Kelch13 (PfK13, encoded by PF3D7_1343700) is a 726-residue BTB/POZ plus six-bladed Kelch-repeat beta-propeller protein of the intraerythrocytic malaria parasite Plasmodium falciparum. It carries a disordered N-terminal region and a coiled coil ahead of the BTB domain (residues 350-452) and the C-terminal propeller, and it self-assembles: the BTB-propeller fragment crystallizes as a tight dimer and the full-length protein forms an N-terminus-driven hexamer in solution. The protein concentrates in discrete foci at the parasite periphery, the so-called Kelch13 compartment, adjacent to cytostomes, and is additionally seen at the endoplasmic reticulum and on Rab-positive vesicles. Its established cellular role is at the uptake step of host nutrient acquisition: together with a set of Kelch13-compartment partners it is required for endocytosis of host erythrocyte cytosol, principally haemoglobin, which is internalized at cytostomal invaginations of the parasite plasma membrane and delivered to the digestive (food) vacuole for proteolysis into amino acids. The amount of functional Kelch13 sets the rate of this endocytosis, so the protein governs how much haemoglobin-derived material, including free haem, reaches the parasite. Because artemisinins are activated by haem released from digested haemoglobin, propeller substitutions such as M476I, R539T and C580Y, or reduced Kelch13 abundance, lower drug activation in ring stages and produce the partial artemisinin resistance phenotype measured by the ring-stage survival assay; these alleles carry a fitness cost proportional to the resistance they confer. Kelch13 has also been reported to bind the parasite phosphatidylinositol 3-kinase PfPI3K (PF3D7_0515300), which was interpreted as a cullin-RING ligase substrate adaptor role by analogy with mammalian KEAP1, but no catalytic activity of its own is known.
Definition: An endocytosis process in which an intracellular parasite internalizes cytosol of its host cell through a cytostome, a stable invagination of the parasite plasma membrane, producing vesicles that deliver the host material to a digestive compartment. An example is the uptake of host erythrocyte haemoglobin by intraerythrocytic Plasmodium, which requires Kelch13 and its compartment partners and delivers haemoglobin to the food vacuole.
Justification: GO:0006897 endocytosis has nine children, none of which covers ingestion of host cell cytosol through a cytostome, so apicomplexan proteins of this pathway can only be annotated to the bare parent. The process is the best-studied route of nutrient acquisition in malaria parasites, is genetically separable from clathrin- and actin-dependent endocytosis, and is the pathway through which artemisinin resistance arises, so a dedicated term would let the growing set of Kelch13-compartment proteins be annotated precisely.
Parent term: endocytosis
Supporting Evidence:
Definition: A focal cytoplasmic structure at the periphery of an intraerythrocytic Plasmodium parasite, adjacent to cytostomes, at which Kelch13 and its interaction partners assemble and from which endocytosis of host cell cytosol is initiated. It is distinct from the endoplasmic reticulum and from the cytostome itself.
Justification: Imaging and proximity-labelling studies define a discrete, reproducible site occupied by Kelch13 and a growing list of Kelch13 interaction candidates, but GO offers only GO:0031910 cytostome, to which these proteins are adjacent rather than resident, or uninformative parents such as cell periphery. A dedicated component term would let the localization of this protein set be stated without over-asserting cytostome membership.
Parent term: cell periphery
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:25874676 A molecular mechanism of artemisinin resistance in Plasmodiu... | REMOVE | Summary: The IntAct-curated interaction partner UniProtKB:Q8I3V5 is PF3D7_0515300, the parasite phosphatidylinositol 3-kinase PfPI3K. Co-immunoprecipitation showed PfPI3K bound to wild-type Kelch13 and reduced binding to the C580Y propeller mutant, which the authors interpreted as Kelch13 acting as a substrate adaptor delivering PfPI3K for ubiquitin-dependent degradation. Reason: Removal is a judgement about the term, not about the experiment: the PfPI3K interaction is accepted as real and remains recorded in IntAct, but GO:0005515 states nothing about what Kelch13 does and so adds no functional information to this review. The obvious informative replacement, GO:1990756 ubiquitin-like ligase-substrate adaptor activity, cannot be substituted: the adaptor model was argued by analogy with the mammalian KEAP1 orthologue rather than demonstrated with a parasite cullin-RING ligase, and an independent Kelch13 interactome recovered endoplasmic-reticulum proteins and Rab GTPases rather than ligase components. With no supportable molecular function to modify to, the bare binding row is dropped and the open question is recorded in suggested_questions. Supporting Evidence: PMID:25874676 Further analysis revealed that immunopurified (IP) PfPI3K was bound to PfKelch13WT and this was reduced by the C580Y mutation (Fig. 2e). PMID:25874676 The mammalian orthologue of PfKelch13 functions is a substrate adapter for an E3 ubiquitin ligase PMID:32310999 Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. |
| GO:0009410 response to xenobiotic stimulus | IDA PMID:24352242 A molecular marker of artemisinin-resistant Plasmodium falci... | MARK AS OVER ANNOTATED | Summary: The cited study selected an artemisinin-resistant line under long-term drug pressure, found the PF3D7_1343700 M476I substitution, and correlated propeller alleles in Cambodian isolates with ring-stage survival and in vivo clearance. What it establishes is that Kelch13 genotype determines susceptibility to the drug, not that the protein executes a cellular response to a xenobiotic. Reason: Resistance here is a loss of Kelch13 activity, not an induced response to the compound: mutant or depleted Kelch13 takes up less host haemoglobin, so less haem is liberated to activate artemisinin, and the parasite survives a pulse of drug it never responded to. Wild-type Kelch13 performs no step of a xenobiotic response, is not induced by the drug, and the phenotype is a property of particular alleles rather than of the gene product's normal function. The term is not flatly wrong, since the protein does determine the outcome of drug exposure, so it is marked as over-annotated rather than removed, and the function that actually underlies the phenotype is added below as endocytosis of host cell cytosol. Supporting Evidence: PMID:24352242 These data indicate that the PF3D7_1343700 M476I mutation increased the resistance of F32-Tanzania to DHA in the RSA0–3h. PMID:24352242 Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. PMID:31896710 ARTs are activated by degradation products of hemoglobin. Hence, reduced activity of Kelch13 and its interactors diminishes hemoglobin endocytosis and thereby ART activation, resulting in parasite resistance. |
| GO:0051260 protein homooligomerization | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: The InterPro2GO call derives from the T1-type BTB domain (IPR003131), a module that mediates self-association, and it is confirmed directly for this protein: the BTB-propeller fragment forms tight crystallographic dimers and solution scattering shows a stable N-terminus-driven hexamer. Reason: The electronic inference is right and is independently corroborated by structural data on Kelch13 itself, so it is kept. Self-assembly is nonetheless an architectural property that underpins the protein's scaffolding role rather than the biological process the gene product exists to carry out, which is endocytic uptake of host cell cytosol; it is therefore retained as non-core. The term is as precise as the evidence supports, so no refinement is proposed. Supporting Evidence: PMID:35092154 PfKelch13 forms a stable hexamer in P6 symmetry, (b) interactions of the N-termini drive the hexameric assembly, and (c) the six KRP domains project independently in space, forming a cauldron-like architecture. PMID:35092154 To date, only two crystal structures of the BTB/POZ-KRP domains as tight dimers are available, which limits structure-based predictions and interpretation of its role(s) in inducing clinical artemisinin resistance. file:PLAF7/K13/K13-uniprot.txt DOMAIN 350..452 |
| GO:0006897 endocytosis | IMP PMID:31896710 A Kelch13-defined endocytosis pathway mediates artemisinin r... | NEW | Summary: Conditional inactivation of Kelch13 reduces endocytosis of haemoglobin from the host erythrocyte, as does a resistance-conferring propeller mutation, and the cellular level of Kelch13 sets the rate at which this endocytosis proceeds. The protein acts at the uptake step, at cytostomal invaginations of the parasite plasma membrane, in company with the other Kelch13-compartment proteins. Reason: This is the function that the whole Kelch13 literature converges on and it is absent from GOA. Kelch13 does part of the work of the process rather than merely being required for it: it is a resident component of the compartment at which endocytosis of host cytosol is initiated, its partners there carry vesicle trafficking and endocytosis domains, and titrating Kelch13 titrates the rate of uptake, which is the behaviour of machinery rather than of cargo or of an upstream permissive factor. GO:0006897 is the most specific existing term: none of its children (receptor-mediated, pinocytosis, phagocytosis, caveolin-mediated, clathrin- and caveolae-independent, ubiquitin-dependent, sperm entry, pre- and postsynaptic) describes cytostome-based ingestion of host cell cytosol, so a child term is proposed below. Supporting Evidence: PMID:31896710 Functional analysis showed that these proteins are required for endocytosis of hemoglobin from the host cell. Parasites with inactivated Kelch13 or a resistance-conferring Kelch13 mutation displayed reduced hemoglobin endocytosis. PMID:38700363 K13 and its compartment neighbors (KICs), including UBP1, are involved in endocytosis of host cell cytosol. PMID:38700363 Titration of K13 and KIC7 indicated that the cellular levels of these proteins determined resistance through the rate of endocytosis. PMID:38039338 vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance or endocytosis (or both), comprising one group of K13 compartment proteins. |
| GO:0044002 acquisition of nutrients from host | IMP PMID:31896710 A Kelch13-defined endocytosis pathway mediates artemisinin r... | NEW | Summary: The material Kelch13-dependent endocytosis internalizes is host erythrocyte cytosol, overwhelmingly haemoglobin, which is carried to the digestive vacuole and proteolysed to supply the parasite with amino acids. Kelch13 therefore participates in the symbiont's acquisition of nutrients from its host cell, at the uptake step of that route. Reason: This is the host-aware framing of the same machinery and is not an ancestor or descendant of GO:0006897: the endocytosis term says how the material gets in, this term says that the material is host-derived nutrient. The uptake step is performed by Kelch13 and its compartment partners, while the downstream proteolysis is done by the digestive-vacuole proteases, so only the uptake portion is claimed here. The term is appropriate for an intracellular parasite and the host, the erythrocyte, is the larger partner as the definition requires. Supporting Evidence: PMID:31896710 Functional analysis showed that these proteins are required for endocytosis of hemoglobin from the host cell. PMID:38039338 Hemoglobin trafficking to the parasite food vacuole is believed to be initiated at membrane invaginations called cytostomes [30,42,48,49] followed by vesicular transport from the parasite plasma membrane (PPM) to the food vacuole [42,50], likely in an actin-myosin motor dependent manner [48,51–53]. PMID:32310999 We also saw evidence of K13 associating with cytostomes that traffic host-endocytosed hemoglobin from the parasitophorous vacuolar space to the DV (Fig 1E). |
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Download this section (compressed HTML)Q: What molecular step does Kelch13 perform within endocytosis of host cell cytosol: does the Kelch propeller recruit specific partners to the cytostome site, or does the oligomer act structurally in forming or constricting the invagination?
Suggested experts: Tobias Spielmann, Jakob Birnbaum
Q: Is the reported Kelch13-PfPI3K interaction part of a genuine parasite cullin-RING ligase, with an identified cullin and RING partner, or an interaction without an E3-adaptor function? Resolving this determines whether GO:1990756 ubiquitin-like ligase-substrate adaptor activity should ever be asserted for Kelch13.
Suggested experts: Kasturi Haldar, David A. Fidock
Q: Does the hexameric assembly seen in solution exist in the parasite at the Kelch13 compartment, and do resistance substitutions act by changing assembly, partner binding, or protein stability and abundance?
Suggested experts: Tobias Spielmann
Q: Why is Kelch13 localized partly to the endoplasmic reticulum and to Rab-positive vesicles, and does any of that pool contribute to uptake, or does it represent biosynthetic transit and a second, unrelated role?
Suggested experts: David A. Fidock
Experiment: Use conditional knock-sideways of endogenous Kelch13 in lines expressing tagged Kelch13-compartment proteins, and score by quantitative live imaging which partners lose focal localization and on what timescale, alongside a haemoglobin-uptake readout. Complement with propeller point mutants, including resistance alleles, to map the surface required for each partner.
Hypothesis: Kelch13 acts as the recruitment platform of the host-cytosol uptake site, so loss of the propeller surface displaces specific compartment proteins without dissolving the site.
Type: conditional mislocalization with quantitative colocalization imaging
Experiment: Perform crosslinking immunoprecipitation mass spectrometry on tagged Kelch13 in isogenic wild-type and C580Y lines, searching specifically for cullin, RING and neddylation machinery, and measure PfPI3K half-life by cycloheximide chase with and without a neddylation inhibitor in both backgrounds.
Hypothesis: The Kelch13-PfPI3K association is not an E3 substrate-adaptor relationship, so no parasite cullin-RING ligase co-purifies with Kelch13 and PfPI3K turnover is unchanged when cullin function is blocked.
Type: interaction proteomics with protein turnover assay
Experiment: Titrate wild-type Kelch13 with an inducible degradation or translational-control system across a range of expression levels in an isogenic background, and measure at each level the rate of host-cytosol endocytosis, ring-stage survival after a dihydroartemisinin pulse, and asexual growth rate, then compare the resulting curves with those of lines carrying M476I, R539T and C580Y.
Hypothesis: Resistance-conferring substitutions act by lowering the amount of functional Kelch13 at the uptake site rather than by creating a new activity, so graded knockdown of wild-type Kelch13 reproduces the full resistance-versus-fitness relationship of the mutant alleles.
Type: graded protein depletion with paired resistance and fitness assays
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