Molecular Factors Responsible for Host Cell Recognition and Invasion in Plasmodium falciparum1

ABSTRACT. In Plasmodium falciparum. the rhoptries involved in the invasion process are a pair of flask-shaped organelles located at the apical tip of invading stages. They, along with the more numerous micronemes and dense granules, constitute the apical complex in Plasmodium and other members of the phylum Apicomplexa. Several proteins of varying molecular weight have been identified in P. falciparum rhoptries. These include the 225-, 140/130/110-, 80/60/40-, RAP-1 80-, AMA-1 80-, QF3 80-, and 55-kDa proteins. Some of these proteins are lost during schizont rupture and release of merozoites. Others such as the 140/130/110-kDa complex are transferred to the erythrocyte membrane during invasion. The ring-infected surface antigen (RESA). a 155-kDa polypeptide located in dense granules also associates with the erythrocyte membrane during invasion. Erythrocyte-binding studies have demonstrated that both the 140/130/110-kDa rhoptry complex and RESA bind to inside-out-vesicles (IOVs) prepared from human erythrocytes. The 140/130/110-kDa complex also binds to erythrocyte membranes prepared by hypotonic lysis. These proteins, however, do not bind to intact human erythrocytes. In a heterologous erythrocyte model, both the 140/130/110-kDa complex and RESA are shown to bind directly to mouse erythrocytes. Other studies have shown that RESA associates with spectrin in the erythrocyte cytoskeleton. We have recently developed a liposome-binding assay to demonstrate the lipophilic binding properties of the P. falciparum rhoptry complex of 140/130/110 kDa. The rhoptry complex binds to liposomes containing neutrally, positively, and negatively charged phospholipids. However, liposomes containing phosphatidylethanolamine compete effectively for rhoptry protein binding to mouse erythrocytes. The rhoptry complex also binds to membrane and inside-out-vesicles prepared from human erythrocytes and erythrocytes from other species. The rhoptry complex associated with the erythrocyte membrane in ring-infected erythrocytes is accessible to cleavage by phospholipase A. Studies are in progress to identify the molecular epitopes on the individual proteins within the complex responsible for lipid interaction in the erythrocyte bilayer and to determine the specificity of the phospholipid interaction using erythrocyte phospholipids.     

Interaction of the 140/130/110 kDa rhoptry protein complex of Plasmodium falciparum with the erythrocyte membrane and liposomes

During Plasmodium falciparum merozoite invasion into human and mouse erythrocytes, a 110-kDa rhoptry protein is secreted from the organelle into the erythrocyte membrane. In the present study our interest was to examine the interaction of rhoptry proteins of P. falciparum with the erythrocyte membrane. It was observed that the complex of rhoptry proteins of 140/130/110 kDa bind directly to a trypsin sensitive site on intact mouse erythrocytes, and not human, saimiri, or other erythrocytes. However, when erythrocytes were disrupted by hypotonic lysis, rhoptry proteins of 140/130/110 kDa were found to bind to membranes and inside-out vesicles prepared from human, mouse, saimiri, rhesus, rat, and rabbit erythrocytes. A binding site on the cytoplasmic face of the erythrocyte membrane suggests that the rhoptry proteins may be translocated across the lipid bilayer during merozoite invasion. Furthermore, pretreatment of human erythrocytes with a specific peptide derived from MSA-1, the major P. falciparum merozoite surface antigen of MW 190,000-200,000, induced binding of the 140/130/110-kDa complex. The rhoptry proteins bound equally to normal human erythrocytes and erythrocytes treated with neuraminidase, trypsin, and chymotrypsin indicating the binding site was independent of glycophorin and other major surface proteins. The rhoptry protein complex also bound specifically to liposomes prepared from different types of phospholipids. Liposomes containing PE effectively block binding of the rhoptry proteins to mouse cells, suggesting that there are two binding sites on the mouse membrane for the 140/130/110-kDa complex, one protein and a second, possibly lipid in nature. The results of this study suggest that the 140/130/110 kDa protein complex may interact directly with sites in the lipid bilayer of the erythrocyte membrane.     

Plasmodium falciparum Rhoptry Proteins of 140/130/110 kd (Rhop-H) Are Located in an Electron Lucent Compartment in the Neck of the Rhoptries

ABSTRACT. To investigate in more detail the structure of the high molecular weight rhoptry protein complex of Plasmodium falciparum, Rhop-H (140/130/110 kd), the complex was affinity purified from parasite extracts using rhoptry protein specific antisera prepared against Rhop-H proteins bound to and eluted from Balb/c mouse erythrocytes, using 0.5 M NaCl. The individual proteins (140 kd/Rhop-1, 130 kd/Rhop-2, and 110 kd/Rhop-3) were separated, electroeluted, and monospecific polyclonal antisera prepared against the individual proteins, and against the affinity purified complex. Immunofluorescence assays and immunoelectron microscopic studies were performed to verify the subcellular localization of the Rhop-H epitopes. Immunoblotting and immunoprecipitation assays were also performed. We report novel findings regarding the localization of the rhoptry proteins to an electron lucent compartment in the neck of the rhoptries. Analysis of the amino acid composition of the individually purified Rhop-H proteins demonstrated a predominance of negatively charged (E, D) as well as hydrophobic residues (L, A, P, S) in the three proteins. The percentage of negatively charged residues was high for all three proteins. Similarities in amino acid composition for the three proteins supports the previous data demonstrating shared properties such as erythrocyte and liposome binding, for the three proteins. Results of antibody characterizations using rhoptry protein specific antisera demonstrate the immunodominance of the Rhop-H complex.     

Identifying and characterising the Plasmodium falciparum RhopH3 Plasmodium vivax homologue

Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.     

Characterization of a membrane-associated rhoptry protein of Plasmodium falciparum

Invasive forms of apicomplexan parasites contain secretory organelles called rhoptries that are essential for entry into host cells. We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptry-associated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion. RAMA is synthesized as a 170-kDa protein in early trophozoites, several hours before rhoptry formation and is transiently localized within the endoplasmic reticulum and Golgi within lipid-rich microdomains. Regions of the Golgi membrane containing RAMA bud to form vesicles that later mature into rhoptries in a process that is inhibitable by brefeldin A. Other rhoptry proteins such as RhopH3 and RAP1 are found in close apposition with RAMA suggesting direct protein-protein interactions. We suggest that RAMA is involved in trafficking of these proteins into rhoptries. In rhoptries, RAMA is proteolytically processed to give a 60-kDa form that is anchored in the inner face of the rhoptry membrane by means of the glycosylphosphatidylinositol anchor. The p60 RAMA form is discharged from the rhoptries of free merozoites and binds to the red blood cell membrane by its most C-terminal region. In early ring stages RAMA is found in association with the parasitophorous vacuole.     

Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites

The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth.     

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.     

Sequence analysis of the Rhop-3 gene of Plasmodium yoelii

The 110 kDa/Rhop-3 rhoptry protein of Plasmodium falciparum is non-covalently associated with two other proteins, the 140 kDa Rhop-1 and the 130 kDa Rhop-2. cDNAs encoding Rhop-3 from Plasmodium yoelii were isolated using rhoptry-specific antisera from Plasmodium falciparum, P. yoelii, and Plasmodium chabaudi. The cDNAs encoded peptides with partial homology to the C-terminal region (residues 541-861) of P. falciparum Rhop-3. Core regions of homology to the P. falciparum gene will be useful in determining the biological role of Rhop-3 and its potential as a vaccine candidate for malaria.     

Plasmodial ortholog of Toxoplasma gondii rhoptry neck protein 3 is localized to the rhoptry body

The proteins in apical organelles of Plasmodium falciparum merozoite play an important role in invasion into erythrocytes. Several rhoptry neck (RON) proteins have been identified in rhoptry proteome of the closely-related apicomplexan parasite, Toxoplasma gondii. Recently, three of P. falciparum proteins orthologous to TgRON proteins, PfRON2, 4 and 5, were found to be located in the rhoptry neck and interact with the micronemal protein apical membrane antigen 1 (PfAMA1) to form a moving junction complex that helps the invasion of merozoite into erythrocyte. However, the other P. falciparum RON proteins have yet to be characterized. Here, we determined that "PFL2505c" (hereafter referred to as pfron3) is the ortholog of the tgron3 in P. falciparum and characterized its protein expression profile, subcellular localization, and complex formation. Protein expression analysis revealed that PfRON3 was expressed primarily in late schizont stage parasites. Immunofluorescence microscopy (IFA) showed that PfRON3 localizes in the apical region of P. falciparum merozoites. Results from immunoelectron microscopy, along with IFA, clarified that PfRON3 localizes in the rhoptry body and not in the rhoptry neck. Even after erythrocyte invasion, PfRON3 was still detectable at the parasite ring stage in the parasitophorous vacuole. Moreover, co-immunoprecipitation studies indicated that PfRON3 interacts with PfRON2 and PfRON4, but not with PfAMA1. These results suggest that PfRON3 partakes in the novel PfRON complex formation (PfRON2, 3, and 4), but not in the moving junction complex (PfRON2, 4, 5, and PfAMA1). The novel PfRON complex, as well as the moving junction complex, might play a fundamental role in erythrocyte invasion by merozoite stage parasites.     

RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum

Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.     

Identification of a stomatin orthologue in vacuoles induced in human erythrocytes by malaria parasites. A role for microbial raft proteins in apicomplexan vacuole biogenesis

When the human malaria parasite Plasmodium falciparum infects erythrocytes, proteins associated with host-derived detergent-resistant membrane (DRM) rafts are selectively recruited into the newly formed vacuole, but parasite proteins that contribute to raft-based vacuole development are unknown. In mammalian cells, DRM-associated integral membrane proteins such as caveolin-1 and flotillin-1 that form oligomers have been linked to the formation of DRM-based invaginations called caveolae. Here we show that the P. falciparum genome does not encode caveolins or flotillins but does contain an orthologue of human band 7 stomatin, a protein known to oligomerize, associate with non-caveolar DRMs and is distantly related to flotillins. Stomatins are members of a large protein family conserved in evolution and P. falciparum (Pf) stomatin appears to be a prokaryotic-like molecule. Evidence is presented that it associates with DRMs and may oligomerize, suggesting that these features are conserved in the stomatin family. Further, Pfstomatin is an integral membrane protein concentrated at the apical end of extracellular parasites, where it co-localizes with invasion-associated rhoptry organelles. A resident rhoptry protein, RhopH2 also resides in DRMs. This provides the first evidence that rhoptries of an apicomplexan parasite contain DRM rafts. Further, when the parasite invades erythrocytes, rhoptry Pfstomatin and RhopH2 are inserted into the newly formed vacuole. Thus, like caveolin-1 and flotillin-1, a stomatin may also associate with non-clathrin coated, DRM-enriched vacuoles. We propose a new model of invasion and vacuole formation involving DRM-based interactions of both host and parasite molecules.     

Plasmodium falciparum: analysis of the interaction of antigen Pf155/RESA with the erythrocyte membrane

The location of the Plasmodium falciparum vaccine candidate antigen Pf155/RESA in the membrane of infected erythrocytes was analzyed by means of selective surface radioiodination and immunofluorescence of surface-modified cells. The lack of radiolabel in Pf155/RESA as well as its localization by immunofluorescence similar to that of the N-terminal region of erythrocyte band 3 suggests that the antigen is associated with the cytoplasmic phase of the erythrocyte membrane. In concordance with this, Pf155/RESA was detected by immunofluorescence on the surface of inside out membrane vesicles from P. falciparum-infected erythrocytes. Pf155/RESA from spent culture medium also bound to inside out membrane vesicles of normal erythrocytes as well as to cytoskeletal shells of such vesicles, but failed to bind to sealed right-side out membrane vesicles. Depletion of spectrin from the vesicles abolished antigen binding, suggesting that Pf155/RESA association with the erythrocyte cytoskeleton is mediated by spectrin.     

Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites

The rhoptries of Plasmodium species participate in merozoite invasion and modification of the host erythrocyte. However, only a few rhoptry proteins have been identified using conventional gene identification protocols. To investigate the protein organization of this organelle and to identify new rhoptry proteins, merozoite rhoptries from three different Plasmodium rodent species were enriched by sucrose density gradient fractionation, and subjected to proteome analysis using multidimensional protein identification technology (MudPIT); 148 proteins were identified. To distinguish abundant cellular contaminants from bona fide organellar proteins, a differential analysis comparing the proteins in the rhoptry-enriched fractions to proteins identified from whole cell lysates of P. berghei mixed asexual blood stages was undertaken. In addition, the proteins detected were analyzed for the presence of transmembrane domains, secretory signal peptide, cell adhesion motifs, and/or rhoptry-specific tyrosine-sorting motifs. Combining the differential analysis and bioinformatic approaches, a set of 36 proteins was defined as being potentially located to the Plasmodium rhoptries. Among these potential rhoptry proteins were homologues of known rhoptry proteins, proteases, and enzymes involved in lipid metabolism. Molecular characterization and understanding of the supramolecular organization of these novel potential rhoptry proteins may assist in the identification of new intervention targets for the asexual blood stages of malaria.     

Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.     

Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented alpha-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%-85%, suggesting that MSP-3 protein's role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens.     

P. falciparum pro-histoaspartic protease (proHAP) protein peptides bind specifically to erythrocytes and inhibit the invasion process in vitro

Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasite's invasion of erythrocytes; however, this protein's role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 (101LKNYIKESVKLFNKGLTKKS120) and 30610 (121YLGSEFDNVELKDLANVLSF140 ). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26- and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro .     

Characterisation of Plasmodium falciparum RESA-like protein peptides that bind specifically to erythrocytes and inhibit invasion

The Plasmodium falciparum ring-erythrocyte surface antigen (RESA)-like putative protein was identified and characterised. PCR and RT-PCR assays revealed that the gene encoding this protein was both present and being transcribed in P. falciparum strain FCB-2 16 h after erythrocyte invasion. Indirect immunofluorescence studies detected this protein in infected erythrocyte (IE) cytosol in dense fluorescent granules similar to Maurer's clefts at 16-20 h (parasites in ring and trophozoite stages) and very strongly on IE membranes at 22 h, suggesting that it is synthesised during early ring stages (16 h) and transported to the infected red blood cell (RBC) membrane surface during the trophozoite stage (22 h). Western blotting showed that antisera produced against polymerised synthetic peptides of this protein recognised a 72-kDa band in P. falciparum schizont lysate. P. falciparum RESA-like peptides used in normal RBC binding assays revealed that peptides 30326 ((101)NAEKI LGFDD KNILE ALDLFY(120)), 30334 ((281)RVTWK KLRTK MIKAL KKSLTY(300)) and 30342 ((431)SSPQR LKFTA GGGFC GKLRNY(450)) bind with high activity and saturability, presenting nM affinity constants. These peptides contain alpha-helical structural elements, as determined by circular dichroism, and inhibit P. falciparum in vitro invasion of normal RBCs by up to 91%, suggesting that some RESA-like protein regions are involved in intra-erythrocyte stage P. falciparum invasion.     

Plasmodium rhoptries: how things went pear-shaped

Plasmodium parasites have three sets of specialised secretory organelles at the apical end of their invasive forms--rhoptries, micronemes and dense granules. The contents of these organelles are responsible for or contribute to host cell invasion and modification, and at least four apical proteins are leading vaccine candidates. Given the unusual nature of Plasmodium invasion, it is not surprising that unique proteins are involved in this process. Nowhere is this more evident than in rhoptries. We have collated data from several recent studies to compile a rhoptry proteome. Discussion is focussed here on rhoptry content and function.     

The Plasmodium rhoptry associated protein complex is important for parasitophorous vacuole membrane structure and intraerythrocytic parasite growth

Plasmodium parasites must invade erythrocytes in order to cause the disease malaria. The invasion process involves the coordinated secretion of parasite proteins from apical organelles that include the rhoptries. The rhoptry is comprised of two compartments: the neck and the bulb. Rhoptry neck proteins are involved in host cell adhesion and formation of the tight junction that forms between the invading parasite and erythrocyte, whereas the role of rhoptry bulb proteins remains ill‐defined due to the lack of functional studies. In this study, we show that the rhoptry‐associated protein (RAP) complex is not required for rhoptry morphology or erythrocyte invasion. Instead, post‐invasion when the parasite is bounded by a parasitophorous vacuolar membrane (PVM), the RAP complex facilitates the survival of the parasite in its new intracellular environment. Consequently, conditional knockdown of members of the RAP complex leads to altered PVM structure, delayed intra‐erythrocytic growth, and reduced parasitaemias in infected mice. This study provides evidence that rhoptry bulb proteins localising to the parasite–host cell interface are not simply by‐products of the invasion process but contribute to the growth of Plasmodium in vivo.     

Plasmodium falciparum AMA1 binds a rhoptry neck protein homologous to TgRON4, a component of the moving junction in Toxoplasma gondii

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) coimmunoprecipitates with the Plasmodium homologue of TgRON4, a secreted rhoptry neck protein of Toxoplasma gondii that migrates at the moving junction in association with TgAMA1 during invasion. PfRON4 also originates in the rhoptry necks, suggesting that this unusual collaboration of micronemes and rhoptries is a conserved feature of Apicomplexa.