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• W2071285311 abstract "Article1 April 2002free access Intramembrane cleavage of microneme proteins at the surface of the apicomplexan parasite Toxoplasma gondii Corinna Opitz Corinna Opitz Zentrum für Molekulare Biologie, Universität Heidelberg, INF282, D-69120 Heidelberg, Germany Search for more papers by this author Manlio Di Cristina Manlio Di Cristina Department of Biological Science, Imperial College of Science, Technology and Medicine, London, UK Search for more papers by this author Matthias Reiss Matthias Reiss Zentrum für Molekulare Biologie, Universität Heidelberg, INF282, D-69120 Heidelberg, Germany Department of Biological Science, Imperial College of Science, Technology and Medicine, London, UK Search for more papers by this author Thomas Ruppert Thomas Ruppert Zentrum für Molekulare Biologie, Universität Heidelberg, INF282, D-69120 Heidelberg, Germany Search for more papers by this author Andrea Crisanti Andrea Crisanti Department of Biological Science, Imperial College of Science, Technology and Medicine, London, UK Search for more papers by this author Dominique Soldati Corresponding Author Dominique Soldati Zentrum für Molekulare Biologie, Universität Heidelberg, INF282, D-69120 Heidelberg, Germany Department of Biological Science, Imperial College of Science, Technology and Medicine, London, UK Search for more papers by this author Corinna Opitz Corinna Opitz Zentrum für Molekulare Biologie, Universität Heidelberg, INF282, D-69120 Heidelberg, Germany Search for more papers by this author Manlio Di Cristina Manlio Di Cristina Department of Biological Science, Imperial College of Science, Technology and Medicine, London, UK Search for more papers by this author Matthias Reiss Matthias Reiss Zentrum für Molekulare Biologie, Universität Heidelberg, INF282, D-69120 Heidelberg, Germany Department of Biological Science, Imperial College of Science, Technology and Medicine, London, UK Search for more papers by this author Thomas Ruppert Thomas Ruppert Zentrum für Molekulare Biologie, Universität Heidelberg, INF282, D-69120 Heidelberg, Germany Search for more papers by this author Andrea Crisanti Andrea Crisanti Department of Biological Science, Imperial College of Science, Technology and Medicine, London, UK Search for more papers by this author Dominique Soldati Corresponding Author Dominique Soldati Zentrum für Molekulare Biologie, Universität Heidelberg, INF282, D-69120 Heidelberg, Germany Department of Biological Science, Imperial College of Science, Technology and Medicine, London, UK Search for more papers by this author Author Information Corinna Opitz1, Manlio Di Cristina2, Matthias Reiss1,2, Thomas Ruppert1, Andrea Crisanti2 and Dominique Soldati 1,2 1Zentrum für Molekulare Biologie, Universität Heidelberg, INF282, D-69120 Heidelberg, Germany 2Department of Biological Science, Imperial College of Science, Technology and Medicine, London, UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1577-1585https://doi.org/10.1093/emboj/21.7.1577 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Apicomplexan parasites actively secrete proteins at their apical pole as part of the host cell invasion process. The adhesive micronemal proteins are involved in the recognition of host cell receptors. Redistribution of these receptor–ligand complexes toward the posterior pole of the parasites is powered by the actomyosin system of the parasite and is presumed to drive parasite gliding motility and host cell penetration. The microneme protein protease termed MPP1 is responsible for the removal of the C-terminal domain of TgMIC2 and for shedding of the protein during invasion. In this study, we used site-specific mutagenesis to determine the amino acids essential for this cleavage to occur. Mapping of the cleavage site on TgMIC6 established that this processing occurs within the membrane-spanning domain, at a site that is conserved throughout all apicomplexan microneme proteins. The fusion of the surface antigen SAG1 with these transmembrane domains excluded any significant role for the ectodomain in the cleavage site recognition and provided evidence that MPP1 is constitutively active at the surface of the parasites, ready to sustain invasion at any time. Introduction The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. The apical secretory organelles, called micronemes, release numerous soluble and transmembrane proteins at the surface of the parasite during invasion (Tomley and Soldati, 2001). TgMIC2, an adhesin secreted during host cell penetration (Carruthers et al., 1999a), belongs to the family of thrombospondin-related anonymous proteins (TRAPs) previously shown to play an essential role in host cell invasion by Plasmodium sporozoites and ookinetes (Sultan et al., 1997; Dessens et al., 1999; Yuda et al., 1999; Templeton et al., 2000). The C-terminal domain of TgMIC2 was recently demonstrated to functionally replace the equivalent domain of TRAP in Plasmodium (Kappe et al., 1999). TgMIC2 has also been reported recently to form a complex with TgM2AP (Rabenau et al., 2001). Other transmembrane microneme proteins described in T.gondii, such as TgMIC6 and TgMIC8, serve as escorters for the soluble adhesins TgMIC1, TgMIC4 and TgMIC3, respectively (Reiss et al., 2001; Meissner et al., 2002). The stimulation of micronemes discharge is induced by a rise in intracellular Ca2+, and can be mimicked by treatment with Ca2+ ionophore or ethanol (Carruthers and Sibley, 1999; Carruthers et al., 1999b). Microneme proteins undergo complex proteolytic processing events that have been best documented in T.gondii. TgMIC3, TgMIC5, TgMIC6 and TgM2AP are proteolytically cleaved intracellularly during transport to the micronemes (Brydges et al., 2000; Garcia-Reguet et al., 2000; Rabenau et al., 2001; Reiss et al., 2001). In addition, TgMIC2, TgMIC4, TgMIC6 and TgMIC8 are processed post-exocytosis (Carruthers et al., 2000; Brecht et al., 2001; Reiss et al., 2001; Meissner et al., 2002). Two types of proteases, termed MPP1 and MPP2, have been reported to process TgMIC2 upon release at the parasite surface (Carruthers et al., 2000). Recently, the profile of protease inhibitors that prevent the N-terminal processing of TgMIC4 suggested that MPP2 is likely to cleave this protein as well (Brecht et al., 2001). MPP2-dependent cleavage precedes the MPP1 cleavage, and these maturation events on TgMIC2 have been shown to change the adhesive properties of the protein drastically (Carruthers et al., 2000). The C-terminal cleavage of TgMIC2, TgMIC6 and TgMIC8 is expected to cause the release of the adhesin complexes from the parasite surface. Presumably, this event constitutes an essential step during host cell invasion, ensuring the dissociation between the parasite and the host plasma membrane at the end of the penetration process. Little is known about the sequence requirement, the localization and the characteristics of the protease responsible for this activity. In this study, we have mapped the site of the MPP1-dependent processing at the C-terminus of TgMIC2, TgMIC6 and a newly characterized microneme protein TgMIC12. The cleavage, which appears to be essential for parasite survival, occurs within the transmembrane domain and does not require sequence information from the ectodomain. The protease is constitutively active at the surface of the parasites and conserved throughout the phylum of Apicomplexa, including in Plasmodium. Results The amino acid sequences within the membrane-spanning domain of apicomplexan microneme proteins are conserved An alignment of the transmembrane and cytoplasmic domains of T.gondii, P.falciparum, P.berghei, Eimeria tenella and Sarcocystis muris microneme proteins revealed a striking conservation of the amino acid sequences within their C-terminal membrane-spanning domain (Figure 1A). In T.gondii, TgMIC2 is processed upon secretion, during host cell invasion by an as yet unidentified protease called MPP1 (Carruthers et al., 2000). A very similar processing event occurs on TgMIC6 (Reiss et al., 2001), TgMIC8 (Meissner et al., 2002), TgAMA-1 (Donahue et al., 2000; Hehl et al., 2000) and TgMIC12 (this study), which most probably involves the same protease activity. Taking advantage of the easy accessibility of T.gondii to transfection, we have examined the sequence requirements of this processing event. Some of the most conserved residues across the phylum of Apicomplexa (boxed in grey in Figure 1A) were chosen for the site-specific mutagenesis. The constructs generated and analysed in this study are depicted in Figure 1B. Figure 1.(A) Amino acid sequence alignment of the TM domains of the apicomplexan microneme proteins TgMIC2, TgMIC6, TgMIC12, PbTRAP, PfTRAP, EtMIC1, Sm70, TgMIC8, TgAMA1, PfCTRP and EtMIC4. The strictly conserved amino acids are in black boxes, and the conserved residues chosen for the mutagenesis are boxed in grey. (B) Schematic representation of the constructs used in this study: MIC2myc1/2, MIC2myc2mut, MIC6Ty, MIC6Tymut and the chimeric fusions SAG1TyTMCDMIC2, SAG1TyTMCDMIC2mut, SAG1TyTMCDMIC6, SAG1TyTMCDMIC12, SAG1TyTMCDMIC12mut, SAG1TyTMCDMIC12* and SAG1TyTMCDMIC12*mut. c-myc or Ty-1 were used as epitope tags and are boxed. Tg, T.gondii; Pb, P.berghei; Pf, P.falciparum; Et, E.tenella; Sm, S.muris; AMA1, apical membrane antigen 1; CTRP, circumsporozoite TRAP-related protein. Download figure Download PowerPoint Point mutations within the transmembrane domain of TgMIC2 and TgMIC6 prevent processing and release of these proteins The insertion of a c-myc epitope tag at different positions in the cytoplasmic region of TgMIC2 previously allowed the mapping of sequences relevant for sorting to the micronemes (Di Cristina et al., 2000). The presence of a c-myc tag close to the transmembrane domain, on the luminal side of TgMIC2, compromised the C-terminal processing of MIC2myc1 (data not shown), while a tag inserted a few amino acids further upstream in MIC2myc2 was processed and secreted like wild-type TgMIC2 (Figure 2A). Based on the sequence alignment described above, we decided to insert four point mutations by site-specific mutagenesis within the transmembrane region of TgMIC2 (MIC2myc2), to generate MIC2myc2mut. The replacement of AGGVIGG with VALVIGV in MIC2myc2mut completely hampered the post-exocytotic cleavage at the C-terminus of the protein as analysed by western blot (Figure 2A). Nevertheless, parasites transiently transfected with MIC2myc2mut showed correct targeting of the transgene product to micronemes by indirect immunofluorescence assay (IFA; Figure 2B). While stably transformed clones expressing MIC2myc2 were easily obtained, we failed to isolate recombinant parasites stably expressing MIC2myc2mut, presumably because the lack of processing was deleterious to parasite survival. When five point mutations were introduced in the same region of TgMIC6 (VALVIVL), the corresponding mutant MIC6Tymut was targeted accurately to the micronemes (data not shown) and, as in the case of MIC2myc2mut, no proteolytic cleavage occurred at the C-terminus. In contrast to MIC2myc2mut, stable cell lines expressing MIC6Tymut in the MIC knockout (mic6ko) could be selected over a short period of time, allowing this mutant to be analysed by secretion assay. While TgMIC6Ty behaves like wild-type MIC6, TgMIC6Tymut was not cleaved and consequently not released in the cell culture supernatant (Figure 2C). Figure 2.Site-specific mutagenesis within the transmembrane domain of TgMIC2 and TgMIC6 abolished the C-terminal processing but did not affect proper sorting to the micronemes. (A) Western blot analysis of the parasite pellet and ESA of parasites stably transfected with pMMIC2myc2 using mAb 9E10 against the c-myc epitope. Right panel: total cell lysates of parasites 24 h after transfection with pMMIC2myc2 or pMMIC2myc2mut and stimulated for microneme secretion with ethanol treatment. The fully processed form of TgMIC2 with the C-terminal domain released is not detectable in MIC2myc2mut. The partial processing at the N-terminus of TgMIC2 is detectable (see arrow pointing to the band below proMIC2). (B) Double immunofluorescence analysis by confocal microscopy of intracellular parasites transiently expressing MIC2myc2 or MIC2myc2mut and detected with anti-myc antibodies. MIC2myc2mut did co-localize with the microneme marker MIC6 (left panel anti-MIC6). (C) Western blot analysis of secretion assays (parasite pellet and ESA) from stably transformed parasites expressing MIC6Ty or MIC6Tymut. The antibodies used were mAb BB2 recognizing the Ty-1 epitope and T3 4A11 anti-MIC2 as a control. The C-terminal processing of MIC6Tymut did not occur and, consequently, the protein was not released in the ESA. Download figure Download PowerPoint Mapping of the C-terminal cleavage site of TgMIC6 To determine with precision the MPP1 site of cleavage, recombinant parasites expressing TgMIC6Ty were used to purify large amounts of the processed form of TgMIC6 and to analyse it by mass spectrometry. The mature form of TgMIC6 was isolated from 5 × 109 freshly released parasites after stimulation of microneme secretion by ethanol and purification by immunoprecipitation using the monoclonal antibody (mAb) BB2 specific for the Ty-1 epitope. The 25 kDa processed product was separated finally on SDS–PAGE and digested with trypsin. The resulting peptides were analysed by mass spectrometry to identify a peptide whose C-terminus was not generated by trypsin cleavage. A doubly charged peptide ion matching the mass of the peptide EEGSGHAGAIA (m/z = 499.9) was detected. This peptide ion was fragmented by collision-induced dissociation and the fragment ions were recorded under conditions optimized for the mass range 500–1200 to confirm the amino acid sequence. The major fragment peaks are assigned to a bo series with the C-terminal peptide sequence AGAIA (Figure 3). Additionally, the fragments labelled as y9 and y10 are consistent with the two N-terminal amino acids EE of the peptide EEGSGHAGAIA. Figure 3.Mapping of the processing site at the C-terminus of TgMIC6 by identification of the peptide EEGSGHAGAIA by MS/ESI. After in-gel digestion of MIC6Ty, the tryptic peptides were bound to Poros R2 and Poros oligoR3 material. In the eluate from the R3 column, a doubly charged peptide ion matching the mass of the peptide EEGSGHAGAIA (m/z = 499.9) was detected. This peptide was fragmented and major fragment peaks are assigned to a bo series with the C-terminal peptide sequence AGAIA. Two additional peaks labelled as y9 and y10 are consistent with the two N-terminal amino acids EE. The amino acid sequence conservation around the cleavage site in TgMIC2, TgMIC6, TgMIC12 and PbTRAP is depicted below, with the tryptic peptide of MIC6 in bold. Download figure Download PowerPoint MPP1 processing is conserved for micronemal proteins of the Apicomplexa phylum The precise determination of the MPP1 cleavage site on TgMIC6 revealed the striking conservation of the amino acid sequence flanking this site in all transmembrane micronemal proteins of the Apicomplexa. In Eimeria, EtMIC1 is processed at the C-terminus upon microneme secretion (F.Tomley, personal communication). In Neospora caninum, the homologue of TgMIC2, NcMIC2, also lacks the C-terminal domain (Lovett et al., 2000). In Plasmodium species, both the redistribution of TRAP from the anterior to the posterior pole and the shedding of the protein by parasites, which presumably involves the C-terminal proteolytic cleavage of TRAP, are crucial for sporozoite gliding motility (Kappe et al., 1999). In a previous study, P.berghei TRAP protein was expressed in T.gondii (Di Cristina et al., 2000). We compared the expression of PbTRAP and PbTRAP lacking the transmembrane and cytoplasmic tail (PbTRAPΔTMCD) by transient transfection in T.gondii. The evidence for C-terminal processing of the full-length TRAP was obtained by western blot analysis (Figure 4). Consistent with the removal of the TMCD domain, the processed form co-migrated with the PbTRAPΔTMCD while the deleted mutant remained unprocessed. The C-terminal cleavage of TRAP in T.gondii reinforces the notion that this proteolytic event is ubiquitous in the Apicomplexa and the components of the machinery are functionally conserved. Figure 4.When expressed in T.gondii, PbTRAP is processed at the C-terminus. Western blot (reducing conditions) analysis of total protein lysates of tachyzoites transiently transformed with PbTRAP wild-type (TRAP), TRAP devoid of the transmembrane domain and cytoplasmic tail (TRAPΔTMCD) and wild-type parasites (control) using mAb anti-PbTRAP. Download figure Download PowerPoint The minimal amino acid sequence information required for MPP1 cleavage does not include the ectodomains The C-terminal domains of TgMIC2 and TgMIC6 carry sufficient information for sorting to the appropriate organelles. When the major surface antigen SAG1 lacking its glycosylphosphatidylinositol (GPI) anchor signal was fused to the transmembrane and cytoplasmic domains of TgMIC2 or TgMIC6, the chimeric products were targeted to the micronemes (Di Cristina et al., 2000; Reiss et al., 2001). These SAG1 fusions exhibited a typical type I membrane topology as demonstrated by transient permeabilization followed by proteinase K treatment (Figure 5A). This type I topology was shown previously to be adopted by wild-type TgMIC6 (Meissner et al., 2002) and TgMIC2 (data not shown). Consequently, the sorting signals present in the C-terminal tail are facing the cytosol and are accessible to the adaptor complex machinery implicated in vesicular trafficking (Hoppe et al., 2000). Analysis of the recombinant parasites stably expressing SAG1TMCDMIC2 by secretion assays revealed that SAG1 is secreted upon ethanol stimulation, processed at the C-terminus and released in the cell supernatant in just the same manner as wild-type TgMIC2 (Figure 5B). To demonstrate unambiguously that the same processing event took place on these SAG1 fusions, the point mutations previously introduced in the transmembrane segment of TgMIC2 and TgMIC6 were included in the pSAG1TMCDMIC2 construct. The SAG1TMCDMIC2mut fusion was sorted accurately to the micronemes and, as expected, the C-terminal cleavage of SAG1TMCDMIC2mut was completely abrogated. Identical results were obtained with SAG1TMCDMIC6mut (data not shown). As in the case of MIC2mut, we failed to obtain stable lines expressing SAG1TMCDMIC2mut (Figure 5C). A schematic representation of the products obtained either after proteinase K digestion or upon stimulation of secretion is depicted in Figure 5D. Figure 5.The minimal requirement for processing to occur involves the luminal region of the MICs. SAG1TyTMCDMIC2 and SAG1TyTMCDMIC6 fusions are targeted to the micronemes and subjected to processing upon release by the micronemes. (A) The type I membrane protein topology of the SAG1TyTMCDMIC2 fusion was determined by partial permeabilization and proteinase K treatment. (B) Western blot analysis of total lysates and ESAs from recombinant parasites expressing SAG1TyTMCDMIC2 or SAG1TyTMCDMIC6. The membrane was probed with anti-Ty-1 antibodies. (C) Western blot analysis of recombinant parasites expressing SAG1TyTMCDMIC2 and SAG1TyTMCDMIC2mut. The presence of the four point mutations prevents the cleavage of the chimeric protein. The membranes were probed with either anti-myc or anti-Ty-1. (D) The construct SAG1TyTMCDMIC2 shown schematically as the full protein form with two Ty-1 epitope tags before the transmembrane domain (TM) and the c-myc epitope tag within the cytoplasmic domain. The smaller form protected from proteinase K treatment and the secreted processed form are shown below. The last cartoon shows the construct SAG1TyTMCDMIC2mut schematically. The localization of only one Ty-1 epitope tag before the TM, as well as the localization of the four point mutations within the TM, is shown. Download figure Download PowerPoint TgMIC12 is a novel microneme protein, which also contains targeting signals to the organelle in the cytoplasmic tail TgMIC12 is a novel microneme protein recently identified in T.gondii and partially sequenced (accession number AAK58479). Antibodies raised against the C-terminus of this protein stained the small apical microneme organelles and detected a product of 50 kDa by western blotting analysis (F.Stavru and D.Soldati, unpublished). The C-terminal region of TgMIC12 exhibited a strong homology to members of the TRAP family (Figure 1) and showed the closest resemblance to the partial sequence of a 70 kDa S.muris microneme protein (Tomley and Soldati, 2001). Consistent with previous observations, the fusion of the TMCD domain of TgMIC12 to the SAG1 ectodomain also localized the chimeric protein to the micronemes (Figure 6A). This SAG1TMCDMIC12 fusion is excluded from the parasitophorous vacuole, as illustrated by the absence of co-localization with the dense granule marker, GRA3. In contrast, an inadvertent mutant generated at the extreme C-terminus of SAG1TMCDMIC12 caused a complete mistargeting of the fusion protein to the dense granules and to the parasitophorous vacuolar space as seen by extensive co-localization with GRA3 (Figure 6B). In this SAG1TMCDMIC12* mutant, the last four amino acids, ADMD, have been replaced by the 10 amino acids QTLLKWIRWH (Figure 1B). The reason for this mistargeting is not understood but might involve an incorrect folding of the cytoplasmic tail, which potentially masks the sorting signals and prevents their interaction with the adaptor complex machinery. Although dense granules were described previously as the default pathway taken only by soluble proteins and not by transmembrane or GPI-anchored proteins in T.gondii (Karsten et al., 1998), SAGTMCDMIC12* is the first example of a transmembrane protein to follow this route by default. Figure 6.SAG1TyTMCDMIC12 is targeted to the micronemes while SAG1TyTMCDMIC12* is re-routed to the dense granules and into the parasitophorous vacuole. Double immunofluorescence analysis by confocal microscopy was carried out with anti-MIC6 and anti-GRA3 as microneme and dense granule/parasitophorous vacuole markers, respectively. (A) SAG1TyTMCDMIC12 was detected with anti-Ty-1 (in red) and co-localized with MIC6 (in green) (B) SAG1TyTMCDMIC12*, carrying an out-of-frame mutation changing the sequence at the extreme C-terminus, was detected with anti-Ty-1 (in red) and co-localized with GRA3 (in green). Download figure Download PowerPoint MPP1 is constitutively active at the plasma membrane of the parasites and does not require the substrate to traffic through the micronemes Despite the fact that SAG1TMCDMIC12* accumulated in the vacuole, the protein was processed accurately at its C-terminus (Figure 7A). However, in contrast to the proteins targeted to the micronemes, including SAG1TMCDMIC12, which are only processed after organelle secretion, the cleavage of SAG1TMCDMIC12* occurred constitutively during the intracellular cycle of parasite replication. Since dense granules are constitutively discharging their contents into the vacuole, this result suggested that MPP1-dependent processing occurred either at the parasite surface or in dense granules. To distinguish between the two possibilities, lysates were prepared from extensively washed extracellular parasites and subsequently stimulated for organelle secretion at 37°C for 60 min and analysed by western blot. Both SAG1TMCDMIC12 and SAG1TMCDMIC12* were processed only upon secretion by micronemes and dense granules, respectively (Figure 7B). This result clearly established that the cleavage of SAG1TMCDMIC12* occurred only when the proteins reached the cell surface, and not during storage in the dense granules. Finally, the inhibitory effect of the presence of the five point mutations on SAG1TMCDMIC12mut and SAG1TMCDMIC12*mut confirmed that the cleavage observed on the mistargeted SAG1TMCDMIC12* was identical to that occurring on the same protein targeted to the micronemes (Figure 7C). The subcellular distribution of SAG1TMCDMIC12mut and SAG1TMCDMIC12*mut was examined by immunofluorescence on confocal microscopy after transient transfection. As expected, the mutations introduced in SAG1TMCDMIC12mut did not affect targeting to the micronemes, while in the case of SAG1TMCDMIC12*mut the absence of cleavage leads to the persistence of the protein at the parasite surface (Figure 7D), instead of an accumulation in the vacuolar space (Figure 6B). Figure 7.Despite mistargeting to the vacuole, SAG1TyTMCDMIC12* is processed accurately at the C-terminus. (A) Western blot analysis of total lysates from intracellular parasites (Int.) stably transformed with SAG1TyTMCDMIC12 and SAG1TyTMCDMIC12* showed constitutive processing at the C-terminus of SAG1TyTMCDMIC12*. (B) Western blot analysis of total lysates from extracellular (Ext.) parasites as well as pellet (P) and secreted material (ESA). For both SAG1TyTMCDMIC12 and SAG1TyTMCDMIC12*, the processing occurred after release from micronemes and dense granules, respectively. (C) Western blot analysis of parasites transiently transfected with SAG1TyTMCDMIC12, SAG1TyTMCDMIC12mut, SAG1TyTMCDMIC12* and SAG1TyTMCDMIC12*mut established that processing occurred only with the non-mutated constructs and thus they are generated by the MPP1-dependent cleavage. (D) Overlays of double immunofluorecence analysis by confocal microscopy on intracellular parasites transiently expressing SAG1TyTMCDMIC12mut and SAG1TyTMCDMIC12*mut. SAG1TyTMCDMIC12mut detected by anti-Ty (in green) co-localized with anti-MIC6 (in red) while SAG1TyTMCDMIC12*mut is present at the plasma membrane of the parasites (in green) and did not co-localize with GRA3 (in red). Download figure Download PowerPoint Discussion Transmembrane microneme proteins are presumed to play a dual essential role during host cell invasion by the Apicomplexa. The adhesin domains present on the luminal side of these proteins or, indirectly, their association with soluble adhesins establish tight and specific interactions with host cell receptors while their cytoplasmic tail presumably is connected to the actomyosin system of the parasite. Only transmembrane proteins are expected to transmit the mechanical force across the plasma membrane and thus to power parasite gliding motion. Upon stimulation of microneme secretion, some proteins were shown previously to redistribute over the entire surface of the parasites and, as invasion proceeds, these proteins are excluded from the forming vacuole, accumulate at the posterior pole of the parasites and are shed. This has been documented previously for TgMIC2 (Carruthers et al., 1999a) and TgMIC3 (Garcia-Reguet et al., 2000). The proteolytic processing occurring on these proteins and causing their rapid release from the cell surface is anticipated to be an essential step to accomplish the penetration process. In a preliminary experiment, we observed that the insertion of a c-myc tag in close proximity to the transmembrane-spanning domain of TgMIC2 hampered its processing at the C-terminus. Subsequently, we identified by site-specific mutagenesis a few conserved amino acids within the transmembrane domain which were necessary for the cleavage of TgMIC2 to take place. Expression of a non-processed TgMIC2 mutant (MIC2myc2mut, Figures 1B and 2A) appeared to compromise the survival of the parasites as all attempts to generate stable transformants remained unsuccessful. The deleterious effect observed due to the absence of cleavage on TgMIC2 underlines the importance of this event for a successful invasion. In the absence of processing, the exocytosed TgMIC2 might create a persistent bridge between the host cell and parasite surface, thus interfering with the proper sealing of the newly formed parasitophorous vacuole and preventing the parasite from replicating inside the vacuole. The introduction of equivalent point mutations on TgMIC6 provoked the same phenotype and was slightly better tolerated by the parasite, but the stable clones could be maintained for only a few passages. TgMIC6 does not bind directly to the host cell surface receptors but functions as an escorter and, presumably, the lack of processing does not lead to such dramatic consequences. Moreover, we have shown previously that TgMIC6 is dispensable, as we could generate mic6ko by homologous recombination but, in contrast, multiple attempts to disrupt the TgMIC2 gene have been unsuccessful so far. This suggests that unlike TgMIC6, TgMIC2 fulfils a more substantial role. In Plasmodium species, by disruption of the genes coding for several members of the TRAP family it was shown that they are a prerequisite for parasite motility and host cell invasion (Sultan et al., 1997; Dessens et al., 1999; Yuda et al., 1999; Templeton et al., 2000). Previously, fusions of SAG1 to the TMCD domains of microneme proteins TgMIC2 and TgMIC6 established that the sorting signal to the micronemes are confined within the short cytoplasmic tails. In this study, we provide compelling evidence that the short C-terminal domain of these proteins is sufficient to serve as a substrate for MPP1, excluding an essential role for luminal domains, which contain diverse combinations of adhesive motifs (Tomley and Soldati, 2001). The striking and unexpected conservation within the transmembrane domain combined with the results of the mutagenesis suggested to us that MPP1 functio" @default.
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• W2071285311 date "2002-04-01" @default.
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• W2071285311 title "Intramembrane cleavage of microneme proteins at the surface of the apicomplexan parasite Toxoplasma gondii" @default.
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