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• W2110366566 abstract "Article16 June 2005free access Trafficking of STEVOR to the Maurer's clefts in Plasmodium falciparum-infected erythrocytes Jude M Przyborski Jude M Przyborski Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Susanne K Miller Susanne K Miller The Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia Search for more papers by this author Judith M Pfahler Judith M Pfahler Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Philipp P Henrich Philipp P Henrich Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Petra Rohrbach Petra Rohrbach Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Brendan S Crabb Brendan S Crabb The Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia Search for more papers by this author Michael Lanzer Corresponding Author Michael Lanzer Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Jude M Przyborski Jude M Przyborski Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Susanne K Miller Susanne K Miller The Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia Search for more papers by this author Judith M Pfahler Judith M Pfahler Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Philipp P Henrich Philipp P Henrich Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Petra Rohrbach Petra Rohrbach Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Brendan S Crabb Brendan S Crabb The Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia Search for more papers by this author Michael Lanzer Corresponding Author Michael Lanzer Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Author Information Jude M Przyborski1, Susanne K Miller2, Judith M Pfahler1, Philipp P Henrich1, Petra Rohrbach1, Brendan S Crabb2 and Michael Lanzer 1 1Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany 2The Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia *Corresponding author. Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany. Tel.: +49 6221 567845; Fax: +49 6221 564643; E-mail: [email protected] The EMBO Journal (2005)24:2306-2317https://doi.org/10.1038/sj.emboj.7600720 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The human malarial parasite Plasmodium falciparum exports proteins to destinations within its host erythrocyte, including cytosol, surface and membranous profiles of parasite origin termed Maurer's clefts. Although several of these exported proteins are determinants of pathology and virulence, the mechanisms and trafficking signals underpinning protein export are largely uncharacterized—particularly for exported transmembrane proteins. Here, we have investigated the signals mediating trafficking of STEVOR, a family of transmembrane proteins located at the Maurer's clefts and believed to play a role in antigenic variation. Our data show that, apart from a signal sequence, a minimum of two addition signals are required. This includes a host cell targeting signal for export to the host erythrocyte and a transmembrane domain for final sorting to Maurer's clefts. Biochemical studies indicate that STEVOR traverses the secretory pathway as an integral membrane protein. Our data suggest general principles for transport of transmembrane proteins to the Maurer's clefts and provide new insights into protein sorting and trafficking processes in P. falciparum. Introduction The human malarial parasite Plasmodium falciparum is responsible for several hundred million clinical cases, and 2–3 million deaths annually, putting a huge economic burden upon the affected countries, not to mention the suffering the infected individuals endure (World Health Organization, 2000). The high morbidity and mortality associated with falciparum malaria relate to the intraerythrocytic stages of the parasite (Miller et al, 2002). During asexual development within human erythrocytes, P. falciparum radically changes its host cell. Electron dense structures, termed knobs, appear underneath the plasma membrane of the infected erythrocyte (Kilejian, 1979) and membranous networks, extending from the parasitophorous vacuolar membrane (PVM) toward the erythrocyte periphery, fill the host cell cytoplasm (Haldar et al, 2002; Wickert et al, 2003b, 2004). Concomitantly, infected erythrocytes acquire adhesive properties and sequester in the deep vascular bed of inner organs, resulting in a broad spectrum of pathology ranging from localized hypoxia to inflammatory reactions and the syndromes of cerebral and maternal malaria (Miller et al, 2002). To change the morphological and functional properties of its host erythrocyte, the parasite exports proteins into the host cell cytosol, and beyond to the erythrocyte plasma membrane. For example, the knob-associated histidine-rich protein (KAHRP), a major constituent of knobs (Culvenor et al, 1987; Pologe et al, 1987), is secreted into the erythrocyte cytoplasm to form a structure that anchors parasite-encoded immunovariant adhesins to the erythrocyte cytoskeleton (Crabb et al, 1997; Waller et al, 1999, 2002). Other exported proteins such as the skeletal binding protein 1 (PfSBP1) (Blisnick et al, 2000), exported protein 1 (PfEXP1) (Kara et al, 1988; Gunther et al, 1991), subtelomeric variable open reading frame (STEVOR) (Cheng et al, 1998; Kaviratne et al, 2002) and P. falciparum homologues of COPII proteins (Albano et al, 1999; Adisa et al, 2001; Wickert et al, 2003a) are associated with the membranous network or regions thereof close to the erythrocyte plasma membrane, referred to as Maurer's clefts. The mechanisms underpinning protein transport to defined destinations within the host erythrocyte have remained largely enigmatic, despite its uniqueness. As the erythrocyte is denucleated, it consequently lacks the secretory apparatus present in eukaryotic cells. Thus, the parasite is forced to create its own protein transport machinery outside its plasma membrane. The generation of an ‘extracellular’ secretory system poses a challenge not met by any other organism studied thus far. Previous studies have suggested that entry into the parasite's default secretory pathway (bulk flow), probably via the endoplasmic reticulum (ER), is mediated by a canonical (Waller et al, 2000; Burghaus and Lingelbach, 2001) or an unconventional N-terminal signal sequence (Wickham et al, 2001). The default pathway allows trafficking of proteins across the parasite's plasma membrane to the lumen of the parasitophorous vacuole (Wickham et al, 2001; Lopez-Estrano et al, 2003). Recent studies have identified a recessed host cell targeting (HCT) signal, which appears to be conserved in a large number of predicted secreted proteins, for transport across the PVM (Hiller et al, 2004; Marti et al, 2004). Interestingly, the same HCT signal appears to play a role in the export of both soluble and transmembrane proteins (Hiller et al, 2004; Marti et al, 2004), suggesting that, for integral membrane proteins, there must be additional sequence elements mediating their sorting to parasite-derived membrane profiles within the host erythrocyte cytoplasm, such as the Maurer's clefts. As a model for integral membrane proteins targeted to the Maurer's clefts, we have studied trafficking of STEVOR. First reported as 7h8 (Limpaiboon et al, 1991), STEVOR proteins are encoded by a subtelomerically located multigene family composed of 30–40 members, dependent on parasite strain (Blythe et al, 2004), and represent, together with the erythrocyte surface located RIFINS, a large superfamily of variant antigens (Cheng et al, 1998). STEVOR variants are 30–40 kDa in size and are predicted to contain a signal sequence and two transmembrane domains flanking a hypervariable loop region (Cheng et al, 1998; Sam-Yellowe et al, 2004). A similar domain structure has been reported for RIFIN and PfMC-2TM (Sam-Yellowe et al, 2004). Transcription of stevor in asexual stages occurs within a tight developmental window 22–32 h after invasion, with peak transcription coinciding with the mid-trophozoite stage (Kaviratne et al, 2002). Single parasites transcribe more than one stevor; however, only a subset of stevor is transcribed at any given time in cultured parasites (Kaviratne et al, 2002). From the trophozoite stage on, STEVOR can be localized to the Maurer's clefts (Kaviratne et al, 2002). STEVOR is also expressed in gametocytes and sporozoites, suggesting that it fulfills a function at different time points throughout the parasite life cycle (McRobert et al, 2004). The small size of STEVOR and its transmembranous nature make it an amenable and relevant tool with which to study export and trafficking of integral membrane proteins in P. falciparum-infected erythrocytes. Our data show that trafficking of STEVOR to the Maurer's clefts is a complex process requiring multiple signals. Results A full-length STEVOR-GFP fusion protein is targeted to Maurer's clefts Initially, we examined targeting of full-length STEVOR fused to the green fluorescent protein (GFP) (STEVORfull). Confocal laser scanning microscopy of parasites expressing STEVORfull revealed a dotted fluorescence pattern within the host erythrocyte cytoplasm, characteristic of the Maurer's clefts (Figure 1A). A control parasite line expressing GFP alone revealed a fluorescence signal confined within the boundaries of the parasite plasma membrane (Figure 1A). To verify a localization of STEVORfull to Maurer's clefts, we performed immunofluorescence colocalization studies using antisera recognizing the established Maurer's clefts marker PfSBP1 (Blisnick et al, 2000) as well as PfEMP1 (Wickert et al, 2003b), which transiently associates with Maurer's clefts on its way to the erythrocyte plasma membrane (Wickham et al, 2001; Kriek et al, 2003; Wickert et al, 2003b). Both PfSBP1 and PfEMP1 colocalize with the chimeric STEVORfull protein (Figure 1B), consistent with previous studies (Kaviratne et al, 2002). These data indicate that a full-length STEVOR-GFP fusion protein, unlike GFP alone, is exported from the parasite and properly targeted to the Maurer's clefts, suggesting that all signals necessary for transport are contained within the STEVOR primary sequence. Figure 1.Subcellular localization of STEVORfull and GFP in P. falciparum-infected erythrocytes. (A) The upper row represents control (GFP alone) and the lower row STEVORfull. The left image shows differential interference contrast (DIC), middle image GFP fluorescence and the right image overlay. The GFP-only control reveals fluorescence only within the boundaries of the parasite plasma membrane. STEVORfull exhibits a dotted fluorescence pattern within the host erythrocyte cytoplasm, characteristic of the Maurer's clefts. Scale refers to the residue number of STEVOR. Red, signal sequence; blue, predicted transmembrane domains; yellow, bulk of STEVOR; green, GFP. Bar, 4 μm. (B) Colocalization of STEVORfull (αGFP) with PfSBP1 (αPfSBP1) and/or PfEMP1 (αPfEMP1) by immunofluorescence microscopy. Overlay of signals is shown in the right panel. Bar, 3 μm. Download figure Download PowerPoint An N-terminal signal sequence and a recessed host cell targeting signal are necessary for STEVOR export into the erythrocyte cytosol To investigate signals mediating targeting of STEVOR to the Maurer's clefts, we generated C-terminal nested deletions. Transfectant lines expressing up to and including the first 20 amino acids of STEVOR exhibit patterns of fluorescence identical to the GFP-only control line, with the fluorescence signals confined within the body of the parasite (Figure 2). Upon adding a further 5 amino acids, the fluorescence signal changed significantly, with the chimera being exported to the lumen of the parasitophorous vacuole. Consistent with this interpretation, we occasionally observed fluorescent protrusions of the PVM. These data indicate that the first 25 amino acids of STEVOR constitute a functional signal sequence, which allows entry into the secretory system and export across the parasite plasma membrane into the parasitophorous vacuole. Figure 2.Nested deletional analysis of STEVOR. The confocal images show the subcellular localization of different STEVOR-GFP chimera in P. falciparum-infected erythrocytes: STEVOR1–20 (top row), STEVOR1–25 (second row), STEVOR1–60 (third row), STEVOR1–70 (fourth row), STEVOR1–80 (fifth row) and STEVOR1–170 (bottom row). Parasites exporting soluble chimera to the parasitophorous vacuole frequently display fluorescent protrusions of the PVM. The predicted signal sequence of STEVOR is shown in red (left side). Green, GFP. Bar, 4 μm. Download figure Download PowerPoint We next created further lines expressing the first 35, 40, 45, 50, 60, 70 and 80 N-terminal amino acids of STEVOR fused to GFP. All transfectant lines expressing up to and including the first 60 amino acids of the STEVOR N-terminal region show a pattern of fluorescence indistinguishable from that of STEVOR1–25, that is, GFP fluorescence is confined to the lumen of the parasitophorous vacuole and only little fluorescence is seen within the parasite body (Figure 2). No fluorescence is evident within the host erythrocyte cytosol (Figure 2). The addition of a further 10 amino acids (STEVOR1–70) changes the distribution of the fluorescence signal. Now, a population of the chimeric protein is transported to the cytosol of the erythrocyte, as is evident by the diffuse fluorescence pattern throughout the entire erythrocyte cytosol (Figure 2). Fluorescence can also be seen within a ring structure surrounding the body of the parasite, corresponding to the lumen of the parasitophorous vacuole, and within the parasite's body, including the large food vacuole (Figure 2). Food vacuolar GFP fluorescence is commonly seen in transfectant lines exporting fluorescent chimera to the host cell cytosol (Waller et al, 2000; Wickham et al, 2001), and has been interpreted as resulting from the uptake of the erythrocyte cytosol by the parasite during feeding. The fluorescence signal within the erythrocyte cytosol substantially increased when the STEVOR N-terminal sequence was extended to include the first 80 amino acids (Figure 2). No further changes in the fluorescence patterns were observed in transfectant lines expressing the first 170 amino acids of STEVOR fused to GFP (Figure 2). To further delineate the first 80 N-terminal amino acids of STEVOR, we deleted amino acids 26–50, which contain conserved HCT motifs (Figure 3A) (Hiller et al, 2004; Marti et al, 2004). The resulting chimera STEVORΔ26–50 remained confined to the parasitophorous vacuole (Figure 3B). We next mutated within the HCT motif residues K46, R48 and Q52. Interestingly, all three mutants revealed different phenotypes. Replacing Q52 by an alanine completely abrogated export into the host erythrocyte, with the chimeric protein accumulating in the parasitophorous vacuolar lumen (Figure 4). In mutant R48A, export into the host erythrocyte was also blocked; however, the mutated protein remained in the parasite with some of the protein being found in a perinuclear compartment indicative of the ER (Figure 4). In the case of mutant K46A, the protein was exported to the host erythrocyte cytoplasm, yet some of it accumulated in the ER (Figure 4). Figure 3.Dissecting the HCT signal of STEVOR. (A) An alignment of the first 21–60 amino acids of STEVOR with the PEXEL (Plasmodium export element) (Marti et al, 2004) and the VTS (vacuolar transport signal) (Hiller et al, 2004). Predicted chaperone binding sites are indicated by an upper bar (see Supplementary Figure S1). The color code for amino acids is as follows: black, hydrophobic; blue, acidic; green, polar; red, basic. (B) Subcellular localization of STEVORΔ26–50 in P. falciparum-infected erythrocytes. In this chimera, the signal sequence of STEVOR (red, amino acids 1–25) was fused to amino acids 51–80, thereby deleting amino acids 26–50 (dotted line). The fluorescence signal is largely retained in a ring structure surrounding the body of the parasite, corresponding to the lumen of the parasitophorous vacuole. Fluorescence is also detected in the parasite's food vacuole. Left image, DIC; middle image, GFP; right image, overlay. Bar, 4 μm. Download figure Download PowerPoint Figure 4.Mutational analysis of the STEVOR HCT signal. Residues in superscript were replaced by an alanine and the subcellular localizations of the resulting GFP chimera (derived from STEVOR1–80) were detected by confocal fluorescence microscopy. H59A revealed fluorescence in the host erythrocyte cytoplasm. In the case of Q52A, the fluorescence signal is largely retained in a ring structure surrounding the body of the parasite, corresponding to the lumen of the parasitophorous vacuole. Mutant R48A revealed fluorescence in the parasite including the ER (indicated by an arrow). Mutants K46A, R42A and R34A showed fluorescence in the host erythrocyte cytoplasm and in a perinuclear compartment (indicated by an arrow). First column, DIC; second column, GFP; third column, nuclear staining with Hoechst; fourth column, overlay of green and blue channels. Bar, 4 μm. Download figure Download PowerPoint We further mutated charged amino acids surrounding the HCT motif. Charged amino acids have been implicated in protein trafficking to organelles, including chloroplast mitochondria and apicoplast (Neupert and Brunner, 2002; Foth et al, 2003; Soll and Schleiff, 2004). Mutants R42A and R34A showed a phenotype similar to that of mutant K46A, that is, export of the protein into erythrocyte host cytosol and accumulation of some protein in the ER (Figure 4). Replacing H59, H61, D63, E65, K67, E68, D71, K72, E75, D76, K79 or K80 by an alanine had no effect on protein export into the host erythrocyte (Figure 4 and data not shown). The apparent indifference of residues downstream of the HCT motif seemed to contrast with the subcellular localization of STEVOR1–60, which remained in the parasitophorous vacuole in spite of a complete HCT motif (see Figure 2 for comparison). We therefore wondered whether downstream sequences may only be necessary to spatially separate the HCT motif from the GFP reporter. Indeed, adding an alanine linker immediately following amino acid 60 or 70 (yielding STEVOR1–69A and STEVOR1–70A) now resulted in complete export of the chimeric proteins to the erythrocyte cytosol (Figure 5), confirming that sequences following amino acid 60 play no specific role in export of STEVOR to the host erythrocyte cytosol. Figure 5.Spatial requirements between the HCT signal and the GFP reporter. A 10-residue alanine linker was added between the STEVOR sequences at the residue indicated and the GFP reporter. The subcellular localizations of the resulting chimera are shown by confocal fluorescence microscopy. Compare the subcellular localization of STEVOR1–60A with that of STEVOR1–60 in Figure 2. Left image, DIC; middle image, GFP; right image, overlay. Bar, 4 μm. Download figure Download PowerPoint Further transport of STEVOR to the Maurer's clefts is mediated by a transmembrane domain Inclusion of the first 217 amino acids of STEVOR, which contain a predicted transmembrane domain (amino acids 178–194), drastically changes the distribution of the fluorescence signal within the host cell. In transfectants expressing STEVOR1–217, discreet foci of fluorescence are observed within the erythrocyte cytoplasm, characteristic of a Maurer's clefts localization (Figure 6A). Extending the STEVOR-GFP fusion protein to include the first 260 amino acids showed a fluorescence pattern identical to STEVOR1–217 (Figure 6A). Figure 6.Effect of a transmembrane domain on targeting of STEVOR to the Maurer's clefts. (A) Subcellular localization of STEVOR1–217 and STEVOR1–260. A punctuate GFP fluorescence is observed within the cytosol of the host erythrocyte, consistent with a Maurer's clefts localization. Left image, DIC; middle image, GFP; right image, overlay. Bar, 4 μm. (B) Subcellular localization of the minimal functional chimera STEVORTM1 (top row) and STEVORTM2 (lower row). These chimera are composed of the first 80 N-terminal amino acids of STEVOR including the signal sequence (red) fused to either the first or second STEVOR transmembrane domain (blue) and the associated charged residues. A punctuate GFP fluorescence is observed within the cytosol of the host erythrocyte, consistent with a localization in Maurer's clefts. Some fluorescence is also noted associated with the parasite. (C) Colocalization of STEVORTM1 (αGFP) and PfSBP1 (αPfSBP1) by immunofluorescence microscopy. Bar, 3 μm. Download figure Download PowerPoint These data suggest that targeting of STEVOR to the Maurer's clefts requires a minimum of three distinct signals: an N-terminal signal sequence, a recessed HCT signal and a transmembrane domain. To verify this, we generated two minimal constructs containing the first 80 amino acids of STEVOR, followed by either the first or second STEVOR transmembrane domain, fused to GFP (STEVORTM1 and STEVORTM2). In both cases, the chimeric proteins are targeted to the Maurer's clefts (Figure 6B), as verified by immunofluorescence colocalization with PfSBP1 (Figure 6C). STEVOR enters the secretory system at the ER as an integral membrane protein To investigate whether STEVOR traffics via the ER, we fused STEVOR1–80 to the ER retention signal KDEL (Figure 7A). In the resulting transfectant line (STEVOR1–80KDEL), a distinct ring of fluorescence surrounding the parasite's nucleus is observed, consistent with an ER localization (Figure 7A). Figure 7.Analysis of the intraparasitic trafficking pathway of STEVOR. (A) Subcellular localization of STEVOR1–80KDEL containing the ER retention signal KDEL. Fluorescence is confined to a perinuclear compartment corresponding to the parasitic ER. First column, DIC; second column, GFP; third column, nuclear staining with Hoechst; fourth column, overlay of green and blue channels. Bar, 4 μm. (B) STEVORfull transport is brefeldin A sensitive. Highly synchronized ring-stage parasites were treated with brefeldin A prior to observation of the GFP fluorescence signal. The STEVORfull chimera accumulates within the body of the parasite, with no fluorescence being seen within the host erythrocyte cytoplasm. Bar, 4 μm. Download figure Download PowerPoint Previous studies have shown that secretion through the ER/Golgi-mediated pathway in P. falciparum is sensitive to brefeldin A (Benting et al, 1994; Wickham et al, 2001). We reasoned that, if STEVOR is exported by this pathway, brefeldin A should abrogate its transport. To investigate this, highly synchronized ring-stage parasites expressing STEVORfull were treated with brefeldin A for 16 h, resulting in the retention of the chimeric protein in an intraparasitic compartment (Figure 7B). This compartment is commonly seen in brefeldin A-treated parasites, and has been shown to represent the plasmodial ER (Wiser et al, 1997; Wickham et al, 2001). To investigate in which state STEVORfull traverses the secretory pathway, membrane fractions from the brefeldin A-treated parasites were prepared and subjected to a carbonate wash, to remove peripheral proteins from the membrane, followed by urea extraction to differentiate between proteins anchored in the membrane by protein–protein or protein–lipid interactions (Papakrivos et al, 2005). A Western analysis using anti-GFP antibodies revealed a signal of the expected size of 60 kDa for STEVORfull exclusively in the final membrane pellet, and not in the water-soluble supernatant, the carbonate wash or the urea extract (Figure 8), consistent with STEVORfull being transported as an integral membrane protein. As controls, we investigated the soluble protein PfSERP and the integral membrane protein PfEXP1. PfSERP mainly associates with the water-soluble fraction, yet some PfSERP is also observed in the carbonate wash (Figure 8), consistent with previous data (Papakrivos et al, 2005). PfEXP1 is mainly found in the final membrane pellet, although some PfEXP1 is also found in the carbonate wash (Figure 8), possibly because prolonged treatment of the cells with brefeldin A resulted in improper cotranslational membrane insertion of this protein. Figure 8.Extraction profile of STEVOR in the parasite's secretory pathway. Highly synchronized ring-stage parasites expressing STEVORfull were treated with brefeldin A for 16 h to arrest protein export. Parasites were lysed, yielding a total lysate (T), and the membranes were separated from the supernatant (S1). The total membrane fraction was then washed with carbonate (S2) and extracted with urea (S3). The nonextractable fraction yielded the final membrane pellet (P). Extracts from 2 × 107 parasites were examined per lane by Western analysis using antibodies against GFP (αGFP recognizing STEVORfull), PfSERP (αSERP) and PfEXP1 (αEXP1). Protein size standards are indicated in kDa. A representative example of three independent sets of experiments is shown. Download figure Download PowerPoint STEVORfull in the Maurer's clefts is partly urea extractable A previous study has indicated that PfEMP1, a family of immunovariant antigens that transiently associates with the Maurer's clefts on their way to the erythrocyte surface (Craig and Scherf, 2001), is anchored in its target membrane by protein–protein interactions, as suggested by the extractability of PfEMP1 from membranes by urea (Papakrivos et al, 2005). To investigate the membrane association of STEVOR at its final destination within the Maurer's clefts, we prepared total membrane fractions from parasites expressing STEVORfull, which were then subjected to extraction with urea. As a control, cells expressing STEVOR1–60 were examined in parallel. As shown in Figure 9, STEVORfull is associated with the membrane pellet fraction, although a significant portion is urea extractable. In comparison, STEVOR1–60 is found mainly in the water-soluble fraction, as was PfSERP (Figure 9). PfEMP1 was only found in the urea-soluble fraction, and human glycophorin B and PfEXP1 in the membrane pellet (Figure 9), consistent with a previous report (Papakrivos et al, 2005). Figure 9.Extraction profile of STEVOR in the Maurer's clefts. Mature-stage infected erythrocytes expressing STEVORfull or STEVOR1–60 were lysed and the membranes separated from the supernatant (S1). The total membrane fraction was extracted with urea (S2). The nonextractable fraction yielded the final membrane pellet (P). Extracts from 2 × 107 parasites were examined per lane by Western analysis using antibodies against GFP (αGFP recognizing STEVORfull), PfSERP (αSERP), PfEXP1 (αEXP1), human glycophorin B (αGlyc B) and PfEMP1 (αPfEMP1). Protein size standards are indicated in kDa. The results were verified for two independent preparations. Download figure Download PowerPoint Membrane topology of STEVOR To determine the topology of STEVOR in the Maurer's clefts, erythrocytes infected with parasites expressing STEVORfull and STEVOR1–260 were permeabilized with streptolysin O and then incubated with an anti-GFP antibody. In the case of STEVORfull, both immunofluorescence (anti-GFP antibody) and GFP fluorescence colocalized in the Maurer's clefts (Figure 10A), suggesting that the C-terminal GFP-containing domain of the protein is exposed to the erythrocyte cytosol (Figure 10B). In comparison, the C-terminus of STEVOR1–260, containing only one transmembrane domain, appears to lie within the lumen of the Maurer's cleft, as the GFP tail was not accessible to the anti-GFP antibody (Figure 10A and B). Figure 10.Membrane topology of STEVORfull and STEVOR1–260. (A) Erythrocytes infected with mature-stage parasites expressing STEVORfull or STEVOR1–260 were permeabilized with streptolysin O and incubated with mouse anti-GFP antibodies followed by an anti-mouse Cy2-conjugated secondary antibody. Cy2 fluorescence (αGFP) and GFP fluorescence (FGFP) were observed by confocal laser scanning microscopy. Bar, 4 μM. (B) Model of membrane topology of STEVOR-GFP chimera in the Maurer's clefts. Green, GFP; yellow, STEVOR; blue, STEVOR transmembrane domain; RBC, red blood cell; MC, lumen of the Maurer's cleft. Download figure Download PowerPoint Discussion Our data suggest that export of STEVOR to the P. falciparum Maurer's clefts is a multistep process that requires a minimum of three defined signals contained within the STEVOR primary sequence. This includes (1) an N-terminal signal sequence for entry into the secretory pathway and se" @default.
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• W2110366566 date "2005-06-16" @default.
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• W2110366566 title "Trafficking of STEVOR to the Maurer's clefts in Plasmodium falciparum-infected erythrocytes" @default.
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• W2110366566 doi "https://doi.org/10.1038/sj.emboj.7600720" @default.
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