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FCP binds PI3P specifically. A, a schematic of the FYVE domain is shown. Basic residues mutated to test functional binding to phosphoinositides included K55 (blue) changed to alanine or three arginine residues Arg-52, Arg-54, and Arg-59 (red) simultaneously changed to alanine. B, binding of recombinant wild type FCP or its mutants (K55A) or (R52 R54 R59 to A) to varying concentrations of phospho (P)-inositides were assayed. WT, wild type.
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Phosphatidylinositol 3-phosphate (PI3P) is a key ligand for recruitment of endosomal regulatory proteins in higher eukaryotes.
Subsets of these endosomal proteins possess a highly selective PI3P binding zinc finger motif belonging to the FYVE domain
family. We have identified a single FYVE domain-containing protein in Plasmodium falciparum which we...
Contexts in source publication
Context 1
... Specifically Interacts with PI3P-To better understand phosphoinositide interactions with FCP, we performed lipid Traffic to the Malaria Parasite Food Vacuole APRIL 13, 2007 • VOLUME 282 • NUMBER 15 dot-blot assays using recombinant wild type and site-directed mutants of FCP. Wild type FCP bound specifically to PI3P (Fig. 2B). Deletion of the FYVE domain (data not shown) resulted in a complete loss of phosphoinositide binding as did a site-directed substitution K55A (Fig. 2B), which resides within the conserved motif R(R/K)HHCR (Figs. 1B and 2A). The crystal structure of the EEA1 FYVE domain bound to inositol 1,3-diphosphate has revealed that amino acid ...
Context 2
... to the Malaria Parasite Food Vacuole APRIL 13, 2007 • VOLUME 282 • NUMBER 15 dot-blot assays using recombinant wild type and site-directed mutants of FCP. Wild type FCP bound specifically to PI3P (Fig. 2B). Deletion of the FYVE domain (data not shown) resulted in a complete loss of phosphoinositide binding as did a site-directed substitution K55A (Fig. 2B), which resides within the conserved motif R(R/K)HHCR (Figs. 1B and 2A). The crystal structure of the EEA1 FYVE domain bound to inositol 1,3-diphosphate has revealed that amino acid residues form a coordination network with the 1 and 3 phosphates of PI3P, and it has been hypothesized that these residues may lend specificity to FYVE-PI3P ...
Context 3
... This three-amino acid substitution resulted in a loss in specificity for PI3P as indicated by increased binding to phosphoinositides, PI 3,4,5-triphosphate and PI 4,5-diphosphate. This loss in PI3P binding specificity demonstrated that indeed a critical interaction does exist between these basic residues and the 1 and 3 phosphates of PI3P (Figs. 2, A and ...
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Citations
... In order to identify possible Rbsn5 candidates, we conducted in silico searches. Rbsn5 from other organisms contain an FYVE-type zinc finger but only a single FYVEdomain containing protein was previously detected in the P. falciparum genome and named FYVE-containing protein (FCP) [36]. BLAST searches using human Rbsn5 (Q9H1K0) identified FCP as the top hit. ...
... As PfRbsn5L localized only with some of the PI3P positive areas in the parasite, it is possible that the FYVE domain alone does not suffice for PI3P binding, permitting a more nuanced, function-dependent targeting. In contrast, FCP, which may correspond to the parasite's EEA1, is located around the food vacuole [36], does contain a fully canonical FYVE domain (S1A Fig). It is therefore possible that these differences underlie a functional separation of the 2 FYVE domain proteins of the parasite. ...
... Sequence logo of the conserved residues in A was generated from the sequences of all FYVE domains found in H. sapiens, S. cerevisiae, T. brucei, A. thaliana, G. intestinalis, P. falciparum, and T. gondii. Red asterisks show amino acids important for PI3P binding or specificity that are not conserved in the FYVE domains of PfRbsn5L and human Protrudin (which contains an FYVE domain not binding PI3P but other phosphoinositides) while they are conserved in PF3D7_1460100 (FCP), the likely PfEEA1 [36]. In B, similarity according to clustal omega (dots and asterisks) are indicated below the alignment; residues involved in hydrogen bonds that are conserved between both Rab-binding domains of human Rbsn5, according to PDB 1z0k and 1z0j, were marked with + above. ...
Vesicular trafficking, including secretion and endocytosis, plays fundamental roles in the unique biology of Plasmodium falciparum blood-stage parasites. Endocytosis of host cell cytosol (HCC) provides nutrients and room for parasite growth and is critical for the action of antimalarial drugs and parasite drug resistance. Previous work showed that PfVPS45 functions in endosomal transport of HCC to the parasite’s food vacuole, raising the possibility that malaria parasites possess a canonical endolysosomal system. However, the seeming absence of VPS45-typical functional interactors such as rabenosyn 5 (Rbsn5) and the repurposing of Rab5 isoforms and other endolysosomal proteins for secretion in apicomplexans question this idea. Here, we identified a parasite Rbsn5-like protein and show that it functions with VPS45 in the endosomal transport of HCC. We also show that PfRab5b but not PfRab5a is involved in the same process. Inactivation of PfRbsn5L resulted in PI3P and PfRab5b decorated HCC-filled vesicles, typical for endosomal compartments. Overall, this indicates that despite the low sequence conservation of PfRbsn5L and the unusual N-terminal modification of PfRab5b, principles of endosomal transport in malaria parasite are similar to that of model organisms. Using a conditional double protein inactivation system, we further provide evidence that the PfKelch13 compartment, an unusual apicomplexa-specific endocytosis structure at the parasite plasma membrane, is connected upstream of the Rbsn5L/VPS45/Rab5b-dependent endosomal route. Altogether, this work indicates that HCC uptake consists of a highly parasite-specific part that feeds endocytosed material into an endosomal system containing more canonical elements, leading to the delivery of HCC to the food vacuole.
... In order to identify possible Rbsn5 candidates we conducted in silico searches. Rbsn5 from other organisms contain a FYVE-type zinc finger but only a single FYVE-domain containing protein was previously detected in the P. falciparum genome and named FYVE-containing protein (FCP) [36]. BLAST searches using human Rbsn5 (Q9H1K0) identified FCP as the top hit. ...
Vesicular trafficking, including secretion and endocytosis, plays fundamental roles in the unique biology of P. falciparum blood-stage parasites. Endocytosis of host cell cytosol (HCC) provides nutrients and room for parasite growth and is critical for the action of antimalarial drugs and parasite drug resistance. Previous work showed that PfVPS45 functions in endosomal transport of HCC to the parasite’s food vacuole, raising the possibility that malaria parasites possess a canonical endolysosomal system. However, the seeming absence of VPS45-typical functional interactors such as rabenosyn 5 (Rbsn5) and the re-purposing of Rab5 isoforms and other endolysosomal proteins for secretion in apicomplexans question this idea. Here we identified the likely parasite Rbsn5 and show that it functions with VPS45 in the endosomal transport of HCC. We also show that PfRab5b but not PfRab5a is involved in the same process. Inactivation of PfRbsn5 resulted in PI3P and PfRab5b decorated HCC-filled vesicles, typical for endosomal compartments. Overall this indicates that despite the low sequence conservation of PfRbsn5 and the unusual N-terminal modification of PfRab5b, principles of endosomal transport in malaria parasite are similar to that of model organisms. Using a conditional double protein inactivation system, we further provide evidence that the PfKelch13 compartment, an unusual apicomplexa-specific endocytosis structure at the parasite plasma membrane, is connected upstream of the Rbsn5/VPS45/Rab5b-dependent endosomal route. Altogether, this work indicates that HCC-uptake consists of a highly parasite-specific part that feeds endocytosed material into a more canonical endosomal system, leading to the delivery of HCC to the food vacuole.
... The Fab1, YOTB, Vac1, EEA1 (FYVE)-containing protein (PfFCP) localizes to the DV lumen and binds PI3P through its FYVE domain. However, this is not required for PfFCP localization to the DV so the role that this protein plays is unclear (48). While Autophagyrelated protein 18 localizes to the DV membrane in a PI3P-dependent manner (49,50), its conditional knockdown does not impact Hb trafficking, resulting instead in apicoplast inheritance defects (51). ...
... A role for PfPI3K and its product PI3P in Hb trafficking was proposed previously (17,47,48) but what this role would be and the identity of PPI binding effectors involved remained elusive. Our work has identified a PX domain-containing protein that likely binds PI3P. ...
Malaria represents an enormous burden for a significant proportion of humanity and the lack of vaccines and problems with drug resistance to all antimalarials demonstrate the need to develop new therapeutics. Inhibitors of phosphoinositide metabolism are currently being developed as antimalarials but our understanding of this biological pathway is incomplete.
... Since partial ingestion of live prey is the definition of trogocytosis, the cytostome-cytopharynx related phenomenon resembles trogocytosis. For instance, a subsequent association of Rab5A and PtdIns3P binding proteins (a FYVE domain containing protein, FCP, and Atg18) with the food vacuole, in the course of cytostome and trogosome formation, and inhibition of cytostome and trogosome formation by a PI3K inhibitor [24,[93][94][95] are shared mechanisms by both of the processes. It has not yet been demonstrated that P. falciparum is able to cross-dress a protein that originated from the PV membrane. ...
Trogocytosis is a mode of internalization of a part of a live cell by nibbling and is mechanistically distinct from phagocytosis, which implies internalization of a whole cell or a particle. Trogocytosis has been demonstrated in a broad range of cell types in multicellular organisms and is also known to be involved in a plethora of functions. In immune cells, trogocytosis is involved in the “cross-dressing” between antigen presenting cells and T cells, and is thus considered to mediate intercellular communication. On the other hand, trogocytosis has also been reported in a variety of unicellular organisms including the protistan (protozoan) parasite Entamoeba histolytica. E. histolytica ingests human T cell line by trogocytosis and acquires complement resistance and cross-dresses major histocompatibility complex (MHC) class I on the cell surface. Furthermore, trogocytosis and trogocytosis-like phenomena (nibbling of a live cell, not previously described as trogocytosis) have also been reported in other parasitic protists such as Trichomonas, Plasmodium, Toxoplasma, and free-living amoebae. Thus, trogocytosis is conserved in diverse eukaryotic supergroups as a means of intercellular communication. It is depicting the universality of trogocytosis among eukaryotes. In this review, we summarize our current understanding of trogocytosis in unicellular organisms, including the history of its discovery, taxonomical distribution, roles, and molecular mechanisms.
... Protein prenyltransferases including geranylgeranyltransferase 1 (GGT1), Rab geranylgeranyltransferase (RabGGT), and farnesyltransferase (FT) catalyze the protein prenylation process [94]. The genetic disruption and the pharmacological inhibition of the protein prenylation mechanism suggested novel perspectives into the etiology and pathobiology of diseases in which prenylation is involved like in P. falciparum malaria [95]. FYVE-containing coiled-coil protein (FCP), as a prenylated protein, is conserved in Plasmodium spp. ...
... and is mostly localized in parasite specialized vacuoles in which host hemoglobin is digested probably playing major roles in vesicle targeting. The use of farnesyl transferase inhibitors (THQ class farnesyltransferase inhibitor BMS-386914) abolished FCP prenylation and led to its cytosolic mislocalization [95,96]. Other P. falciparum substrates for farnesyltransferases like Ykt6 proteins are considered the most versatile fusogens in eukaryotic cells [97]. ...
There are important challenges when investigating individual post-translational modifications (PTMs) or protein interaction network and delineating if PTMs or their changes and cross-talks are involved during infection, disease initiation or as a result of disease progression. Proteomics and in silico approaches now offer the possibility to complement each other to further understand the regulatory involvement of these modifications in parasites and infection biology. Accordingly, the current review highlights key expressed or altered proteins and PTMs are invisible switches that turn on and off the function of most of the proteins. PTMs include phosphorylation, glycosylation, ubiquitylation, palmitoylation, myristoylation, prenylation, acetylation, methylation, and epigenetic PTMs in P. falciparum which have been recently identified. But also other low-abundant or overlooked PTMs that might be important for the parasite's survival, infectivity, antigenicity, immunomodulation and pathogenesis. We here emphasize the PTMs as regulatory pathways playing major roles in the biology, pathogenicity, metabolic pathways, survival, host-parasite interactions and the life cycle of P. falciparum. Further validations and functional characterizations of such proteins might confirm the discovery of therapeutic targets and might most likely provide valuable data for the treatment of P. falciparum, the main cause of severe malaria in human.
... In other studies, K13 also localizes to PI3P-labeled structures implicated in modulation of artemisinin susceptibility (25,26). In Plasmodium, PI3P is a membrane component of endocytic vesicles (24,51). Thus, our findings and those of other investigators support a role for endocytosis, hemoglobin ingestion, and more generally intracellular traffic in artemisinin susceptibility. ...
The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 . Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum , we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.
IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum . In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.
... One FYVE domain of E. histolytica, is associated with phagosomes indicating a role of this lipid in phagocytosis (Powell et al. 2006). The apicomplexans, Plasmodium falciparum and Toxoplasma gondii, also possess one and two FYVE domain-containing proteins, respectively (McIntosh et al. 2007;Daher et al. 2015). In contrast to other organisms, the FYVE domaincontaining protein of P. falciparum is localized to a lysosomal compartment, known as food vacuole, rather than to any endocytic organelle through specific binding to PtdIns-3-P (McIntosh et al. 2007). ...
... The apicomplexans, Plasmodium falciparum and Toxoplasma gondii, also possess one and two FYVE domain-containing proteins, respectively (McIntosh et al. 2007;Daher et al. 2015). In contrast to other organisms, the FYVE domaincontaining protein of P. falciparum is localized to a lysosomal compartment, known as food vacuole, rather than to any endocytic organelle through specific binding to PtdIns-3-P (McIntosh et al. 2007). Putative T. gondii FYVE domain-containing effector proteins are localized to the vacuolar compartments indicating that a pool of PtdIns-3-P is associated with this subcellular compartment (Daher et al. 2015). ...
... Intracellular eukaryotic parasites rely on their own PI metabolism to regulate signalling and trafficking (McIntosh et al. 2007;Vaid et al. 2010;Tawk et al. 2011) as well as host cell PI metabolism to establish and maintain infection. Some parasite-derived PIbinding proteins have been identified, including Toxoplasma gondii TgPH1 (PtdIns(3,5)P 2 ) (Daher et al. 2016) and Phytophthora sojae Avr1b, Avh5, and Avh331 (PtdIns3P) (Lu et al. 2013); however, the roles of these proteins during infection remain largely unknown. ...
Signalling through phosphoinositide lipids is essential for regulating many cellular processes, including endosomal trafficking. A number of intracellular pathogens have found ways to subvert host trafficking pathways via exploitation of endosomal phosphoinositides. This review will discuss how pathogens such as bacteria, viruses, and eukaryotic parasites depend on endosomal phosphoinositides for infection, as well as the mechanisms through which some are able to actively manipulate these signalling lipids to facilitate invasion, survival, replication, and immune evasion.
... PI3P effector proteins containing FYVE-or PX-domains are scarce in apicomplexan parasites: Plasmodium contains one FYVE-and two PX-domain proteins and T. gondii two FYVE-and four PX-domain proteins ( Table 3). The P. falciparum FYVEcontaining protein (FCP) has been shown to bind specifically to PI3P and has been detected inside the parasite food vacuole; however this localisation was independent of the presence of the FYVEdomain (McIntosh et al., 2007). In T. gondii, one of the FYVEdomain proteins corresponds to the PI(3,5)P 2 producing PIKfyve enzyme that will be described in Section 4.4. ...
Phosphoinositides are the phosphorylated derivatives of the structural membrane phospholipid phosphatidylinositol. Single or combined phosphorylation at the 3, 4 and 5 positions of the inositol ring gives rise to the seven different species of phosphoinositides. All are quantitatively minor components of cellular membranes but have been shown to have important functions in multiple cellular processes. Here we describe our current knowledge of phosphoinositide metabolism and functions in apicomplexan parasites, mainly focusing on Toxoplasma gondii and Plasmodium spp. Even though our understanding is still rudimentary, phosphoinositides have already shown their importance in parasite biology and revealed some very particular and parasite-specific functions. Not surprisingly, there is a strong potential for phosphoinositide synthesis to be exploited for future anti-parasitic drug development.
... What functions the individual PIPs might play has recently become the focus of a number of studies. PI3P is thought to play a role in processes such as hemoglobin uptake to the food vacuole (Elabbadi et al., 1994;McIntosh et al., 2007;Wengelnik and Vial, 2007;Tawk et al., 2010;Vaid et al., 2010), biogenesis of the apicoplast (Tawk et al., 2010(Tawk et al., , 2011, resistance to artemisinin (Mbengue et al., 2015) and export of proteins to the erythrocyte (Bhattacharjee et al., 2012) although the latter data have recently been challenged (Boddey et al., 2016). Inhibition of a P. falciparum PI4kinase with imidazopyrazines and quinoxalines has revealed that PI4P was likely critical for proper plasma membrane ingression during schizogony (McNamara et al., 2013). ...
Despite representing a small percentage of the cellular lipids of eukaryotic cells, phosphoinositides (PIPs) are critical in various processes such as intracellular trafficking and signal transduction. Central to their various functions is the differential distribution of PIP species to specific membrane compartments through the actions of kinases, phosphatases and lipases. Despite their importance in the malaria parasite lifecycle, the subcellular distribution of most PIP species in this organism is still unknown. We here localise several species of PIPs throughout the erythrocytic cycle of Plasmodium falciparum. We show that PI3P is mostly found at the apicoplast and the membrane of the food vacuole, that PI4P associates with the Golgi apparatus and the plasma membrane and that PI(4,5)P2, in addition to being detected at the plasma membrane, labels some cavity-like spherical structures. Finally, we show that the elusive PI5P localises to the plasma membrane, the nucleus and potentially to the transitional endoplasmic reticulum (ER). Our map of the subcellular distribution of PIP species in P. falciparum will be a useful tool to shed light on the dynamics of these lipids in this deadly parasite.
