PfCRT is a 10-transmembrane integral membrane protein of the drug/metabolite transporter superfamily that resides in the membrane of the digestive (food) vacuole of the intraerythrocytic malaria parasite. The digestive vacuole is the acidic compartment in which endocytosed host erythrocyte cytosol, chiefly haemoglobin, is proteolysed. PfCRT exports the resulting host-derived peptides of roughly four to eleven residues from the vacuolar lumen into the parasite cytosol in a proton-dependent, saturable manner, thereby supplying amino acids for parasite metabolism and relieving the osmotic load that accumulating peptides would otherwise impose on the organelle; loss or impairment of this activity swells the vacuole and slows asexual blood-stage growth. Purified protein in proteoliposomes additionally exports basic amino acids, polyamines and organic cations down a proton electrochemical gradient, although these substrates have not been corroborated in the parasite. Field-derived polymorphisms, above all K76T, reshape the central substrate cavity so that the transporter also effluxes protonated quinoline drugs, including chloroquine, quinine, quinidine, lumefantrine and mefloquine, out of the vacuole and away from their haem target; these same substitutions reduce peptide transport capacity, which is the structural basis of the fitness cost that accompanies chloroquine resistance. Trafficking of the protein to the vacuolar membrane passes through the secretory pathway and depends on phosphorylation of a C-terminal threonine.
Definition: The lipid bilayer surrounding a food vacuole, the vacuole within a parasite used for digestion of host cell cytoplasm. An example of this component is the digestive vacuole membrane of intraerythrocytic Plasmodium, which harbours transporters including PfCRT and PfMDR1 and the vacuolar proton pumps that acidify the lumen.
Justification: GO contains GO:0020020 food vacuole but no term for its bounding membrane, so apicomplexan digestive-vacuole membrane proteins can only be annotated to the generic GO:0005774 vacuolar membrane. The membrane is the functional site of several of the best-studied antimalarial-resistance determinants, and a dedicated term would let those annotations state the compartment precisely.
Parent term: vacuolar membrane
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005774 vacuolar membrane | IEA GO_REF:0000044 | ACCEPT | Summary: PfCRT is a multi-pass protein of the digestive (food) vacuole membrane, the compartment in which host haemoglobin is digested and in which chloroquine acts. The UniProt subcellular-location mapping is correct. Reason: Digestive-vacuole membrane residence is established experimentally (PMID:11090624, PMID:20015114) and is the functional site of peptide export (PMID:32764664). GO has GO:0020020 food vacuole but no "food vacuole membrane" child of vacuolar membrane, so GO:0005774 is the most precise membrane term currently available and is kept rather than modified; a food vacuole membrane term is proposed below. Supporting Evidence: PMID:11090624 This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. PMID:20015114 Our data establish PfCRT as a phosphoprotein and suggest that phosphorylation of threonine 416 is a possible deciding signal for the sorting of PfCRT to the digestive vacuolar membrane. |
| GO:0005783 endoplasmic reticulum | IDA PMID:20015114 Trafficking of the phosphoprotein PfCRT to the digestive vac... | KEEP AS NON CORE | Summary: Localization to the endoplasmic reticulum was observed in the course of dissecting how PfCRT is sorted to the digestive vacuolar membrane; the ER is the entry point of the secretory route taken by this multi-pass membrane protein. Reason: The curator of this IDA read the full text of a dedicated trafficking study, so the observation is retained. It is nonetheless a biosynthetic transit compartment rather than a site of function: the transporter does its work in the digestive vacuole membrane, which that same study identifies as its destination. The term is as precise as the evidence allows, so it is kept as non-core rather than refined. No quotation is attached: only the abstract of PMID:20015114 is cached and it does not mention the endoplasmic reticulum, so the IDA rests on full-text data the curator read and we cannot quote. |
| GO:0009410 response to xenobiotic stimulus | IDA PMID:15240840 The malaria parasite's chloroquine resistance transporter is... | KEEP AS NON CORE | Summary: PfCRT variants mediate efflux of antimalarial drugs from the digestive vacuole, which is a cellular response to those xenobiotics; the capability is strongly allele-dependent and is not the protein's natural role. Reason: Drug handling by PfCRT is real and well documented for resistance-conferring isoforms (PMID:25378409, PMID:35507548) and, for purified protein in proteoliposomes, also for the chloroquine-sensitive form (PMID:25733858), so the claim is kept. It is peripheral to the transporter's physiological function, which is export of host-derived peptides from the vacuole (PMID:32764664). The cited reference is a sequence-analysis paper that performed no transport experiment, so the IDA evidence code is a poor fit; the claim is supported by the later experimental literature instead, and the term is kept as non-core rather than removed. Supporting Evidence: PMID:25378409 The work described here provides direct evidence of the ability of a chloroquine resistance-conferring form of PfCRT to transport quinine, quinidine, and verapamil. PMID:15240840 The mutation is predicted to alter the selectivity of the protein such that it is able to transport the cationic (protonated) form of chloroquine down its steep concentration gradient, out of the acidic vacuole, and therefore away from its site of action. |
| GO:0016020 membrane | TAS PMID:15240840 The malaria parasite's chloroquine resistance transporter is... | TOO GENERAL | Summary: A generic membrane assertion taken from the statement that PfCRT is an integral membrane protein with ten transmembrane domains. Reason: The claim is true but conveys nothing beyond what GO:0005774 vacuolar membrane, accepted in this review, already entails. No action is needed on the underlying biology; the term is simply uninformative here. Supporting Evidence: PMID:15240840 The malaria parasite's chloroquine resistance transporter (CRT) is an integral membrane protein localized to the parasite's acidic digestive vacuole. |
| GO:0020020 food vacuole | IDA PMID:11090624 Mutations in the P. falciparum digestive vacuole transmembra... | ACCEPT | Summary: Immuno-localization in the paper that identified pfcrt as the determinant of chloroquine resistance placed the protein in the parasite's digestive (food) vacuole, the apicomplexan compartment in which ingested host cytosol is digested. Reason: This is the core, correctly apicomplexan location term, confirmed by subsequent work on vacuolar morphology in PfCRT mutants (PMID:26420308) and by the in-situ peptide-transport study (PMID:32764664). Supporting Evidence: PMID:11090624 This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. PMID:26420308 causing the development of enlarged food vacuoles. |
| GO:0022857 transmembrane transporter activity | IBA GO_REF:0000033 | MODIFY | Summary: The phylogenetic inference that this CRT-like family member is a transmembrane transporter is correct, and PfCRT's own experimental characterization is among the descendant evidence behind the ancestral node. Reason: Transport across a membrane is certainly right, but the substrate class is now known: PfCRT exports host-haemoglobin-derived peptides of four to eleven residues from the digestive vacuole, demonstrated both in oocytes and in parasites (PMID:32764664), and takes up polypeptides in proteoliposomes (PMID:25733858). The evidence supports the descendant term GO:0035673 oligopeptide transmembrane transporter activity, which is where this row belongs. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN001263463 SUPPORTS TRANSFER The ancestral CRT-like node correctly asserts transmembrane transport, a claim the target's own experimental data support; only the granularity is at issue, since the substrate class is now established for this member. Proposed replacements: oligopeptide transmembrane transporter activity Supporting Evidence: PMID:32764664 We show that PfCRT functions to export host-derived peptides 4–11 residues in length from the parasite’s DV, and that the protein does not behave as a non-specific transporter of other metabolites and/or ions. PMID:25733858 All PfCRTs tested actively took up tetraethylammonium, verapamil, CQ, basic amino acids, polypeptides, and polyamines at the expense of an electrochemical proton gradient. |
| GO:0034635 glutathione transport | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Glutathione transport is propagated to PfCRT from the plant CRT-like (CLT) proteins, plastid-membrane glutathione transporters, together with the oocyte result obtained with mutant pfcrt alleles. Reason: The plant donors sit on a plastid-targeted branch of the CRT-like family whose substrate preference need not extend to the apicomplexan digestive-vacuole member, and the one experimental result in P. falciparum concerns resistance-conferring alleles rather than the reference 3D7 protein (PMID:23256874). The most thorough substrate analysis screened glutathione directly, in oocytes and in parasites, and concluded that PfCRT is a peptide exporter that does not transport other metabolites non-specifically (PMID:32764664). The claim is not simply false, since mutant isoforms did move GSH in a heterologous system, so it is flagged as an over-annotation of the family and of the resistant alleles rather than removed. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN001263463 SUPPORTS SOURCE BUT NOT TARGET The ancestral CRT-like node carries glutathione transport largely on the strength of the Arabidopsis CLT plastid transporters; in-situ work on the apicomplexan digestive-vacuole member does not support the substrate for this target. Supporting Evidence: PMID:32764664 We screened many solutes, including Fe2+, Fe3+ and glutathione, but were unable to identify the co-substrate (Supplementary Data 1). PMID:23256874 We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. |
| GO:0034635 glutathione transport | IDA PMID:23256874 Glutathione transport: a new role for PfCRT in chloroquine r... | MARK AS OVER ANNOTATED | Summary: Xenopus oocytes expressing mutant pfcrt alleles from chloroquine-resistant parasites took up glutathione selectively, which the authors interpret as GSH redistribution into the digestive vacuole contributing to resistance. Reason: The experiment is sound but the authors themselves attribute the activity to the mutant alleles, not to the wild-type 3D7 protein that this entry represents, and the proposed role is a resistance mechanism rather than a physiological function. A later study that screened glutathione explicitly, in oocytes and in parasites, found peptides to be the substrates and no evidence for non-specific metabolite transport (PMID:32764664). Retained as an allele-specific over-annotation rather than removed, since the oocyte transport measurement itself stands. Supporting Evidence: PMID:23256874 We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. PMID:32764664 a glutathione transporter32, a non-specific transporter of inorganic and organic cations as well as of many other metabolites33, or an Fe2+/Fe3+ transporter34. |
| GO:0042908 xenobiotic transport | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: The InterPro family signature for PfCRT carries xenobiotic transport, reflecting the transporter's well-documented role in moving antimalarial drugs out of the digestive vacuole. Reason: Drug efflux is genuine for resistance-conferring isoforms (PMID:25378409, PMID:31776516, PMID:35507548) and chloroquine uptake was also measured with purified chloroquine-sensitive protein (PMID:25733858), so the process term is defensible for the gene product. It is a secondary, largely allele-dependent capability rather than the natural function, which is peptide export (PMID:32764664), so it is kept as non-core. Supporting Evidence: PMID:31776516 The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. PMID:25733858 All PfCRTs tested actively took up tetraethylammonium, verapamil, CQ, basic amino acids, polypeptides, and polyamines at the expense of an electrochemical proton gradient. |
| GO:0042910 xenobiotic transmembrane transporter activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Family-level assignment of drug transporter activity, matching the experimentally demonstrated transport of chloroquine and related antimalarials by PfCRT isoforms. Reason: Supported by direct transport measurements on the protein (PMID:25378409, PMID:31776516, PMID:35507548) and consistent with the IMP row for the same term, but an acquired, polymorphism-dependent activity rather than the core evolved function of the transporter. Supporting Evidence: PMID:35507548 demonstrate that these PfCRT isoforms transport all 3 drugs, whereas wild-type PfCRT does not. PMID:31776516 The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. |
| GO:0042910 xenobiotic transmembrane transporter activity | IMP PMID:25378409 Multiple drugs compete for transport via the Plasmodium falc... | KEEP AS NON CORE | Summary: Kinetic analysis in the Xenopus oocyte system showed that mutant PfCRT carries chloroquine, quinine, quinidine and verapamil, with the drugs competing at distinct but interacting sites, establishing the protein as a multidrug carrier. Reason: The experimental support is strong, and the activity is kept. It is nonetheless peripheral to the physiological role: the capability is conferred by resistance-associated substitutions, the wild-type form did not transport chloroquine in this system, and the same substitutions reduce the transporter's capacity for its natural peptide substrates (PMID:32764664). Supporting Evidence: PMID:25378409 mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. PMID:25378409 chloroquine away from its target, whereas the wild-type form of PfCRT does not. |
| GO:0055085 transmembrane transport | IEA GO_REF:0000108 | MODIFY | Summary: Process term inferred automatically from the transmembrane transporter activity annotation. Reason: Correct but less specific than the evidence supports. The transported cargo is known: host-haemoglobin-derived peptides moved from the digestive vacuole lumen to the parasite cytosol, shown in vitro and in situ (PMID:32764664). GO:0015833 peptide transport is the supported descendant and replaces the generic term. Proposed replacements: peptide transport Supporting Evidence: PMID:32764664 the PfCRT-mediated transport of peptides from the DV to the cytosol serves to (1) provide a source of amino acids to support the parasite’s high growth rate and (2) reduce peptide levels within the DV and thereby prevent the osmotic stress, swelling and dysfunction of this organelle. |
| GO:0035673 oligopeptide transmembrane transporter activity | IDA PMID:32764664 The natural function of the malaria parasite's chloroquine r... | NEW | Summary: PfCRT expressed in Xenopus oocytes transports host-derived peptides of four to eleven residues in a saturable, proton-dependent manner, and the corresponding peptides accumulate in parasites whose PfCRT has reduced transport capacity. Reason: The protein itself performs the translocation step, measured directly on the purified or heterologously expressed transporter, so this is participation and not mere necessity. No existing annotation names the physiological substrate class; this term supplies it and is the proposed replacement for the generic GO:0022857 IBA row. Supporting Evidence: PMID:32764664 We show that PfCRT functions to export host-derived peptides 4–11 residues in length from the parasite’s DV, and that the protein does not behave as a non-specific transporter of other metabolites and/or ions. PMID:25733858 All PfCRTs tested actively took up tetraethylammonium, verapamil, CQ, basic amino acids, polypeptides, and polyamines at the expense of an electrochemical proton gradient. |
| GO:0015833 peptide transport | IDA PMID:32764664 The natural function of the malaria parasite's chloroquine r... | NEW | Summary: Export of haemoglobin-derived peptides from the digestive vacuole to the cytosol is the physiological process PfCRT carries out, providing amino acids for growth and preventing osmotic swelling of the organelle. Reason: PfCRT executes the translocation step of this transport process itself, both in oocytes and in parasites, so it is a participant rather than a substrate or a merely required factor. The process is not covered by any existing row except the generic GO:0055085, for which it is the proposed replacement. Supporting Evidence: PMID:32764664 the PfCRT-mediated transport of peptides from the DV to the cytosol serves to (1) provide a source of amino acids to support the parasite’s high growth rate and (2) reduce peptide levels within the DV and thereby prevent the osmotic stress, swelling and dysfunction of this organelle. PMID:26420308 causing the development of enlarged food vacuoles. |
Loading supporting content…
Download this section (compressed HTML)Q: What is the cytosolic co-substrate whose counter-movement drives peptide export by PfCRT, and is it exchanged stoichiometrically with the peptide?
Suggested experts: Rowena E. Martin, Sarah H. Shafik
Q: Do the basic amino acids, polyamines and organic cations transported by purified PfCRT in proteoliposomes represent genuine physiological substrates in the parasite, or in-vitro promiscuity of a peptide carrier reconstituted without its co-substrate?
Suggested experts: Yoshinori Moriyama, Rowena E. Martin
Q: Is glutathione movement across the digestive vacuole membrane in chloroquine-resistant parasites mediated by mutant PfCRT itself, or by another transporter whose activity the mutant alleles alter indirectly?
Suggested experts: Sylke Mueller, Rowena E. Martin
Q: How much of the amino-acid supply of the intraerythrocytic parasite flows through PfCRT-mediated peptide export versus intravacuolar proteolysis to free amino acids followed by other carriers?
Experiment: Reconstitute purified 3D7 PfCRT into proteoliposomes of defined internal composition and screen candidate counter-substrates by trans-stimulation of radiolabelled peptide uptake, combining an untargeted metabolomic comparison of digestive-vacuole and cytosolic fractions to shortlist candidates. Confirm hits by measuring coupled fluxes and the apparent stoichiometry as a function of the imposed pH gradient and membrane potential.
Hypothesis: Peptide export by PfCRT is obligatorily coupled to counter-transport of a specific cytosolic solute, and identifying it will explain the protein's proton dependence.
Type: reconstituted transport assay with metabolomics
Experiment: Use conditional knockdown or a rapid chemical-genetic shutdown of PfCRT in synchronous parasites and quantify, by isotope-labelled metabolomics of isolated digestive vacuoles and cytosol, which of the proposed substrates accumulate in the vacuole on loss of transport. Peptides should accumulate; free basic amino acids and polyamines should not if their transport is an artefact of reconstitution.
Hypothesis: The amino acids and polyamines transported by purified PfCRT are not carried in the parasite, so the in-vitro polyspecificity reflects the reconstituted system.
Type: inducible knockdown with compartment-resolved metabolomics
Experiment: Compare glutathione concentrations in isolated digestive vacuoles of isogenic lines carrying wild-type, Dd2 and 7G8 pfcrt alleles, using a vacuole-targeted genetically encoded glutathione redox sensor, and test whether PfCRT inhibitors that block peptide transport also abolish the difference. A persistent difference under transport blockade would place the glutathione effect downstream of PfCRT rather than on it.
Hypothesis: Glutathione flux into the digestive vacuole of chloroquine-resistant parasites does not require PfCRT-mediated transport.
Type: compartment-targeted redox-sensor imaging in isogenic lines
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)