Mapping the binding domains involved in the interaction between the Plasmodium falciparum knob-associated histidine-rich protein (KAHRP) and the cytoadherence ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1).

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) clusters at electron-dense knob-like structures on the surface of malaria-infected red blood cells and mediates their adhesion to the vascular endothelium. In parasites lacking knobs, vascular adhesion is less efficient, and infected red cells are not able to immobilize successfully under hemodynamic flow conditions even though PfEMP1 is still present on the exterior of the infected red cell. We examined the interaction between the knob-associated histidine-rich protein (KAHRP), the parasite protein upon which knob formation is dependent, and PfEMP1, and we show evidence of a direct interaction between KAHRP and the cytoplasmic region of PfEMP1 (VARC). We have identified three fragments of KAHRP which bind VARC. Two of these KAHRP fragments (K1A and K2A) interact with VARC with binding affinities (K(D(kin))) of 1 x 10(-7) M and 3.3 x 10(-6) M respectively, values comparable to those reported previously for protein-protein interactions in normal and infected red cells. Further experiments localized the high affinity binding regions of KAHRP to the 63-residue histidine-rich and 70-residue 5' repeats. Deletion of these two regions from the KAHRP fragments abolished their ability to bind to VARC. Identification of the critical domains involved in interaction between KAHRP and PfEMP1 may aid development of new therapies to prevent serious complications of P. falciparum malaria.     

Genetic ablation of a Maurer's cleft protein prevents assembly of the Plasmodium falciparum virulence complex

The malaria parasite Plasmodium falciparum assembles knob structures underneath the erythrocyte membrane that help present the major virulence protein, P. falciparum erythrocyte membrane protein-1 (PfEMP1). Membranous structures called Maurer's clefts are established in the erythrocyte cytoplasm and function as sorting compartments for proteins en route to the RBC membrane, including the knob-associated histidine-rich protein (KAHRP), and PfEMP1. We have generated mutants in which the Maurer's cleft protein, the ring exported protein-1 (REX1) is truncated or deleted. Removal of the C-terminal domain of REX1 compromises Maurer's cleft architecture and PfEMP1-mediated cytoadherance but permits some trafficking of PfEMP1 to the erythrocyte surface. Deletion of the coiled-coil region of REX1 ablates PfEMP1 surface display, trapping PfEMP1 at the Maurer's clefts. Complementation of mutants with REX1 partly restores PfEMP1-mediated binding to the endothelial cell ligand, CD36. Deletion of the coiled-coil region or complete deletion of REX1 is tightly associated with the loss of a subtelomeric region of chromosome 2, encoding KAHRP and other proteins. A KAHRP-green fluorescent protein (GFP) fusion expressed in the REX1-deletion parasites shows defective trafficking. Thus, loss of functional REX1 directly or indirectly ablates the assembly of the P. falciparum virulence complex at the surface of host erythrocytes.     

Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes

After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob-associated histidine-rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite-derived structures, known as Maurer's clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.     

Trafficking determinants for PfEMP3 export and assembly under the Plasmodium falciparum-infected red blood cell membrane

During the maturation of intracellular asexual stages of Plasmodium falciparum parasite-encoded proteins are exported into the erythrocyte cytosol. A number of these parasite proteins attach to the host cell cytoskeleton and facilitate transformation of a disk-shaped erythrocyte into a rounded and more rigid infected erythrocyte able to cytoadhere to the vasculature. Knob formation on the surface of infected erythrocytes is critical for this cytoadherence to the host endothelium. P. falciparum proteins have been identified that localize to the parasite-infected erythrocyte membrane: the variant cytoadherence ligand erythrocyte membrane protein 1 (PfEMP1), the knob-associated histidine-rich protein (KAHRP) and the erythrocyte membrane protein 3 (PfEMP3). In this study, we have generated parasites expressing PfEMP3-green fluorescent protein chimeras and identified domains involved in entry to the secretory pathway, export across the parasitophorous vacuolar membrane and attachment to Maurer's clefts and the erythrocyte membrane. Solubility assays, fluorescence photobleaching experiments and immunogold electron microscopy suggest that the exported chimeric proteins are trafficked in a complex rather than in vesicles. This study characterizes elements involved in the tight but transient binding of PfEMP3 to Maurer's clefts and shows that the same elements are necessary for correct assembly under the erythrocyte membrane.     

Clag9 is not essential for PfEMP1 surface expression in non-cytoadherent Plasmodium falciparum parasites with a chromosome 9 deletion

Background

The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1.

Methodology/Principal Findings

Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36.

Conclusions/Significance

Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications.     

School-based participatory health education for malaria control in Ghana: engaging children as health messengers

Background

Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by Plasmodium falciparum parasites to the erythrocyte membrane, but little is known about the kinetics of this antibody-receptor binding reaction or how the topology of PfEMP1 on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target.

Methods

A Quartz Crystal Microbalance biosensor was used to measure the association and dissociation kinetics of VAR2CSA PfEMP1 binding to human monoclonal antibodies. Immuno-fluorescence microscopy was used to visualize antibody-mediated adhesion between the surfaces of live infected erythrocytes and atomic force microscopy was used to obtain higher resolution images of the membrane knobs on the infected erythrocyte to estimate knob surface areas and model VAR2CSA packing density on the knob.

Results

Kinetic analysis indicates that antibody dissociation from the VAR2CSA PfEMP1 antigen is extremely slow when there is a high avidity interaction. High avidity binding to PfEMP1 antigens on the surface of P. falciparum-infected erythrocytes in turn requires bivalent cross-linking of epitopes positioned within the distance that can be bridged by antibody. Calculations of the surface area of the knobs and the possible densities of PfEMP1 packing on the knobs indicate that high-avidity cross-linking antibody reactions are constrained by the architecture of the knobs and the large size of PfEMP1 molecules.

Conclusions

High avidity is required to achieve the strongest binding to VAR2CSA PfEMP1, but the structures that display PfEMP1 also tend to inhibit cross-linking between PfEMP1 antigens, by holding many binding epitopes at distances beyond the 15-18 nm sweep radius of an antibody. The large size of PfEMP1 will also constrain intra-knob cross-linking interactions. This analysis indicates that effective vaccines targeting the parasite's vulnerable adhesion receptors should primarily induce strongly adhering, high avidity antibodies whose association rate constant is less important than their dissociation rate constant.     

Scanning electron microscope-analysis of the protrusions (knobs) present on the surface of Plasmodium falciparum-infected erythrocytes

The nature of the surface deformations of erythrocytes infected with the human malaria parasite Plasmodium falciparum was analyzed using scanning electron microscopy at two stages of the 48-h parasite maturation cycle. Infected cells bearing trophozoite-stage parasites (24-36 h) had small protrusions (knobs), with diameters varying from 160 to 110 nm, and a density ranging from 10 to 35 knobs X micron-2. When parasites were fully mature (schizont stage, 40-44 h), knob size decreased (100-70 nm), whereas density increased (45-70 knobs X micron-2). Size and density of the knobs varied inversely, suggesting that knob production (a) occurred throughout intraerythrocytic parasite development from trophozoite to schizont and (b) was related to dynamic changes of the erythrocyte membrane. Variation in the distribution of the knobs over the red cell surface was observed during parasite maturation. At the early trophozoite stage of parasite development, knobs appeared to be formed in particular domains of the cell surface. As the density of knobs increased and they covered the entire cell surface, their lateral distribution was dispersive (more-than-random); this was particularly evident at the schizont stage. Regional surface patterns of knobs (rows, circles) were seen throughout parasite development. The nature of the dynamic changes that occurred at the red cell surface during knob formation, as well as the nonrandom distribution of knobs, suggested that the red cell cytoskeleton may have played a key role in knob formation and patterning.     

Discovery of a novel and conserved Plasmodium falciparum exported protein that is important for adhesion of PfEMP1 at the surface of infected erythrocytes

Plasmodium falciparum virulence is linked to its ability to sequester in post‐capillary venules in the human host. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant surface antigen implicated in this process. Complete loss of parasite adhesion is linked to a large subtelomeric deletion on chromosome 9 in a number of laboratory strains such as D10 and T9‐96. Similar to the cytoadherent reference line FCR3, D10 strain expresses PfEMP1 on the surface of parasitized erythrocytes, however without any detectable cytoadhesion. To investigate which of the deleted subtelomeric genes may be implicated in parasite adhesion, we selected 12 genes for D10 complementation studies that are predicted to code for proteins exported to the red blood cell. We identified a novel single copy gene (PF3D7_0936500) restricted to P. falciparum that restores adhesion to CD36, termed here virulence‐associated protein 1 (Pfvap1). Protein knockdown and gene knockout experiments confirmed a role of PfVAP1 in the adhesion process in FCR3 parasites. PfVAP1 is co‐exported with PfEMP1 into the host cell via vesicle‐like structures called Maurer's clefts. This study identifies a novel highly conserved parasite molecule that contributes to parasite virulence possibly by assisting PfEMP1 to establish functional adhesion at the host cell surface.     

Role of Plasmepsin V in Export of Diverse Protein Families from the Plasmodium falciparum Exportome

Plasmodium falciparum exports several hundred effector proteins that remodel the host erythrocyte and enable parasites to acquire nutrients, sequester in the circulation and evade immune responses. The majority of exported proteins contain the Plasmodium export element (PEXEL; RxLxE/Q/D) in their N‐terminus, which is proteolytically cleaved in the parasite endoplasmic reticulum by Plasmepsin V, and is necessary for export. Several exported proteins lack a PEXEL or contain noncanonical motifs. Here, we assessed whether Plasmepsin V could process the N‐termini of diverse protein families in P. falciparum. We show that Plasmepsin V cleaves N‐terminal sequences from RIFIN, STEVOR and RESA multigene families, the latter of which contain a relaxed PEXEL (RxLxxE). However, Plasmepsin V does not cleave the N‐terminal sequence of the major exported virulence factor erythrocyte membrane protein 1 (PfEMP1) or the PEXEL‐negative exported proteins SBP‐1 or REX‐2. We probed the substrate specificity of Plasmepsin V and determined that lysine at the PEXEL P3 position, which is present in PfEMP1 and other putatively exported proteins, blocks Plasmepsin V activity. Furthermore, isoleucine at position P1 also blocked Plasmepsin V activity. The specificity of Plasmepsin V is therefore exquisitely confined and we have used this novel information to redefine the predicted P. falciparum PEXEL exportome .     

Characterization of a Plasmodium falciparum PHISTc protein,PF3D7_0801000, in blood- stage malaria parasites

During intraerythrocytic development Plasmodium falciparum deploys numerous proteins to support erythrocyte invasion, intracellular growth and development, as well as host immune evasion. Since these proteins are key for parasite intraerythrocytic survival and propagation, they represent attractive targets for antimalarial vaccines. In this study we sought to characterize a member of the PHISTc family of proteins, PF3D7_0801000, as a potential vaccine target. Using the wheat germ cell-free system we expressed the N-terminal region of PF3D7_0801000 (G₉₃-L₄₉₄, PF3D7_0801000N) and generated specific immune sera. We observed that PF3D7_0801000 localizes in merozoites, and antibodies against PF3D7_0801000N modestly inhibit P. falciparum parasite growth in in vitro culture. Sliding window analysis of the coding sequence revealed that pf3d7_0801000n is relatively conserved among African parasite isolates. Antibody profiles in a malaria-exposed Ugandan population revealed that PF3D7_0801000N is strongly immunoreactive with antibody acquisition increasing with age. Taken together, these findings suggest the need for further evaluation of PF3D7_0801000 for its role in merozoite invasion and utility as an asexual blood-stage vaccine candidate antigen.     

Host chaperones are recruited in membrane-bound complexes by Plasmodium falciparum.

The ability of malarial parasite to deploy proteins at the surface of infected erythrocytes is well known. After their synthesis within the parasite, the cargo proteins are exported from the parasite and carried across the erythrocyte cytoplasm to be delivered at the erythrocyte surface. Our knowledge about the mechanisms involved in this complex trafficking path is limited. We have addressed the involvement of chaperones in traffic across erythrocyte cytoplasm. Our analyses of the chaperones available to the parasite indicated that none of the reported chaperones of the parasite origin are present in the erythrocyte cytoplasm. The chaperones of the host (Hsp70, Hsp90, Hop60), on the other hand, were readily detected in the erythrocyte cytosol. Hypotonic lysis and detergent solubilization experiments indicated that unlike their soluble nature in normal erythrocytes, host chaperones are recruited in membrane-bound, detergent-resistant complexes in infected cells. The association of host-Hsp70 with detergent-resistant complexes was ATP-dependent. Importantly, host chaperones could be detected in knob-enriched fractions and could be cross-linked to the knob subunit, PfHRP1, in a large complex at the surface of the infected erythrocytes. Our results implicate host chaperones in the assembly of parasite proteins such as knob subunits at the erythrocyte surface.     

Knobs, knob proteins and cytoadherence in falciparum malaria.

1. The sequestration of trophozoite and schizont infected erythrocytes (IRBC) in post-capillary venules of host internal organs causes most of the morbidity and mortality in falciparum malaria. It is a knob mediated cytoadherence phenomenon where knobs act as the focal junction between IRBC and host endothelial cell. Knobless (K-) parasites, isolated from cultures (not yet isolated from in vivo), do not cause virulent infections. Knobs thus play an important role in pathophysiology of falciparum malaria. 2. The chemical composition of knobs is partly explored, several proteins (Known as knob proteins) have been identified. According to their function they can be classified as (a) knob-inducing protein, "KAHRP" (b) knob-associated cytoadherent proteins, e.g. PFEMP-1, modified band 3 and an antigen recognized by monoclonal 33G2 and (c) knob-associated structural protein, e.g. PFEMP-2/MESA/PP-300. Most of them show size polymorphism among different isolates. Only KAHRP and MESA/PFEMP-2 have been studied at molecular level. Their chromosomal locations have been identified such as KAHRP on chromosome 2 and MESA/PFEMP-2 on chromosomes 5 and 6. 3. The receptor molecules on endothelial cells for knob ligands have been identified and partially characterized. 4. Knob ligands and their receptor molecules can play an important role in developing the immunotherapeutic reagents. 5. Based on the available data a tentative hypothesis has been proposed about the loss of knobs in vitro. Nevertheless, this needs further support from other experimental evidence. 6. Future work should be directed towards the structure and function of knob proteins and their interactions with each other as well as with host proteins. Regulation of expression of knobs and knob protein(s), evaluation of knob antigens for immunotherapy of severe falciparum malaria and for a malaria vaccine also require further investigations.     

Plasmodium falciparum-infected erythrocytes: qualitative and quantitative analyses of parasite-induced knobs by atomic force microscopy

We used the combination of an atomic force microscope and a light microscope equipped with epifluorescence to serially image Plasmodium falciparum-infected erythrocytes. This procedure allowed us to determine unambiguously the presence and developmental stage of the malaria parasite as well as the number and size of knobs in singly, doubly, and triply infected erythrocytes. Knobs are not present during the ring stage of a malaria infection but a lesion resulting from invasion by a merozoite is clearly visible on the erythrocyte surface. This lesion is visible into the late trophozoite stage of infection. Knobs begin to form during the early trophozoite stage of infection and have a single-unit structure. Our data suggest the possibility that a two-unit structure of knobs, which was reported by Aikawa et al. (1996, Exp. Parasitol. 84, 339-343) using atomic force microscopy, appears to be a double-tipped image. The number of knobs per unit of host cell surface area is directly proportional to parasite number in both early and late trophozoite stages. These results indicate that knob formation by one parasite does not influence knob formation by other parasites in a multiply infected erythrocyte. In addition, knob volume is not influenced by either parasite stage or number at the late trophozoite stage, indicating that the number of component molecules per knob is constant throughout the parasite maturation process.     

Functional Evaluation of Plasmodium Export Signals in Plasmodium berghei Suggests Multiple Modes of Protein Export

The erythrocytic stage development of malaria parasites occurs within the parasitophorous vacuole inside the infected-erythrocytes, and requires transport of several parasite-encoded proteins across the parasitophorous vacuole to several locations, including the cytosol and membrane of the infected cell. These proteins are called exported proteins; and a large number of such proteins have been predicted for Plasmodium falciparum based on the presence of an N-terminal motif known as the Plasmodium export element (PEXEL) or vacuolar transport signal (VTS), which has been shown to mediate export. The majority of exported proteins contain one or more transmembrane domains at the C-terminus and one of three types of N-terminus domain architectures. (1) The majority, including the knob-associated histidine rich protein (KAHRP), contain a signal/hydrophobic sequence preceding the PEXEL/VTS motif. (2) Other exported proteins, including the P. berghei variant antigen family bir and the P. falciparum skeleton binding protein-1, do not appear to contain a PEXEL/VTS motif. (3) The P. falciparum erythrocyte membrane protein-1 (PfEMP1) family lacks a signal/hydrophobic sequence before the motif. These different domain architectures suggest the presence of multiple export pathways in malaria parasites. To determine if export pathways are conserved in plasmodia and to develop an experimental system for studying these processes, we investigated export of GFP fused with N- and C-terminus putative export domains in the rodent malaria parasite P. berghei. Export was dependent on specific N- and C-terminal domains. Constructs with a KAHRP-like or bir N-terminus, but not the PfEMP1 N-terminus, exported GFP into the erythrocyte. The C-terminus of a P. falciparum variant antigen rifin prevented GFP export by the KAHRP-like N-terminus. In contrast, GFP chimeras containing KAHRP-like N-termini and the PfEMP1 C-terminus were exported to the surface of erythrocytes. Taken together, these results suggest that proteins with KAHRP-like architecture follow a common export pathway, but that PfEMP1s utilize an alternative pathway. Functional validation of common putative export domains of malaria parasites in P. berghei provides an alternative and simpler system to investigate export mechanisms.     

Localization and function of a Plasmodium falciparum protein (PF3D7_1459400) during erythrocyte invasion

Nearly 60% of Plasmodium falciparum proteins are still uncharacterized and their functions are unknown. In this report, we carried out the functional characterization of a 45 kDa protein (PF3D7_1459400) and showed its potential as a target for blood stage malaria vaccine development. Analysis of protein subcellular localization, native protein expression profile, and erythrocyte invasion inhibition of both clinical and laboratory parasite strains by peptide antibodies suggest a functional role of PF3D7_1459400 protein during erythrocyte invasion. Also, immunoreactivity screens using synthetic peptides of the protein showed that adults resident in malaria endemic regions in Ghana have naturally acquired plasma antibodies against PF3D7_1459400 protein. Altogether, this study presents PF3D7_1459400 protein as a potential target for the development of peptide-based vaccine for blood-stage malaria.Impact statementPlasmodium falciparum malaria is a global health problem. Erythrocyte invasion by P. falciparum merozoites appears to be a promising target to curb malaria. We have identified and characterized a novel protein that is involved in erythrocyte invasion. Our data on protein subcellular localization, stage-specific protein expression pattern, and merozoite invasion inhibition by α-peptide antibodies suggest a role for PF3D7_1459400 protein during P. falciparum erythrocyte invasion. Even more, the human immunoepidemiology data present PF3D7_1459400 protein as an immunogenic antigen which could be further exploited for the development of new anti-infective therapy against malaria.     

Plasmodium falciparum‐encoded exported hsp70/hsp40 chaperone/co‐chaperone complexes within the host erythrocyte

Malaria parasites modify their host cell, the mature human erythrocyte. We are interested in the molecules mediating these processes, and have recently described a family of parasite‐encoded heat shock proteins (PfHsp40s) that are targeted to the host cell, and implicated in host cell modification. Hsp40s generally function as co‐chaperones of members of the Hsp70 family, and until now it was thought that human Hsp70 acts as the PfHsp40 interaction partner within the host cell. Here we revise this hypothesis, and identify and characterize an exported parasite‐encoded Hsp70, referred to as PfHsp70‐x. PfHsp70‐x is exported to the host erythrocyte where it forms a complex with PfHsp40s in structures known as J‐dots, and is closely associated with PfEMP1. Interestingly, Hsp70‐x is encoded only by parasite species that export the major virulence factor EMP1, implying a possible role for Hsp70‐x in EMP1 presentation at the surface of the infected erythrocyte. Our data strongly support the presence of parasite‐encoded chaperone/co‐chaperone complexes within the host erythrocyte, which are involved in protein traffic through the host cell. The host–pathogen interaction within the infected erythrocyte is more complex than previously thought, and is driven notonly by parasite co‐chaperones, but also by the parasite‐encoded chaperone Hsp70‐x itself.     

A particle-based computational model to analyse remodelling of the red blood cell cytoskeleton during malaria infections

Red blood cells can withstand the harsh mechanical conditions in the vasculature only because the bending rigidity of their plasma membrane is complemented by the shear elasticity of the underlying spectrin-actin network. During an infection by the malaria parasite Plasmodium falciparum, the parasite mines host actin from the junctional complexes and establishes a system of adhesive knobs, whose main structural component is the knob-associated histidine rich protein (KAHRP) secreted by the parasite. Here we aim at a mechanistic understanding of this dramatic transformation process. We have developed a particle-based computational model for the cytoskeleton of red blood cells and simulated it with Brownian dynamics to predict the mechanical changes resulting from actin mining and KAHRP-clustering. Our simulations include the three-dimensional conformations of the semi-flexible spectrin chains, the capping of the actin protofilaments and several established binding sites for KAHRP. For the healthy red blood cell, we find that incorporation of actin protofilaments leads to two regimes in the shear response. Actin mining decreases the shear modulus, but knob formation increases it. We show that dynamical changes in KAHRP binding affinities can explain the experimentally observed relocalization of KAHRP from ankyrin to actin complexes and demonstrate good qualitative agreement with experiments by measuring pair cross-correlations both in the computer simulations and in super-resolution imaging experiments.     

Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component,PTEX150

The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C‐terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C‐terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C‐terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX‐associated proteins, namely PV1, Pf113 and Hsp70‐x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.