PfATP4 is a 1264-residue P2-type (P-type, TC 3.A.3 family) cation-transporting ATPase of the plasma membrane of the intraerythrocytic malaria parasite Plasmodium falciparum. It is an ATP-driven sodium efflux pump (EC 7.2.2.3): hydrolysis of ATP at its cytoplasmic N/P/A domains is coupled to extrusion of Na(+) across the parasite plasma membrane, with a Km for Na(+) of about 16 mM and for ATP of about 0.23 mM. Because the mature trophozoite induces new permeability pathways in the host erythrocyte membrane, the parasite sits in a Na(+)-rich cytosol, and PfATP4 is the principal mechanism by which it keeps its own cytosolic Na(+) low; loss of pump activity leads to Na(+) accumulation, cytosolic alkalinisation and osmotic swelling of the parasite. The protein belongs to a P-type ATPase subclass restricted to apicomplexans and is most closely related to the fungal and plant ENA Na(+)-ATPases, a group whose similarity to SERCA/PMCA Ca(2+) pumps led to early misassignment of PfATP4 as a Ca(2+)-ATPase; Ca(2+) transport has never been demonstrated and the PfATP4-associated ATPase activity is insensitive to Ca(2+) and to K(+). A cryo-EM structure of the endogenous protein shows a SERCA-like architecture with a conserved cation-binding site between TM4, TM5, TM6 and TM8, and an apicomplexan-specific partner protein (PfABP) on which PfATP4 stability depends. PfATP4 is the target of several chemically unrelated antimalarial chemotypes, including the clinical candidates cipargamin (KAE609) and (+)-SJ733, and resistance mutations map throughout the protein.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000002 | MODIFY | Summary: Generic nucleotide-binding term from the InterPro P-type ATPase cytoplasmic N-domain signature. The nucleotide bound by PfATP4 is specifically ATP, and ATP binding is independently annotated and structurally resolved. Reason: True but less specific than the evidence supports: the cryo-EM structure of endogenous PfATP4 resolves the ATP-binding site, and biochemical assays give a Km for ATP. Replace with the ATP binding descendant the gene already carries. Proposed replacements: ATP binding Supporting Evidence: PMID:41115914 Our structure reveals the molecular details of ATP and ion-binding sites in PfATP4 and provides a structural framework for understanding the spatial organization of resistance-conferring mutations. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: PfATP4 binds and hydrolyses ATP at its cytoplasmic nucleotide-binding domain; the ATP site is resolved in the cryo-EM structure and a Km for ATP of 0.23 mM has been measured for the PfATP4-associated membrane ATPase activity. Reason: Correct and as precise as the evidence allows, but ATP binding is an enabling property of the pump rather than its distinguishing activity; the core molecular function is the ATP-driven Na(+) transport captured by GO:0008554. Supporting Evidence: PMID:41115914 Our structure reveals the molecular details of ATP and ion-binding sites in PfATP4 and provides a structural framework for understanding the spatial organization of resistance-conferring mutations. |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: The PAINT node groups PfATP4 with the fungal and plant ENA-type plasma membrane cation ATPases. Direct experimental work in P. falciparum places PfATP4 in the parasite plasma membrane, where it effluxes Na(+) into the host erythrocyte cytosol. Reason: The phylogenetic inference is confirmed by parasite-specific evidence: UniProt records cell membrane localisation from four independent studies, and the transport physiology (efflux of Na(+) out of the parasite against an inward gradient) requires the parasite surface membrane. Note this is the symbiont's own plasma membrane, not the host erythrocyte membrane. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000643667 SUPPORTS TRANSFER The ancestral node for ENA-type plasma membrane cation pumps; the localisation it asserts is independently confirmed for PfATP4 in the parasite. Supporting Evidence: PMID:29986883 These parasites were characterized by mutations in PfATP4, a plasma membrane type II P-type ATPase reported to belong to a subclass unique to apicomplexan parasites ( 3 ). PMID:23414762 The results are consistent with the hypothesis that PfATP4 is a plasma membrane Na+ efflux pump, similar to the ENA Na+-ATPases of other lower eukaryotes, and a target of the spiroindolone class of antimalarials. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping of the curated "Cell membrane; Multi-pass membrane protein" annotation, which is itself based on experimental localisation in P. falciparum. Reason: Agrees with the IBA row and with direct localisation and physiological evidence in the parasite; the two rows reinforce one claim. Supporting Evidence: PMID:29986883 These parasites were characterized by mutations in PfATP4, a plasma membrane type II P-type ATPase reported to belong to a subclass unique to apicomplexan parasites ( 3 ). |
| GO:0006813 potassium ion transport | IBA GO_REF:0000033 | REMOVE | Summary: Phylogenetic transfer from fungal ENA-type pumps, several of which transport K(+) as well as Na(+). PfATP4 has been assayed directly for K(+) dependence and none was found over the full physiological range. Reason: Target-specific evidence argues against this branch of the inherited function in P. falciparum: the cipargamin-sensitive (PfATP4-associated) ATPase activity in parasite membranes is unaffected by K(+) from 0 to 150 mM, and no K(+) transport by PfATP4 has been demonstrated. The slight K(+) stimulation seen in low-Na(+) conditions was attributed by the authors to a different, K(+)-dependent membrane ATPase. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000643667 SUPPORTS SOURCE BUT NOT TARGET The ancestral node legitimately covers ENA-family pumps with K(+) transport activity in fungi, but PfATP4 has been tested directly and is K(+)-independent. Supporting Evidence: PMID:29986883 K + was without effect on PfATP4-associated ATPase activity when present at concentrations ranging from 0 to 150 m m . PMID:29986883 The slight stimulation of membrane ATPase activity in low [Na + ] conditions by [K + ] of 15–120 m m suggests that there may be an active K + -dependent ATPase in parasite membranes. |
| GO:0006814 sodium ion transport | IBA GO_REF:0000033 | MODIFY | Summary: Correct process assignment inherited from the ENA Na(+)-ATPase clade, and directly supported in P. falciparum: PfATP4 extrudes Na(+) from the parasite cytosol across its plasma membrane. Reason: The claim is right but less specific than the evidence supports. PfATP4 performs directional export of Na(+) across the parasite plasma membrane, so the descendant term GO:0036376 sodium ion export across plasma membrane is the correct level. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000643667 SUPPORTS TRANSFER The Na(+) transport function inherited from the ENA node is correct for PfATP4; only the granularity of the term is at issue, since the direction and membrane are known for this gene. Proposed replacements: sodium ion export across plasma membrane Supporting Evidence: PMID:23414762 The results are consistent with the hypothesis that PfATP4 is a plasma membrane Na+ efflux pump, similar to the ENA Na+-ATPases of other lower eukaryotes, and a target of the spiroindolone class of antimalarials. PMID:29986883 Thus, despite its intracellular location, the mature trophozoite finds itself in a high-Na + environment and must efflux Na + ions across its plasma membrane to maintain a low cytosolic Na + concentration ([Na + ] cyt ). |
| GO:0006874 intracellular calcium ion homeostasis | IBA GO_REF:0000033 | REMOVE | Summary: Transfer from the SERCA/PMCA Ca(2+)-pump side of the P2-type ATPase tree. PfATP4 was originally described as a Ca(2+)-ATPase on sequence grounds and on a Ca(2+)-stimulated ATPase activity in Xenopus oocytes, but Ca(2+) transport has never been demonstrated and later work found the PfATP4-associated ATPase activity to be Na(+)-dependent and insensitive to Ca(2+). Reason: This is the classic misassignment the ENA/SERCA sequence similarity produces, and it is explicitly corrected in the primary literature and in the UniProt CAUTION block. PfATP4 is a Na(+) pump of the parasite plasma membrane, not a Ca(2+) pump, and it has no established role in parasite Ca(2+) homeostasis. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN002450634 SUPPORTS SOURCE BUT NOT TARGET The node gathers SERCA/PMCA Ca(2+)-transporting ATPases, for which the Ca(2+) homeostasis role is well established; PfATP4 sits in the ENA-like apicomplexan subclass of the same P2 family and does not share it. Supporting Evidence: PMID:23414762 There is evidence for it having an associated Ca2+-dependent ATPase activity (Krishna et al., 2001); however, this has not been confirmed (Rottmann et al., 2010), and a Ca2+ transport function has not been demonstrated directly. PMID:23414762 The ENA ATPases are closely related to sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) and plasma membrane Ca2+-ATPases (PMCA), leading some Na+-ATPases within this family to be misannotated as Ca2+-ATPases (Benito et al., 2002). PMID:29986883 Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na + transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K + ] or [Ca 2+ ]. |
| GO:0008554 P-type sodium transporter activity | IBA GO_REF:0000033 | ACCEPT | Summary: The defining molecular function: ATP hydrolysis coupled to Na(+) translocation out of the cell (EC 7.2.2.3). Inherited from the ENA Na(+)-ATPase node and confirmed biochemically in parasite membranes, with a Km for Na(+) of ~16 mM and loss of Na(+) affinity in resistance mutants. Reason: Directly supported by Na(+)-dependent, cipargamin-sensitive ATPase assays on P. falciparum membranes, by Na(+) efflux measurements in intact parasites, and by the conserved SERCA-like cation site in the cryo-EM structure. This is the core function of the gene. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000643667 SUPPORTS TRANSFER The ENA Na(+)-ATPase node is the right placement for this function, and the target has its own direct biochemical evidence for it. Supporting Evidence: PMID:29986883 Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na + transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K + ] or [Ca 2+ ]. PMID:23414762 The results are consistent with the hypothesis that PfATP4 is a plasma membrane Na+ efflux pump, similar to the ENA Na+-ATPases of other lower eukaryotes, and a target of the spiroindolone class of antimalarials. |
| GO:0008554 P-type sodium transporter activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 7.2.2.3 (Na(+)-transporting ATPase) mapping. The EC number is itself curated in UniProt from four experimental studies of PfATP4 catalytic activity. Reason: The EC-based row makes the same, correct claim as the IBA row and is grounded in the parasite's own catalytic-activity evidence (Na(+)(in) + ATP + H2O = Na(+)(out) + ADP + phosphate + H(+)). Supporting Evidence: PMID:29986883 Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na + transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K + ] or [Ca 2+ ]. |
| GO:0008556 P-type potassium transmembrane transporter activity | IBA GO_REF:0000033 | REMOVE | Summary: K(+)-transport branch of the ENA-family function, transferred phylogenetically. Direct assays of PfATP4-associated ATPase activity show no K(+) dependence. Reason: No K(+) transport or K(+) dependence has been demonstrated for PfATP4; the cipargamin-sensitive ATPase activity is unaffected by K(+) across 0-150 mM, and all parasite phenotypes of PfATP4 inhibition are Na(+)-driven. The ancestral K(+) capability of fungal ENA pumps should not be asserted for this protein. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000643667 SUPPORTS SOURCE BUT NOT TARGET The node's fungal members (ENA1/ENA2, cta3) transport K(+) as well as Na(+); the apicomplexan members have only Na(+) evidence, and PfATP4 has been tested and found K(+)-independent. Supporting Evidence: PMID:29986883 K + was without effect on PfATP4-associated ATPase activity when present at concentrations ranging from 0 to 150 m m . |
| GO:0016020 membrane | IEA GO_REF:0000120 | MODIFY | Summary: Generic membrane localisation from the P-type ATPase signature and ARBA rules. PfATP4 is a multi-pass protein of the parasite plasma membrane specifically. Reason: True but uninformative compared with the evidence available: the specific compartment, plasma membrane, is established experimentally and is already annotated to this gene. Proposed replacements: plasma membrane Supporting Evidence: PMID:29986883 These parasites were characterized by mutations in PfATP4, a plasma membrane type II P-type ATPase reported to belong to a subclass unique to apicomplexan parasites ( 3 ). |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | MODIFY | Summary: PfATP4 does hydrolyse ATP, but as the energising half-reaction of Na(+) translocation; the measured parasite-membrane activity is a Na(+)-dependent, cipargamin-sensitive ATPase. Reason: Correct but less specific than the evidence supports: the hydrolysis is obligately coupled to Na(+) export, so the P-type transporter activity term the gene already carries (GO:0008554, which entails the coupled ATP hydrolysis) is the right level of description. Proposed replacements: P-type sodium transporter activity Supporting Evidence: PMID:29986883 Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na + transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K + ] or [Ca 2+ ]. |
| GO:0034220 monoatomic ion transmembrane transport | IBA GO_REF:0000033 | MODIFY | Summary: Family-level ion transport term placed at a deep P-type ATPase node spanning plant H(+)-ATPases, animal Na(+)/K(+) and Ca(2+) pumps and fungal ENA pumps. True of PfATP4, which transports Na(+) across its plasma membrane. Reason: The ion transported and the direction are known for this gene, so the term should be refined to the supported descendant rather than left at the family level. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN001943383 SUPPORTS TRANSFER A deep P-type ATPase node whose only safe common claim is ion transmembrane transport; the transfer is sound, but PfATP4 has ion-specific and directional evidence that supports a more informative term. Proposed replacements: sodium ion export across plasma membrane Supporting Evidence: PMID:29986883 Thus, despite its intracellular location, the mature trophozoite finds itself in a high-Na + environment and must efflux Na + ions across its plasma membrane to maintain a low cytosolic Na + concentration ([Na + ] cyt ). |
| GO:0034220 monoatomic ion transmembrane transport | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning assignment of the same family-level ion transport term. Reason: Same claim as the IBA row and reviewed consistently: correct, but refinable to the specific, experimentally supported Na(+) export process. Proposed replacements: sodium ion export across plasma membrane Supporting Evidence: PMID:23414762 The results are consistent with the hypothesis that PfATP4 is a plasma membrane Na+ efflux pump, similar to the ENA Na+-ATPases of other lower eukaryotes, and a target of the spiroindolone class of antimalarials. |
| GO:0035725 sodium ion transmembrane transport | IEA GO_REF:0000108 | MODIFY | Summary: Logical inference from the P-type sodium transporter activity annotation. Correct, and the direction of transport is known: efflux from the parasite. Reason: Refinable to the descendant that captures the established directionality of the pump, sodium ion export across the plasma membrane. Proposed replacements: sodium ion export across plasma membrane Supporting Evidence: PMID:29986883 Thus, despite its intracellular location, the mature trophozoite finds itself in a high-Na + environment and must efflux Na + ions across its plasma membrane to maintain a low cytosolic Na + concentration ([Na + ] cyt ). |
| GO:0071805 potassium ion transmembrane transport | IEA GO_REF:0000108 | REMOVE | Summary: Logical inference from the P-type potassium transporter activity annotation, which is itself an unsupported phylogenetic transfer. Reason: Follows the removal of GO:0008556: PfATP4-associated ATPase activity is K(+)-independent and no K(+) transport has been shown, so the inferred process term has no basis in this organism. Supporting Evidence: PMID:29986883 K + was without effect on PfATP4-associated ATPase activity when present at concentrations ranging from 0 to 150 m m . |
| GO:0036376 sodium ion export across plasma membrane | IDA PMID:23414762 Na(+) regulation in the malaria parasite Plasmodium falcipar... | NEW | Summary: Proposed annotation capturing what PfATP4 actually does: ATP-dependent extrusion of Na(+) from the parasite cytosol across the parasite plasma membrane, measured as Na(+) efflux after an imposed Na(+) load in intact parasites and as Na(+)-dependent, inhibitor-sensitive ATPase activity in parasite membranes. Reason: PfATP4 itself performs the translocation step, so it participates in the process rather than merely being required for it. This is the replacement term proposed for the general transport rows (GO:0006814, GO:0034220 twice, GO:0035725) and is supported by direct measurements in P. falciparum. Supporting Evidence: PMID:23414762 The results are consistent with the hypothesis that PfATP4 is a plasma membrane Na+ efflux pump, similar to the ENA Na+-ATPases of other lower eukaryotes, and a target of the spiroindolone class of antimalarials. PMID:29986883 Thus, despite its intracellular location, the mature trophozoite finds itself in a high-Na + environment and must efflux Na + ions across its plasma membrane to maintain a low cytosolic Na + concentration ([Na + ] cyt ). |
| GO:0006883 intracellular sodium ion homeostasis | IMP PMID:36180431 A G358S mutation in the Plasmodium falciparum Na(+) pump PfA... | NEW | Summary: PfATP4 sets the low resting cytosolic Na(+) concentration of the intraerythrocytic parasite. Point mutations in PfATP4 that lower its Na(+) affinity raise resting cytosolic Na(+), and chemical inhibition causes Na(+) accumulation and osmotic swelling. Reason: The homeostatic set point is established by the pump's own transport activity, so PfATP4 performs the work of this process rather than merely being required for it. Mutations within the protein that change its Na(+) affinity shift the set point, which is participation evidence rather than necessity evidence alone. Supporting Evidence: PMID:36180431 The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite's resting cytosolic [Na+]. PMID:29555632 Here we show that six structurally diverse PfATP4-associated compounds, including the clinical candidate KAE609 (cipargamin), induce swelling of both isolated blood-stage parasites and intact parasitized erythrocytes. |
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Download this section (compressed HTML)Q: Is H(+) the counter-transported ion of PfATP4, and what is the Na(+):H(+):ATP stoichiometry of the transport cycle?
Q: Does PfATP4 function in mosquito and liver stages, where the parasite's ionic environment differs from the Na(+)-rich infected erythrocyte cytosol, or is its role restricted to the asexual blood stage?
Q: Is PfABP a regulatory subunit of PfATP4 (analogous to the Na(+)/K(+)-ATPase beta subunit) or purely a stability factor, and should the PfATP4-PfABP complex be represented as a protein-containing complex in GO?
Experiment: Reconstitute endogenously purified PfATP4 (with and without PfABP) into proteoliposomes and measure ATP-dependent Na(+) flux, internal pH change and membrane potential simultaneously, to establish the counter-ion and the transport stoichiometry.
Experiment: Test for K(+) and Ca(2+) translocation by the reconstituted pump using radiotracer or fluorescent-indicator flux assays, to settle directly the K(+) and Ca(2+) annotations removed here on the basis of ATPase-activity data.
Experiment: Conditional knockdown or stage-specific depletion of PfATP4 in gametocytes, mosquito stages and liver stages, with cytosolic Na(+) imaging, to determine whether Na(+) extrusion by PfATP4 is required outside the asexual blood stage.
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The counter-ion of PfATP4 is unknown. Inhibition of the pump produces a Na(+)-dependent cytosolic alkalinisation, which has been read as a cessation of H(+) import, but no direct measurement of H(+) (or any other ion) counter-transport coupled to Na(+) extrusion exists, so the transport stoichiometry of the pump is undetermined.
BIOLOGY MF_DARK
What is known: That PfATP4 exports Na(+) in an ATP-dependent manner with Km(Na+) ~16 mM, and that its activity increases with increasing pH, is established biochemically in parasite membranes.
Significance: Whether PfATP4 is a Na(+)/H(+)-ATPase or an electrogenic Na(+) pump determines how its inhibition perturbs parasite pH as well as Na(+), and therefore which of the two disruptions kills the parasite; this bears directly on the mechanism of cipargamin and (+)-SJ733.
What would resolve it: Reconstitution of purified PfATP4 (now feasible given the endogenous purification used for the cryo-EM structure) into proteoliposomes with simultaneous measurement of Na(+) movement, pH change and membrane potential.
Gap: The functional role of the apicomplexan-specific partner protein PfABP is unknown: it is required for PfATP4 protein stability, but whether it also modulates the pump's catalytic cycle, ion affinity or trafficking, in the manner of a Na(+)/K(+) ATPase beta subunit, has not been tested.
BIOLOGY MF_DARK
What is known: The cryo-EM structure of endogenous PfATP4 shows a stable PfATP4-PfABP complex with a conserved interface, and conditional knockdown of PfABP reduces PfATP4 levels within 24 h and kills the parasite.
Significance: If PfABP is a regulatory subunit rather than only a stabiliser, the interface is a druggable site distinct from the inhibitor-binding pockets where resistance mutations arise.
What would resolve it: Separation-of-function mutations at the PfATP4-PfABP interface that preserve PfATP4 abundance, combined with Na(+)-dependent ATPase assays and cytosolic Na(+) measurements in the mutant parasites.
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