tuberculosis (TC# 2.A.1.3.43). Residues 257-318 hit TMSs 7 and 8 in FmtC (MrpF), TC#2.A.1.3.37 with a score of 8 e-4. The last 3 TMSs of the N-terminal fused domain of 3.A.3.4.3 and 3.A.3.4.4 are homologous (e-10) to the last 3 TMSs in 2.A.1.3.43. The N-terminal domain is homologous to the 8TMS domains of 9.B.3 family members.
Mg2+-ATPase of Pseudomonas stutzeri (F2N2Z6)
Mg2+ P-type ATPase (1195 aas; 18-20 TMSs) with an extra N-terminal 8-10 TMSs (residues 1-330). Similar to 3.A.3.4.3. The last 3 TMSs of the N-terminal fused domain to 3.A.3.4.3 and 3.A.3.4.4 are homologous (e-10) to the last 3 TMSs in 9.A.30.2.1. The N-terminal domain is homologous to the 8TMS domains of 9.B.3 family members.
Mg2+-ATPase with N-terminal 8-10 TMS domain of ~300 residues of Azotobacter vinelandii (C1DHA2)
Uncharacterized Mg2+-ATPase, MgtA, of 912 aas and 10 TMSs (Pohland and Schneider 2019).
MgtA of Microcystis aeruginosa
Cu+ (Km 0.3 μM), Ag+ transporting ATPase, CopB (Mana-Capelli et al., 2003)
CopB of Archaeoglobus fulgidus (AAB91079)
Chloroplast envelope Cu+-uptake ATPase, PAA1 or HMA1. Essential for growth under adverse light conditions (Seigneurin-Berny et al. 2006).
PAA1 of Arabidopsis thaliana (Q9SZC9)
Chloroplast thylakoid Cu+-ATPase, PAA2/HMA8 (delivers Cu+ to the thylakoid lumen). Degraded by the Clp protease undeer conditions of Cu+ excess (Tapken et al. 2014).
PAA2 of Arabidopsis thaliana (AAP55720)
The archaeal Cu+ efflux pump (CopA)
CopA of Sulfolobus solfataricus (Q97UU7)
The transferable, plasmid-localized, copper sensitivity (uptake) ATPase, TcrA (811aas) (46% identical to 3.A.3.5.1) (Hasman, 2005)
TcrA of Enterococcus faecium (ABA39707)
The transferable, plasmid-localized, copper resistance (efflux) ATPase, TcrB (50% identical to 3.A.3.5.2) (Hasman, 2005)
TcrB of Enterococcus faecium (AAL05407)
Golgi Cu2+ ATPase, Ccc2, retrieves Cu2+ from the metallochaperone Atx1 and transports it to the lumen of Golgi vesicles (Lowe et al., 2004)
Ccc2 of Saccharomyces cerevisiae
The copper resistance ATPase, CopA (Ettema et al., 2006; Lübben et al., 2007; Villafane et al., 2009).
CopA of Bacillus subtilis (O32220)
PacS of Sulfolobus solfataricus (Q97VH4)
Pae1 of Pyrobaculum aerophilum (Q8ZUJ0)
Tro1 of Thermoplasma volcanium (Q978Z8)
Putative Copper P-type ATPase (46% identical to 3.A.3.5.10)
Putative Copper P-type ATPase of Candidatus Korarchaeum cryptofilum (B1L487)
Ape2 of Aeropyrum pernix (Q9YBZ6)
The copper (Cu2+) transporting ATPase, Ccc2
Ccc2 of Schizosaccharomyces pombe (O59666)
Cu+-, Ag+-ATPase (efflux from the cytosol into the secretory pathway) (Barnes et al., 2005); ATP7B (Wilson's disease protein, α-chain) is continuously expressed in Purkinje neurons. It delivers Cu+ to the ferroxidase, ceruloplasmin, in liver. May also transport Fe2+ (Takeda et al., 2005). Critical roles for the COOH terminus of ATP7B in protein stability, trans-Golgi network retention, copper sensing, and retrograde trafficking have been reported (Braiterman et al. 2011). Modeling suggests that Cu+-binding sites HMBDs 5 and 6 are most important for function (Gourdon et al. 2012). ATP7B loads Cu+ into newly synthesized cupro-enzymes in the trans-Golgi network and exports excess copper out of cells by trafficking from the Golgi to the plasma membrane. Mutations causing disease can affect activity, stability or trafficking (Braiterman et al. 2014). Cisplatin is a poor substrate relative to Cu+with a Km of 1 mμM, and copper and cisplatin compete with each other (Safaei et al. 2008). Veratridine can bind to a site at the mouth of the channel pore in the human cardiac sodium channel, NaV1.5 (Gulsevin et al. 2022). ATP7A/B contains a P-type ATPase core consisting of a membrane transport domain and three cytoplasmic domains, the A, P, and N domains, and a unique amino terminus comprising six consecutive metal-binding domains. Bitter et al. 2022 presented a cryo-EM structure of frog ATP7B in a copper-free state. Interacting with both the A and P domains, the metal-binding domains are poised to exert copper-dependent regulation of ATP hydrolysis coupled to transmembrane copper transport. A ring of negatively charged residues lines the cytoplasmic copper entrance that is presumably gated by a conserved basic residue sitting at the center. Within the membrane, a network of copper-coordinating ligands delineates a stepwise copper transport pathway.
Cu+-ATPase, ATP7B, of Homo sapiens
Copper (Cu+) exporting P-ATPase, CopA (3-D structure known to 3.2 Å; PDB# 3RFU; Gourdon et al. 2011). The internal surface of the ATPase interacts with the copper chaparone, CopZ (Padilla-Benavides et al. 2012). A sulfur-lined metal transport pathway has been identified (Mattle et al. 2015). Cu+ is bound at a high-affinity transmembrane-binding site in trigonal-planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Methionine residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport (Mattle et al. 2015).
CopA of Legionella pneumophila (Q5X2N1)
Mycobacterial copper transporter, MctB (Wolschendorf et al., 2011).
MctB of Mycobacterium abscessus (B1MHH7)
Copper-transporting ATPase RAN1 or HMP7 (EC 126.96.36.199) (Protein HEAVY METAL ATPASE 7) (Protein RESPONSIVE TO ANTAGONIST 1). Receptors involved in ethylene signaling can acquire their copper load by different routes and adopt the metal ion from the plasma membrane either by sequential transfer from soluble chaperones of the ATX1-family via the ER-bound copper-transporting ATPase RAN1 or by direct transfer from the soluble chaperones (Hoppen and Groth 2020). ER-anchored SPL7 (Transcription factor) constitutes a cellular mechanism for the regulation of RAN1 in ethylene signaling and lays the foundation for investigating how Cu homeostatic conditions ethylene sensitivity in the developmental context (Yang et al. 2022).
RAN1 of Arabidopsis thaliana
Ca2+ exporting ATPase, CopA. The domain organization and mechanism have been studied (Hatori et al., 2009, Hatori et al., 2008, Hatori et al., 2007). Residues involved in catalysis have been defined (Hatori et al. 2009).
CopA of Thermotoga martima (Q9WYF3)
Cu+ export ATPase, CopA1, required to maintain cytoplasmic copper levels (González-Guerrero et al. 2010; Raimunda et al. 2013).
CopA1 of Pseudomonas aeruginosa
Functionally uncharacterized P-type ATPase. Three proteins from Corynebacteria of 841-976 aas are similar in sequence. Formerly members of the FUPA26 family (Chan et al. 2010).
Uncharacterized ATPase of Corynebacterium diphtheriae (Q6NJJ6)
Functionally uncharacterized P-type ATPase, formerly of family 28 (FUPA28). Two proteins in γ-proteobacteria are similar in sequence; of 847-852 aas (Chan et al. 2010).
P-type ATPase (formerly FUPA28a) of Legionella pneumophila (Q5ZYY0)
Copper exporting ATPase, ATP7 of 1254 aas and 10 - 12 TMSs. DmATP7 is the sole Drosophila melanogaster ortholog of the human MNK and WND copper transporters. A regulatory element drives expression in all neuronal tissues examined and demonstrates copper-inducible, Mtf-1-dependent expression in the larval midgut. Thus, an important functional role for copper transport in neuronal tissues is implied. Regulation of DmATP7 expression is not used to limit copper absorption under toxic copper conditions. The protein localizes to the basolateral membrane of DmATP7 expressing midgut cells, supporting a role in export of copper from midgut cells (Burke et al. 2008).
ATP7 of Drosophila melanogaster (Fruit fly)
Cuprous ion (Cu+) exporter, CopB, of 785 aas and 8 TMSs in a 4 + 2 + 2 arrangement. The copper-transporting P1B-ATPases have been divided traditionally into two subfamilies, the P1B-1-ATPases or CopAs and the P1B-3-ATPases or CopBs. CopAs selectively export Cu+ whereas previous studies have suggested that CopBs are specific for Cu2+ export. Biochemical and spectroscopic characterization of Sphaerobacter thermophilus CopB (StCopB) showed that, while it does bind Cu2+, the binding site is not in the transmembrane domain (Purohit et al. 2018). StCopB exhibits metal-stimulated ATPase activity in response to Cu+, but not Cu2+, indicating that it is actually a Cu+ transporter. Cu+ is coordinated by four sulfur ligands derived from conserved cysteine and methionine residues. The histidine-rich N-terminal region is required for maximal activity, but is inhibitory in the presence of divalent metal ions. P1B-1- and P1B-3-ATPases may therefore all transport Cu+ (Purohit et al. 2018).
CopB of Sphaerobacter thermophilus
Cu+, Zn2+, Cd2+ exporting ATPase of 815 aas and 8 TMSs, CueA. Has two N-terminal metal binding domains that are essential for resistance to these three metal ions (Liang et al. 2016).
CueA of Bradyrhizobium liaoningense
Copper-exporting P-type ATPase of 742 aas and 8 TMSs (Singh et al. 2015).
CopA of Streptococcus mutans
Cuprous ion (Cu+) exporter, CtpA (does not export Co2+, Mn2+, Ni2+, Zn2+ or Cu2+). Km for Cu+ = 0.05 μM (León-Torres et al. 2015).
Cu+ ATPase of Mycobacterium tuberculosis
Cu+, Ag+-ATPase (efflux) (Fan and Rosen, 2002). There are two metal binding domains (MBDs). The distal N-terminal MBD1 possesses a function analogous to the metallochaperones of related prokaryotic copper resistance systems and is involved in copper transfer to the membrane-integral ion binding sites of CopA. In contrast, the proximal domain MBD2 has a regulatory role by suppressing the catalytic activity of CopA in the absence of copper (Drees et al. 2015). The functions of Me2+ exporters are often supported by chaperone proteins, which scavenge the metal ions from the cytoplasm. A CopA chaperone is expressed in E. coli from the same gene that encodes the transporter (Meydan et al. 2017). Some ribosomes translating copA undergo programmed frameshifting, terminate translation in the -1 frame, and generate the 70 aa-long polypeptide CopA(Z), which helps cells survive toxic copper concentrations. The high efficiency of frameshifting is achieved by the combined stimulatory action of a "slippery" sequence, an mRNA pseudoknot, and the CopA nascent chain. Similar mRNA elements are not only found in the copA genes of other bacteria but are also present in ATP7B, the human homolog of copA, and direct ribosomal frameshifting in vivo (Meydan et al. 2017). Cu(i) (Cu+) pumps, of which CopA is an example, are primary-active electrogenic uniporters. The Cu+ translocation cycle does not require proton counter-transport, resulting in electrogenic generation of a transmembrane potential upon translocation of one Cu+ per ATP hydrolysis in the catalytic cycle (Abeyrathna et al. 2020).
CopA of E. coli
Cu+-ATPase, ATP7A (MNK or Mc1) (efflux from the cytosol into the secretory pathway) (Menkes disease protein, α-chain) (Tümer 2013). It plays a role in systemic copper absorption in the gut and copper reabsorption in the kidney. In nonpolarized cells, the metal binding sites in the amino-terminal domain of MNK are required for copper-regulated trafficking from the Golgi to the plasma membrane (Greenough et al. 2004). It is expressed in Purkinje cells early in development and later in Bergmann glia. In melanocytes, it delivers Cu2+ to tyrosinase (Barnes et al., 2005). ATP7A has dual functions: 1) it incorporates copper into copper-dependent enzymes; and 2) it maintains intracellular copper levels by removing excess copper from the cytosol. To accomplish both functions, the protein traffics between different cellular locations, depending on copper levels (Bertini and Rosato, 2008). The lumenal loop Met672-Pro707 of ATP7A binds metals and facilitates copper release from the intramembrane sites (Barry et al., 2011). Modeling suggests that Cu+-binding sites HMBDs 5 and 6 are most important for function (Gourdon et al. 2012). In addition to X-linked recessive Menkes disease, mutations cause occipital horn syndrome and adult-onset distal motor neuropathy (Yi and Kaler 2014). p97/VCP interacts with ATP7A playing a role in motor neuron degeneration (Yi and Kaler 2018). 55 different mutations were located around the six copper binding sites and the ATP binding site. 76.7% of the mothers were carriers. Approximately half of the male siblings of patients with MNK were diagnosed with MNK (Fujisawa et al. 2019). It may play a role in melanosome (melanocyte) function (Wiriyasermkul et al. 2020).
ATP7A of Homo sapiens
Cu+-Ag+-ATPase (efflux), CopA of 804 aas. Exhibits maximal activity at 75˚C (Cattoni et al., 2007). The 3-D structure of the ATP-binding domain has been solved (2HC8_A) (functions with the Cu+ chaperone, CopZ; 130aas) (González-Guerrero and Argüello, 2008). This protein has both N- and C- terminal metal binding domains (MBDs). The N-MBD exhibits a conserved ferredoxin-like fold, binds metals to CXXC, and regulates turnover. The C-MBD interacts with the ATP-binding (ATPB) domain and the actuator (A) domain (Agarwal et al., 2010). Cysteine is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme in the E1 form (Yang et al. 2007).
CopAZ of Archaeoglobus fulgidus:
CopA (PaeS) (O29777)
CopZ (2HU9_A; O29901)
Cu+ transporting ATPase (intracellular, in the trans-Golgi membrane), Ccc2
Ccc2 of Candida albicans
Zn2+-, Cd2+-, Pb2+-ATPase (efflux). The enzyme from S. aureus strain 17810R, of 726 aas, functions as a Cd2+:H+ antiporter, using both the pmf and ATP hydrolysis to drive Cd2+ expulsion (Tynecka et al. 2016).
Bacteria; plants; fungi; protozoa
CadA of Staphylococcus aureus
The heavy metal efflux pump, AztA (exports Zn2+, Cd2+, Pb2+; has two adjacent heavy metal binding domains (Liu et al., 2007)
AztA of Anabaena (Nostoc) sp. PCC7120 (Q8ZS90)
The heavy metal transporter A (HmtA) mediates uptake of copper and zinc but not of silver, mercury, or cadmium (Lewinson et al., 2009).
HmtA of Pseudomonas aeruginosa (Q9I147)
Mac1 of Methanosarcina acetivorans (Q8TJZ4)
Cd2+-selective export ATPase, HMA3 (expressed in root cell tonoplasts wherein Cd2+ is sequestered (Ueno et al., 2010)). HMA3 may play a role in Cd2+ accumulation in rice (Cao et al. 2019).
HMA3 of Oryza sativa (Q8H384)
Cd2+/Zn2+ exporting ATPase, HMA4. (very similar to HMA3; TC# 3.A.3.6.7). Important for Zn2+ nutrition. Has a C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines with a high affinity for Zn2+ and Cd2+ and a capacity to bind 10 Zn2+ ions per C-terminus (Baekgaard et al., 2010). The pathway of translocatioin through the protein has been investigated, and the demonstration that mutations affect Zn2+ and Cd2+ transport differentially has been reported (Lekeux et al. 2018).
HMA4 of Arabidopsis thaliana (O64474)
Ca2+/Zn2+ ATPase, OsHMA2 (Satoh-Nagasawa et al., 2012).
HMA2 of Oryza sativa (E7EC32)
Zn2+-, Cd2+-, Co2+-, Hg2+-, Ni2+-, Cu2+, Pb2+-ATPase (efflux), ZntA, of 732 aas and 8 TMSs (Hou and Mitra, 2003). The first four TMSs in ZntA and presumably other P1B-type ATPases play an important role in maintaining the correct dimer structure (Roberts et al. 2020).
ZntA of E. coli
Cadmium/zinc-transporting ATPase 4, HMA3
Cobalt ion exporting ATPase, slr0797 (Rutherford et al. 1999).
Co-ATPase of Synechocystis PCC6803
Co2+-specific P1B-ATPase, CoaT (Zielazinski et al., 2012).
CoaT of Sulfitobacter sp. NAS-14.1 (A3T2G5)
Heavy metal (Pb2+, Cd2+, Zn2+) export ATPase of 970 aas, PbtA (Hložková et al. 2013; Suman et al. 2014)
PbtA of Achromobacter xylosoxidans
Fur-regulated virulence factor A of 626 aas, FrvA; suggested by the authors to be a heme exporter, but maybe more likely to be an iron exporter (McLaughlin et al. 2012).
FrvA of Listeria monocytogenes
Cd2+/Zn2+/Co2+ export ATPase, ZntA, of 904 aas and 8 TMSs. Expression of the zntA gene is inducible by all three metal ions, with Cd2+ being the most potent, mediated by the MerR-like regulator, ZntR (Chaoprasid et al. 2015). zntA and zntR mutants were highly sensitive to CdCl2 and ZnCl2, and less sensitive to CoCl2. Inactivation of zntA increased the accumulation of intracellular cadmium and zinc and conferred hyper-resistance to H2O2. Thus, ZntA and its regulator, ZntR, are important for controlling zinc homeostasis and cadmium and cobalt detoxification. The loss of either the zntA or zntR gene did not affect the virulence of A. tumefaciens in Nicotiana benthamiana (Chaoprasid et al. 2015).
ZntA of Agrobacterium tumefaciens
Cadmium/zinc resistance efflux pump, CadA of 910 aas and 8 TMSs (Maynaud et al. 2014).
CadA of Mesorhizobium metallidurans
Transition metal efflux ATPase of 829 aas and 6 TMSs, CzcP. Exports Zn2+, Cd2+ and Co2+ efficiently (Scherer and Nies 2009). The side chains of Met254, Cys476, and His807 contribute to Cd2+, Co2+, and Zn2+ binding and transport (Smith et al. 2017).
CzcP of Cupriavidus metallidurans (Ralstonia metallidurans)
Pb2+-ATPase (efflux), PbrA. Mediates resistance to Pb2+, Cd2+ and Zn2+. Lead resistance is facilitated by the phosphatase, PbrB, possibly by allowing complexation of the Pb2+ by phosphate in the periplasm (Hynninen et al. 2009).
PbrA of Ralstonia metallidurans
Mono- and divalent heavy metal (Cu+, Ag+, Zn2+, Cd2+) ATPase, Bxa1. bxa1 gene expression is induced by all four heavy metal ions (Tong et al., 2002). The His-rich domain is essential for both monovalent (Ag+ and Cu+) and divalent ( Cd2+ and Zn2+) metal tolerance (Nakakihara et al. 2009).
Bxa1 ATPase of Oscillatoria brevis
The Cd2+ resistance ATPase, CadA (Wu et al., 2006)
CadA of Listeria monocytogenes (Q60048)
K+-ATPase (uptake), KdpFABC. (KdpA is homologous to other K+ transporters such as KcsA (1.A.1.1.1), KtrB (2.A.38.4.2 and 2.A.38.4.3), and HKT (2.A.38.3.1 and 2.A.38.3.2); KdpB is homologous to P-ATPase α-subunits; KdpC and KdpF may facilitate complex assembly and stabilize the complex (Bramkamp et al., 2007; Haupt et al., 2005; Greie and Altendorf, 2007; Irzik et al., 2011). The KdpFABC acts as a functional and structural dimer with the two KdpB subunits in direct contact, but the enzyme can dissociate to the monomer (Heitkamp et al., 2008). KdpF is part of and stabilizes the KdpABC complex (Gassel et al., 1999). Transcription of the kdp operon is activated by the KdpDE sensor kinase/response regulator pair, and unphosphorylated IIANtr of the PTS (TC# 4.A) binds KdpD to stimulate its activity, thereby enhancing kdp operon expression (Lüttmann et al. 2009, Lüttmann et al. 2015). Transcriptional regulation of the Pseudomonas putida kdpFABC operon by the KdpDE sensor kinase/response regulator by direct interaction of IIANtr of the PTS with KdpD has also been studied (Wolf et al. 2015). The 2.9 Å X-ray structure of the complete Escherichia coli KdpFABC complex with a potassium ion within the selectivity filter of KdpA and a water molecule at a canonical cation site in the transmembrane domain of KdpB has been solved (Huang et al. 2017). The structure reveals two structural elements that appear to mediate the coupling between these two subunits: a protein-embedded tunnel runs between these potassium and water sites, and a helix controlling the cytoplasmic gate of KdpA is linked to the phosphorylation domain of KdpB. A mechanism that repurposes protein channel architecture for active transport across biomembranes was proposed (Huang et al. 2017). The cytoplasmic C-terminal domain of KdpD functions as a K+ sensor (Rothenbücher et al. 2006). Serine phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated via an intracellular pathway between TMSs M1 and M2 which opens in response to the rearrangement of cytoplasmic domains, resulting from phosphorylation (Dubey et al. 2021). This causes pump inhibition in the presence of high K+ resulting in ATP conservation.
KdpABCF of E. coli
High affinity potassium uptake ATPase, KdpABC. Regulated by direct interaction of the IIANtr protein with the sensor kinase/response regulator, KdpDE (Prell et al. 2012).
KdpABC of Rhizobium leguminosarum
Potassium transporter, KdpABC, with 3 subunits: KdpA, B2HPR5, 552 aas and 10 TMSs in a 2 + 2 + 2 + 2 + 2 arrangement; KdpB ATPase, B2HRP6, 693 aas and 7 TMSs in a 2 + 2 + 3 TMS arrangement, and KdpC, D2HRP7, 296 aas with one N-terminal TMS and possibly one C-terminal TMS. A kdpA null mutantion reduced the fraction of persisters after exposure to rifampicin (Liu et al. 2020). kdpA encodes a transmembrane protein that is part of the Kdp-ATPase, an ATP-dependent high-affinity potassium (K+) transport system. kdpA expression is induced under low K+ conditions and is required for pH homeostasis and growth in media with low concentrations of K+. Inactivation of the Kdp system caused hyperpolarization of the membrane potential, increased the proton motive force (PMF) and elevated levels of intracellular ATP. The KdpA mutant phenotype could be complemented with a functional kdpA gene or supplementation with high K+ concentrations. Thus, the Kdp system is required for ATP homeostasis and persister formation. ATP-mediated regulation of persister formation may be a general mechanism in bacteria, and suggest that K+ transporters may play a role in the regulation of ATP levels and persistence (Liu et al. 2020).
KdpABC of Mycobacterium marinum
Golgi Aminophospholipid (phosphatidyl serine and phosphatidyl ethanolamine) translocase (flipping from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry), required for vesicle-mediated protein transport from the Golgi and endosomes. The system has been reconstituted after purification in proteoliposomes. It flips phosphatidyl serine but not phosphatidylcholine or sphinogomyelin (Zhou and Graham, 2009). A unified mechanism of flipping for ABC and P-type ATPases has been proposed (López-Marqués et al. 2014).
ATPase II of Bos taurus
Lipid flippase, Apt1 (involved in stress tolerance and virulence). Deletion of Apt1 causes (1) altered actin distribution, (2) increased sensitivity to stress conditions (oxidative and nitrosative stress) and to trafficking inhibitors, such as brefeldin A and monensin, a reduction in exported acid phosphatase activity, and (3) hypersensitivity to the antifungal drugs amphotericin B, fluconazole, and cinnamycin (Hu and Kronstad, 2010).
Apt1 of Cryptococcus neoformans (Q5KP96)
Phospholipid (e.g., cardiolipin) transporter, Atp8b1. A mutant version is associated with severe
pneumonia in humans and mice. It binds and internalizes cardiolipin from extracellular fluid via a basic residue-enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 gene transfer in mice lessens bacterium-induced lung injury and improves survival (Ray et al., 2010). Mutations have been identified that give rise to progressive familial intrahepatic cholestasis (Stone et al. 2012). This lipid flippase forms a heterodimer with CDC50A/Transmembrane protein 30A (TC# 8.A.27.1.5) and is essential for surface expressioin of the apical Na+-bile acid transporter, Slc10A2/ASBT (TC#2.A.28.1.2) (van der Mark et al. 2014).
Atp8b1 of Homo sapiens (O43520)
Probable phospholipid-transporting ATPase IF (EC 188.8.131.52) (ATPase IR) (ATPase class VI type 11B). Among the ATP10 and ATP11 proteins of P4-ATPases, ATP10A, ATP10D, ATP11A, and ATP11C localize to the plasma membrane, while ATP10B and ATP11B localize to late endosomes and early/recycling endosomes, respectively. The N- or C-terminal cytoplasmic regions of P4-ATPases determine their cellular localization (Okamoto et al. 2020).
ATP11B of Homo sapiens
P4 phospholipid (phosphatidyl serine)-transporting ATPase 8A1 (EC 184.108.40.206) (ATPase class I type 8A member 1) (Chromaffin granule ATPase II). Also found in the liver canicular membrane (Chaubey et al. 2016). The 3-D strcutures of 6 distinct intermediates (2.6 - 3.3 Å resolution) of the complex of this protein with CDC50A (TC# 8.A.27.1.5) have been solved, revealing the transport cycle for lipid flipping (Hiraizumi et al. 2019). ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site in the phosphorylation domain, accompanied by lateral shifts of the first and second TMSs, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment (Hiraizumi et al. 2019).
ATP8A1 of Homo sapiens
ATP11C (ATPIG, ATPIQ) aminophospholipid (phosphatidyl serine and phosphatidyl ethanolamine, but not phosphatidyl choline) flippase of 1132 aas and 10 TMSs. It is dependent on CDC50A (1232 aas and 9 TMSs, Anoctamin-8, Ano8; TC#1.A.17.1.30), for proper localization to the plasma membrane, and possibly also for activity (Segawa et al. 2014). Present in liver basolateral membranes (Chaubey et al. 2016). It is the only phospholipid flipping ATPase in the human red blood cell (Liou et al. 2019). In the cell membrane of erythrocytes, it is required to maintain phosphatidylserine (PS) in the inner leaflet preventing its exposure on the surface. This asymmetric distribution is critical for the survival of erythrocytes in circulation since externalized PS is a phagocytic signal for splenic macrophages (Arashiki et al. 2016). Phospholipid translocation seems also to be implicated in vesicle formation and in the uptake of lipid signaling molecules, and is required for B cell differentiation past the pro-B cell stage It seems to mediate PS flipping in pro-B cells and may be involved in the transport of cholestatic bile acids. Caspase-dependent inactivation of ATP11C is essential for an apoptotic "eat me" signal, phosphatidylserine exposure, which prompts phagocytes to engulf cells. Nakanishi et al. 2020 presented six cryo-EM structures of ATP11C at 3.0-4.0 A resolution in five different states of the transport cycle. A structural comparison revealed phosphorylation-driven domain movements coupled with phospholipid binding. Three structures of phospholipid-bound states visualize phospholipid translocation accompanied by the rearrangement of transmembrane helices and an unwound portion at the occlusion site. They thus detail the basis for head group recognition and the locality of the protein-bound acyl chains in transmembrane grooves. Invariant Lys880 and the surrounding hydrogen-bond network serve as a pivot point for helix bending and precise P domain inclination, which is crucial for dephosphorylation. The structures detail key features of phospholipid translocation by ATP11C; a common basic mechanism for flippases is emerging (Nakanishi et al. 2020).
ATP11C of Homo sapiens
Phospholipid transporting ATPase, Tat1 of 1139 aas. Transports phosphatidylserine from the outer to the inner leaflet of the plasma membrane, thereby maintaining the enrichment of this phospholipid in the inner leaflet. Ectopic exposure of phosphatidylserine on the cell surface may result in removal of living cells by neighboring phagocytes in an apoptotic process (Darland-Ransom et al. 2008). Tat1 regulates lysosome biogenesis and endocytosis as well as yolk uptake in oocytes. It is required at multiple steps of the endolysosomal pathway, at least in part by ensuring proper trafficking of cell-specific effector proteins (Ruaud et al. 2009). TAT-1 and its chaperone, the Cdc50 family protein CHAT-1, maintain membrane phosphatidylserine (PS) asymmetry, which is required for membrane tubulation during endocytic sorting and recycling. Loss of tat-1 and chat-1 disrupts endocytic sorting, leading to defects in both cargo recycling and degradation. Chen et al. 2019 identified the C. elegans aspartyl aminopeptidase DNPP-1, loss of which suppresses the sorting and recycling defects in tat-1 mutants without reversing the PS asymmetry defect.
Tat1 of Caenorhabditis elegans
ATP9A lipid flippase of 1047 aas and 10 TMSs. Present in the liver canicular membrane (Chaubey et al. 2016).
ATP9A of Homo sapiens
Intracellular phospholipid flippase ATP11A (Chaubey et al. 2016). Catalytic component of a P4-ATPase flippase complex which catalyzes the hydrolysis of ATP coupled to the transport of aminophospholipids from the outer to the inner leaflet and ensures the maintenance of asymmetric distribution of phospholipids. Phospholipid translocation seems also to be implicated in vesicle formation and in uptake of lipid signaling molecules. May be involved in the uptake of farnesyltransferase inhibitory drugs, such as lonafarnib (Zhang et al. 2005). A sublethal ATP11A mutation is associated with neurological deterioration due to aberrant phosphatidylcholine flipping in plasma membranes (Segawa et al. 2021).
ATP11A of Homo sapiens
The essential endosomal Neo1 phospholipid flipping ATPase of 1151 aas. Neo1 plays an essential role in establishing phosphatidyl serine (PS) and phosphatidyl ethanolamine (PE) plasma membrane asymmetry in budding yeast (Takar et al. 2016). A common mechanism for substrate recognition in widely divergent P4-ATPases including Neo1 has been proposed (Huang et al. 2019).
Neo1 of Saccharomyces cerevisiae
The Leishmania miltefosine transporter (LMT) is a plasma membrane P4-ATPase that catalyses translocation into the parasite of the leishmanicidal drug, miltefosine as well as phosphatidylcholine and phosphatidylethanolamine analogues. Five highly-conserved amino acids in the cytosolic N-terminal tail (Asn58, Ile60, Lys64, Tyr65 and Phe70) and two (Pro72 and Phe79) in the first TMS were examined, and several of these were important for activity (Perandrés-López et al. 2018). The beta subunit of this system has TC# 8.A.27.1.3.
LMT of Leishmania amazonensis
Golgi aminophospholipid translocase (flipping from the exofacial to the cytosolic leaflet of membranes), required for vesicle-mediated protein transport from the Golgi and endosomal lumen to the cytoplasm, Gea2p (Pomorski et al., 2003). The system has been reconstituted after purification in proteoliposomes. It flips phosphatidyl serine and phosphatidyl ethanolamine, but not phosphatidylcholine or sphingomyelin (Zhou and Graham, 2009). Drs2p (ACT3; ATP8A2) is required for phospholipid translocation across the Golgi membrane. It interacts with CDC50 (TC# 8.A.27) (Bryde et al., 2010). Activated by ArfGEF when bound to the C-terminus (Natarajan et al. 2009). The beta-subunit, CDC50A, allows the stable expression, assembly, subcellular localization, and lipid transport activity of the P4-ATPase ATP8A2 (Coleman and Molday, 2011). Timcenko et al. 2019 described the cryo-EM structure of Drs2p-Cdc50p, It is autoinhibited by the C-terminal tail of Drs2p and activated by the lipid phosphatidylinositol-4-phosphate (PI4P). Three structures were solved that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis revealed sites of autoinhibition and PI4P-dependent activation. A putative lipid translocation pathway involves a conserved PISL motif in TMS 4 and polar residues of TMSs 2 and 5, in particular Lys1018, in the centre of the lipid bilayer (Timcenko et al. 2019). The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, Timcenko et al. 2021 determined cryo-EM structures of Drs2p-Cdc50p covering multiple intermediates of the cycle. They identified several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent (Timcenko et al. 2021). The Arf activator, Gea2p (Uniprot P39993, 1459 aas), and Drs2p interact in the Golgi (Chantalat et al. 2004).
DRS2 of Saccharomyces cerevisiae
Plasma membrane phospholipid flippase of 1656 aas, Dnf3-Crf1. Dnf3 flips phospholipids from the outer leaflet of the membrane to the inner leaflet (Sartorel et al. 2015). Crf1, a non-catalytic subunit, regulates the activity of Dnf3. It is listed under TC# 8.A.27.1.7.
Dnf3/Crf1 of Saccharomyces cerevisiae
Putative lipid-flipping magnesium-transporting ATPase of 922 aas and 10 TMSs (Greiner et al. 2018).
ATPase of Klosneuvirus KNV1
Probable phospholipid-transporting P-type ATPase of 903 aas and 10 TMSs.
ATPase of soda lake Tupanvirus
Possible lipid flipping P-type ATPase of 809 aas and 7 putative TMSs. It is probably C-terminally truncated.
ATPase of Catovirus CTV1
Broad range phospholipid-transporting ATPase 10, ALA10, of 1202 aas and 10 TMSs. A structural model of ALA10 reveals a cavity delimited by TMSs 3, 4 and 5 at a similar position as the cation-binding region in related cation transporting P-type ATPases. Docking of a phosphatidylcholine headgroup in silico showed that the cavity can accommodate a phospholipid headgroup, likely leaving the fatty acid tails in contact with the hydrophobic portion of the lipid bilayer. Mutagenesis data supported this interpretation and suggested that two residues in TMS 4 (Y374 and F375) are important for coordination of the phospholipid headgroup (Jensen et al. 2017). These results point to a general mechanism of lipid translocation by P4 ATPases, which closely resembles that of cation-transporting pumps, through coordination of the hydrophilic portion of the substrate in a central membrane cavity.
Phospholipid-transporting ATPase VD or Atp10d, of 1426 aas and 10 TMSs. It is expressed in placenta and, to a lesser extent, in the kidney (Flamant et al. 2003). It is the catalytic component of a P4-ATPase flippase complex which catalyzes the hydrolysis of ATP coupled to the transport of aminophospholipids from the outer to the inner leaflet of various membranes and ensures the maintenance of asymmetric distribution of phospholipids. Phospholipid translocation has been implicated in vesicle formation and in the uptake of lipid signaling molecules. ATP10D reduces high-fat diet induced obesity and improves insulin sensitivity (Sigruener et al. 2017). It also transports glycosphingolipids including glucosphinolipids (Roland et al. 2019).
Atp10d of Homo sapiens
P-type ATPase of 1499 aas and 10 TMSs, Atp10A, Atp10C, AtpVA, AtpVC. ATP10A transports phosphatidylcholine but not aminophospholipids (Shin and Takatsu 2019). Among the ATP10 and ATP11 proteins of P4-ATPases, ATP10A, ATP10D, ATP11A, and ATP11C localize to the plasma membrane, while ATP10B and ATP11B localize to late endosomes and early/recycling endosomes, respectively. The N- or C-terminal cytoplasmic regions of P4-ATPases determine their cellular localization (Okamoto et al. 2020).
Atp10A of Homo sapiens
Miltefosine/glycerophospholipid uptake translocase and phospholipid uptake flippase, MIL (Pérez-Victoria et al., 2003)
MIL of Leishmania donovani (Q6VXY9)
Inwardly directed phospholipid and lysophospholipid (phosphatidylcholine, phosphatidyl serine and lysophosphoethanolamine) flippase, Dnf1 or ATP11C (functions with the β-subunit, Lem3 or CDC50A (TC# 8.A.27.1.5) (Elvington et al., 2005; Pomorski et al., 2003; Riekhof and Voelker, 2006; Riekhof et al., 2007) Also transports the anti-neoplastic and anti-parasitic ether lipid substrates related to edelfosine (Riekhof and Voelker, 2009) (not required for phosphotidyl serine inwardly directed flipping (Stevens et al. 2008)). Transports diacyl phospholipids in preference to lyso (monoacyl) phospholipids (Baldridge et al. 2013). A conserved asparagine (N220) in the first transmembrane segment specifies glycerophospholipid binding and transport, but specific substitutions at this site allow transport of sphingomyelin (Roland and Graham 2016). It transports glycosphingolipids (Roland et al. 2019). Nakanishi et al. 2020 presented the crystal structures of a human plasma membrane flippase, the ATP11C-CDC50A complex, in a stabilized E2P conformation. The structure revealed a deep longitudinal crevice along transmembrane helices continuing from the cell surface to the phospholipid occlusion site in the middle of the membrane. The extension of the crevice on the exoplasmic side was open, and the complex was therefore in an outward-open E2P state, similar to a cryo-EM structure of the yeast flippase Drs2p-Cdc50p complex. Phosphatidylserines were in the crevice and in its extension to the extracellular side. One was close to the phosphatidylserine occlusion site as previously reported for the human ATP8A1-CDC50A complex, and the other in a cavity at the surface of the exoplasmic leaflet of the bilayer. Substitutions in either of the binding sites or along the path between them impaired ATPase and transport activities. Thus, the crevice is the conduit along which phosphatidylserine traverses the membrane (Nakanishi et al. 2020).
Dnf1 of Saccharomyces cerevisiae (P32660)
Inwardly directed phosphatidylcholine, phosphatidyl serine, and lysophosphoethanolamine flippase, Dnf2 (functions with the β-subunit, Lem3) (Elvington et al., 2005; Pomorski et al., 2003; Riekhof and Voelker, 2006; Riekhof et al., 2007). This plasma membrane P-type ATPase (ACT4) is a phospholipid flippase that contributes to endocytosis, protein transport and all polarity (Hua et al., 2002). Transports monoacyl (lyso) phospholipids much better than diacyl phospholipids, but can be mutated to transport diacyl phospholipids (Baldridge et al. 2013). It transports glycosphingolipids (Roland et al. 2019).
Dnf2 of Saccharomyces cerevisiae (Q12675)
The aminophospholipid ATPase1 (ALA1) (mediate chilling tolerance; Gomes et al., 2000). Promotes antiviral silencing (Guo et al. 2017).
ALA1 of Arabidopsis thaliana (P98204)
The phosphatidylserine flippase in photoreceptor disc membranes, ATP8A2 (Coleman et al., 2009). The beta-subunit, CDC50A (TC#8.A.27.1.5), allows the stable expression, assembly, subcellular localization, and lipid transport activity of ATP8A2 (Coleman and Molday, 2011). Missennse mutations in ATP8A2 are associated with cerebellar atrophy and guadrupedal locomotion (Emre Onat et al. 2012). Asparagine-905 of the mammalian phospholipid flippase ATP8A2 is essential for lipid substrate-induced activation of ATP8A2 dephosphorylation (Mikkelsen et al. 2019). Phosphatidylserine flipping by the P4-ATPase, ATP8A2, is electrogenic (Tadini-Buoninsegni et al. 2019).
ATP8A2 of Mus musculus (P98200)
The phospholipid flipping ATPase (contributes to vesicle biogenesis in the secretory and endocytic pathways). Forms heteromeric complexes with ALIS Cdc50-like β-subunits (ALIS1 = Q9LTW0; TC#8.A.27.1.4) promoting functionality (López-Marqués et al., 2010). The beta-subunit, CDC50A, allows the stable expression, assembly, subcellular localization, and lipid transport activity of the P4-ATPase ATP8A2 (Coleman and Molday, 2011). Promotes antiviral silencing (Guo et al. 2017).
Ala2 of Arabidopsis thaliana (P98205)
Ena1 of Ustilago maydis (B5B9V9)
Ena2 of Ustilago maydis (Q4PI59)
P-type Ca2+ ATPase of 1041 aas and 12 TMSs. Found to be essential for bloodstream-form Trypanosoma brucei through a genome-wide RNAi screen (Schmidt et al. 2018).
P-type Ca2+ ATPase of Trypanosoma brucei
Cation_ATPase_N domain-containing protein of 1084 aas and 10 TMSs in a 2 + 2 + 6 TMS arrangement. This P-type Na+/K+ ATPases essential and nonessential for cellular homeostasis and insect pathogenicity of Beauveria bassiana, respectivelly (Mou et al. 2020). Beauveria bassiana is an insect-pathogenic fungus serving as a main source of fungal insecticides worldwide. ENA1a and ENA2b are involved in both transmembrane and vacuolar activities and are essential for cellular cation homeostasis, insect pathogenicity and multiple stress tolerance in B. bassiana (Mou et al. 2020).
Na+-ATPase of Beauveria bassiana (White muscardine disease fungus) (Tritirachium shiotae)