Article

• The EMBO Journal (2005) 24, 2306 - 2317
• doi:10.1038/sj.emboj.7600720

Published online: 16 June 2005

• Subject Categories:

  • Membranes and Transport | Microbiology and Pathogens

Trafficking of STEVOR to the Maurer's clefts in Plasmodium falciparum-infected erythrocytes

Jude M Przyborski¹, Susanne K Miller², Judith M Pfahler¹, Philipp P Henrich¹, Petra Rohrbach¹, Brendan S Crabb² and Michael Lanzer¹

1. Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany
2. The Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

Correspondence to:

Michael Lanzer, Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany. Tel.: +49 6221 567845; Fax: +49 6221 564643; E-mail: michael_lanzer@med.uni-heidelberg.de

Received 1 December 2004; Accepted 27 May 2005

• Keywords:

  • malaria,
  • protein export,
  • trafficking signal,
  • transmembrane protein

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.

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) (STEVOR^full). Confocal laser scanning microscopy of parasites expressing STEVOR^full 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 STEVOR^full 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 STEVOR^full 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 STEVOR^full and GFP in P. falciparum-infected erythrocytes. (A) The upper row represents control (GFP alone) and the lower row STEVOR^full. 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. STEVOR^full 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 m. (B) Colocalization of STEVOR^full (GFP) with PfSBP1 (PfSBP1) and/or PfEMP1 (PfEMP1) by immunofluorescence microscopy. Overlay of signals is shown in the right panel. Bar, 3 m.

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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: STEVOR^1–20 (top row), STEVOR^1–25 (second row), STEVOR^1–60 (third row), STEVOR^1–70 (fourth row), STEVOR^1–80 (fifth row) and STEVOR^1–170 (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 m.

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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 STEVOR^1–25, 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 (STEVOR^1–70) 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 STEVOR^26–50 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 (Plasmodiumexport 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 STEVOR^26–50 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 m.

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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 STEVOR^1–80) 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 m.

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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 STEVOR^1–60, 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 STEVOR^1–69A and STEVOR^1–70A) 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 STEVOR^1–60A with that of STEVOR^1–60 in Figure 2. Left image, DIC; middle image, GFP; right image, overlay. Bar, 4 m.

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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 STEVOR^1–217, 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 STEVOR^1–217 (Figure 6A).

Figure 6.

Effect of a transmembrane domain on targeting of STEVOR to the Maurer's clefts. (A) Subcellular localization of STEVOR^1–217 and STEVOR^1–260. 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 m. (B) Subcellular localization of the minimal functional chimera STEVOR^TM1 (top row) and STEVOR^TM2 (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 STEVOR^TM1 (GFP) and PfSBP1 (PfSBP1) by immunofluorescence microscopy. Bar, 3 m.

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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 (STEVOR^TM1 and STEVOR^TM2). 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 STEVOR^1–80 to the ER retention signal KDEL (Figure 7A). In the resulting transfectant line (STEVOR^1–80KDEL), 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 STEVOR^1–80KDEL 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 m. (B) STEVOR^full transport is brefeldin A sensitive. Highly synchronized ring-stage parasites were treated with brefeldin A prior to observation of the GFP fluorescence signal. The STEVOR^full chimera accumulates within the body of the parasite, with no fluorescence being seen within the host erythrocyte cytoplasm. Bar, 4 m.

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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 STEVOR^full 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 STEVOR^full 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 STEVOR^full exclusively in the final membrane pellet, and not in the water-soluble supernatant, the carbonate wash or the urea extract (Figure 8), consistent with STEVOR^full 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 STEVOR^full 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 (GFP recognizing STEVOR^full), PfSERP (SERP) and PfEXP1 (EXP1). Protein size standards are indicated in kDa. A representative example of three independent sets of experiments is shown.

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STEVOR^full 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 STEVOR^full, which were then subjected to extraction with urea. As a control, cells expressing STEVOR^1–60 were examined in parallel. As shown in Figure 9, STEVOR^full is associated with the membrane pellet fraction, although a significant portion is urea extractable. In comparison, STEVOR^1–60 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 STEVOR^full or STEVOR^1–60 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 (GFP recognizing STEVOR^full), PfSERP (SERP), PfEXP1 (EXP1), human glycophorin B (Glyc B) and PfEMP1 (PfEMP1). Protein size standards are indicated in kDa. The results were verified for two independent preparations.

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Membrane topology of STEVOR

To determine the topology of STEVOR in the Maurer's clefts, erythrocytes infected with parasites expressing STEVOR^full and STEVOR^1–260 were permeabilized with streptolysin O and then incubated with an anti-GFP antibody. In the case of STEVOR^full, 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 STEVOR^1–260, 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 STEVOR^full and STEVOR^1–260. (A) Erythrocytes infected with mature-stage parasites expressing STEVOR^full or STEVOR^1–260 were permeabilized with streptolysin O and incubated with mouse anti-GFP antibodies followed by an anti-mouse Cy2-conjugated secondary antibody. Cy2 fluorescence (GFP) and GFP fluorescence (F_GFP) were observed by confocal laser scanning microscopy. Bar, 4 M. (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.

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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 secretion across the parasite plasma membrane into the parasitophorous vacuole, (2) an HCT signal for passage through the PVM and entry into the host erythrocyte cytosol and (3) a transmembrane domain for sorting to the Maurer's clefts.

P. falciparum possesses an intracellular secretory machinery including an ER and a functional, albeit not morphologically defined Golgi (Elmendorf and Haldar, 1993, 1994). Our data suggest that STEVOR traffics via this pathway. Treatment of transfected parasites with brefeldin A prevented export of STEVOR^full from the parasite and led to retention within the ER (Figure 7A). Similarly, the addition of the lumenal ER retention signal KDEL resulted in accumulation of STEVOR^1–80KDEL in the ER (Figure 7A).

Previous studies have suggested that entry into the P. falciparum secretory pathway is mediated by a signal sequence, which may be either canonical, as found in PfEXP1 (Burghaus and Lingelbach, 2001) and HRPII (Lopez-Estrano et al, 2003), or functional, as exemplified by KAHRP (Wickham et al, 2001). STEVOR contains a canonical N-terminal signal sequence. Signal sequences are usually cleaved during translocation into the lumen of the ER, as shown in other systems (Blobel and Dobberstein, 1975), and for P. falciparum EXP1 (Burghaus and Lingelbach, 2001) and KAHRP (Wickham et al, 2001). Cleavage of the STEVOR signal sequence is predicted but has to date not been experimentally verified.

N- and C-terminal deletional analysis as well as site-directed mutagenesis revealed the presence of an HCT signal contained between amino acids 26 and 60, which is required for targeting STEVOR into the host cell. This region contains the conserved RxLxE/Q/D, respectively RxSRILAExxx motif (Figure 3A), which, according to previous studies, appears to be required for protein trafficking into the host erythrocyte cytosol (Hiller et al, 2004; Marti et al, 2004). Consistent with this view, deletion of this motif from the STEVOR primary sequence or replacing key amino acids within it (R⁴⁸A and Q⁵²A) completely abrogates export to the host cell cytoplasm. Unexpectedly, the two mutants R⁴⁸A and Q⁵²A accumulate the mutated protein in different subcellular compartments. In mutant Q⁵²A, the protein is located in the lumen of the parasitophorous vacuole. In mutant R⁴⁸A, however, the protein is not retained in the parasitophorous vacuole but within the parasite including the ER, in spite of a functional signal sequence (Figure 4). A similar phenotype was also observed by Hiller et al (2004) and Marti et al (2004) but not commented upon. At present, we can only speculate about the cause of these two different phenotypes. It may be that the HCT motif is not just required for translocation across the PVM, as previously thought (Hiller et al, 2004; Marti et al, 2004), but may also play a role in other events, such as protein stability and/or quality control in the ER.

Residues preceding the conserved HCT motif appear to play an auxiliary role during transport processes. Mutation of either amino acid K⁴⁶, R⁴² or R³⁴ did not ablate trafficking into the host erythrocyte cytoplasm, yet resulted in a significant and clearly discernable portion of the chimeric protein being retained within the parasite's ER (see for comparison Figure 7B). Interestingly, these residues are part of predicted chaperone binding sites (Figure 3A and Supplementary Figure S1). As shown in other systems, translocation processes are often aided by molecular chaperones, which appear to prevent retrograde movement by drawing the protein across the membrane, and facilitate correct protein folding (Neupert and Brunner, 2002).

Residues beyond the conserved HCT motif do not appear to provide specific information for trafficking, as demonstrated by site-directed mutagenesis of 13 different residues between positions 56 and 80, and by bulk replacement of 10 residues by alanines following position 60. In this context, it is interesting to note the requirement for a spacer between the conserved HCT motif and the GFP reporter, as exemplified by STEVOR^1–60, which is retained in the parasitophorous vacuole despite possessing a complete HCT motif. The translocation block is overcome by the insertion of a 10-amino-acid alanine spacer between STEVOR^1–60 and GFP (see STEVOR^1–60A). The need for a spatial separation between the HCT motif and GFP may help explain previous data in which GFP fusion proteins failed to translocate across the PVM despite the presence of a complete HCT signal (Wickham et al, 2001; Lopez-Estrano et al, 2003).

In this study, we furthermore show that a transmembrane domain is crucial for the eventual targeting of STEVOR to the Maurer's clefts. Parasite lines expressing constructs STEVOR^1–217, STEVOR^TM1 and STEVOR^TM2 exhibit dotted fluorescence patterns within the host erythrocyte cytoplasm (Figure 6), identified as Maurer's clefts using antisera to the specific marker PfSBP1 (Figure 6C). Thus, the addition of a predicted transmembrane domain drastically changes the subcellular localization of the corresponding STEVOR chimera within the host erythrocyte cytosol, from an apparently diffusible soluble form to a membrane-bound state.

One can envisage two different models for transport of STEVOR to the Maurer's clefts. The first model imagines that STEVOR traverses the secretory pathway in a soluble, non-membrane-bound form, in a manner similar to that previously described for PfEMP1 (Papakrivos et al, 2005), and only after passage across the PVM would STEVOR insert into parasite-derived membranes, including Maurer's clefts, by virtue of its transmembrane domains. The second model would predict that STEVOR inserts cotranslationally into the ER membrane and traverses the secretory pathway as an integral membrane protein. To differentiate between the two models, we determined the extraction and solubility profile of STEVOR^full after treatment of ring-stage parasites with brefeldin A for 16 h to block protein export from the ER (see Figure 8) (Benting et al, 1994; Wickham et al, 2001). Our finding that STEVOR^full associates with the parasite's membrane fraction, and is not found in the water-soluble fraction, the carbonate wash or the urea extract, would support a model in which STEVOR traverses the secretory pathway as an integral membrane protein. In comparison, PfEMP1 within the secretory pathway can be extracted by carbonate, and it has been suggested that PfEMP1 is translocated into the ER lumen and is secreted as a sodium carbonate-extractable protein (Papakrivos et al, 2005). Thus, STEVOR and PfEMP1 appear to traverse the secretory pathway differently, although both are targeted to the Maurer's clefts. On this note, the signals required for trafficking also appear to differ between these two proteins (Hiller et al, 2004; Marti et al, 2004; Przyborski and Lanzer, 2004; Knuepfer et al, 2005) and PfEMP1 seems to possess an atypical transmembrane domain (Papakrivos et al, 2005). Moreover, there appear to be proteins, including the integral membrane protein PfSBP1, that lack the conserved HCT motif, but are nevertheless targeted to the Maurer's clefts (Blisnick et al, 2000; Vincensini et al, 2005), suggesting alternative trafficking signals and possibly pathways.

At the Maurer's clefts, the extraction profile of STEVOR slightly changes (Figure 9). STEVOR still associates with the membrane fraction, but some protein can be extracted from the membrane with urea. In comparison, PfEMP1 is only found in the urea extract, which has been interpreted in terms of a model in which PfEMP1 is anchored in the membrane by protein–protein rather than protein–lipid interactions (Papakrivos et al, 2005). Whether STEVOR interacts with other transmembrane proteins in the Maurer's clefts is speculative, but would be consistent with its partial extractability with urea. While our study has identified a set of discreet signals necessary for trafficking of an integral membrane protein to the P. falciparum Maurer's clefts, the transport machinery recognizing these signals and the nature of the interactions remain elusive.

In silico analysis

Multiple sequence alignments were carried out at http://www.ebi.ac.uk/clustalw/ (Thompson et al, 1994). Transmembrane domains were predicted using TMHMM v. 2.0 at http://www.cbs.dtu.dk/services/TMHM
M-2.0 (Moller et al, 2001), and signal sequences using SignalP v. 3.0 (Bendtsen et al, 2004). Chaperone binding sites were predicted using a previously described algorithm (Rudiger et al, 1997).

Vector construction

Primers stev^for and stev^rev were designed to PCR amplify a full-length stevor gene from the P. falciparum clone 3D7. The resulting product was cloned into pCR2.1^TOPO (Invitrogen, designated pCR2.1-STEV^full) and individual clones sequenced using M13 F+R primers. The GFPmut2 coding sequence was amplified from plasmid pHC1-ACP_L (Waller et al, 2000) using primers gfp^Afor and gfp^Krev and cloned into the AvrII/KpnI restriction site of the expression vector pARL1a⁺ (Crabb et al, 2004) to create pARL1-GFP. pARL-STEVOR^full: A fragment encoding one entire stevor gene but missing the stop codon was amplified from clone pCR2.1-STEVOR^full using primers stev^Xfor and stev^Arev and cloned into the XhoI/AvrII site of pARL1-GFP. pARL-STEVOR^1–5,1–10: Fragments encoding the first 5 or 10 amino acids of STEVOR were created by annealing complementary oligonucleotides stev^5for, stev^5rev and stev^10for, stev^10rev. These fragments were then ligated to XhoI/AvrII-restricted pARL1-GFP. pARL-STEVOR^1–15: A fragment encoding the first 15 amino acids of STEVOR was created with overlapping oligonucleotides. Primers stev^15for and stev^15rev were annealed, filled in with Klenow, digested with XhoI/AvrII and ligated to XhoI/AvrII-restricted pARL1-GFP. pARL-STEVOR^{(1–20)–(1–260)}: Oligonucleotide primers were designed to allow amplification of fragments encoding between 20 and 260 amino acids of STEVOR. The following primers were used together with primer stev^Xfor: stev^20rev, stev^25rev, stev^30rev, stev^35rev, stev^40rev, stev^45rev, stev^50rev, stev^60rev, stev^70rev, stev^A80rev, stev^170rev, stev^217rev, stev^260rev. Fragments were digested with XhoI/AvrII and ligated to XhoI/AvrII-digested pARL1-GFP. pARL2-STEVOR^{1–25,51–80}: A linker derived from annealed complementary oligonucleotides pl^for and pl^rev was cloned into XhoI/XmaI-digested pARL1a⁺ to create pARL2. GFPmut2 coding sequence was amplified from pARL1-GFP with primers gfp^Kfor and gfp^Xrev and cloned into KpnI/XmaI-digested pARL2 to create pARL2-GFP. A fragment encoding amino acids 51–80 of STEVOR was obtained by PCR from template pARL-STEVOR^full with primers stev^51for and stev^K80rev. A fragment encoding the first 25 amino acids of STEVOR was obtained by XhoI/AvrII digestion of pARL-STEVOR^1–25 and was used in a three-part ligation with AvrII/KpnI-restricted fragment stev^51–80 to XhoI/KpnI-digested pARL2-GFP to create pARL2-STEVOR^26–50. pARL2-STEVOR^TM1/TM2: A fragment encoding STEVOR amino acids 26–80 was amplified with from template pARL-STEVOR^full with primer pair stev^26for and stev^K80rev. A further fragment encoding either the first or the second TM domain of STEVOR was amplified from template pARL-STEVOR^full using primer combinations stev^tm1for, stev^tm1rev and stev^tm2for, stev^tm2rev. The fragment encoding either TM1 or TM2 was then used in a three-part ligation reaction together with fragment stev^26–80 from above to AvrII/KpnI-digested pARL2^-STEVOR^26–50. pARL-STEV^1–80KDEL: A fragment was amplified from template pARL-STEVOR^1–80 with primers stev^Xfor and gfp^KDELrev and cloned into XhoI/KpnI-restricted pARL1a+. pARL-STEVOR^{1–60A,1–70A}: A polylinker was created by annealing oligos 10A^for/10A^rev. Fragments encoding STEVOR^{1–60,1–70} were obtained by XhoI/AvrII digestion of pARL-STEVOR^{1–60/1–70}, and were then used in a three-piece ligation with the polylinker to XhoI/KpnI-restricted pARL2-GFP to give pARL-STEVOR^{1–60A,1–70A}. All constructs were checked by restriction digest and automated sequencing.

Site-directed mutagenesis

A fragment encoding STEVOR^26–80 was amplified from template pARL-STEVOR^full using primer pair stev^BA25for and stev^K80rev, digested with XbaI/KpnI and cloned into pGEM7Zf (Promega) to create pGEM^26–80. Site-directed mutagenenesis was carried out using the GeneTailor kit (Invitrogen) and pGEM^25–80 as template. Fragments containing the required mutations were then released by digestion with AvrII/KpnI and cloned into AvrII/KpnI-restricted pARL-STEVOR^26–50. All constructs were checked by restriction digest and automated sequencing.

Parasite culture and transfection

The P. falciparum clone 3D7 was cultured in 0⁺ human erythrocytes as described previously (Trager and Jensen, 1976) in RPMI media supplemented with 5% 0⁺ human serum, 0.25% Albumax II (Gibco), 200 M hypoxanthine and 20 g/ml gentamycin. Cultures were synchronized by sorbitol lysis (Lambros and Vanderberg, 1979). Parasites were transfected with 100 g plasmid DNA and transfectants selected with 5 nm WR99210 as described previously (Fidock and Wellems, 1997). Resistant parasites were first seen in blood smears between days 13 and 50.

Brefeldin A treatment

Treatment of highly synchronized ring-stage parasites with 5 g/ml brefeldin A (Sigma) was carried out as described previously (Wickham et al, 2001).

Indirect immunofluorescence assay

Immunofluorescence was carried out on air-dried monolayers as described previously (Haeggstrom et al, 2004). Primary antisera and dilutions were as follows: guinea-pig anti-PfEMP1 1:200 (Wickert et al, 2003b), rabbit anti-GFP 1:200 (Clontech), mouse anti-PfSBP1 1:100 (gift of C Braun-Breton). Corresponding secondary antibodies conjugated to either Alexa 488 or 546 were used at a dilution of 1:800. Slides were mounted with mowiol and viewed using an LSM510 laser scanning confocal microscope (Carl Zeiss, Jena). Parasites at the trophozoite stage were investigated.

Membrane preparation and protein analysis

Soluble, carbonate, urea and membrane protein fractions were prepared as described previously (Papakrivos et al, 2005). Samples corresponding to 2 10⁷ parasites/lane were separated on NuPage gels (Invitrogen) and transferred to PVDF as recommended by the manufacturer. Membranes were then probed with -EXP1, -SERP, -glycophorin (gifts of Klaus Lingelbach) and -ExonII antisera as described previously (Wickert et al, 2003b; Papakrivos et al, 2005). Mouse anti-GFP antibodies (Roche) were used at 1:200, PBS/5% skimmed milk, for 2 h at room temperature (RT), followed by 30 min horseradish peroxidase-conjugated goat anti-mouse (Dianova, 1:20 000, PBS/5% skimmed milk). Membranes were developed using the Supersignal Femto kit (Pierce), and exposed to Kodak BioMax XAR film.

Labelling of internal GFP populations

Trophozoite-stage parasites expressing either STEVOR^full or STEVOR^1–60 were purified on a MACS column as described previously (Staalsoe et al, 1999). The purified infected erythrocytes were permeabilized with streptolysin O (a gift from Klaus Lingelbach) as described previously (Ansorge et al, 1996), and incubated with mouse anti-GFP antibodies (Roche, 1:200, PBS/2% FCS, 1 h RT) followed by a Cy2-conjugated donkey anti-mouse secondary antibody (Dianova, 1:1000, PBS/2% FCS, 1 h RT). Parasites were viewed at RT on an LSM510 laser scanning confocal microscope (Carl Zeiss) at the appropriate wavelengths.

Confocal microscopy and image processing

P. falciparum-infected erythrocytes were viewed within 20 min of removal from culture at RT in phenol red free RPMI media in a perfusion chamber on an LSM510 confocal laser scanning microscope (Carl Zeiss, Jena). GFPmut2 and fusion protein derivatives were excited at 488 nm using an argon laser (Argon, laser power 40%, transmission 2%, C-Apochromat 63/1.2 water immersion). Throughout the study, we examined parasites at the trophozoite stage, and the data shown are representative of at least 20 independent observations.

Online supplemental material

Primers and oligonucleotides used in this study are compiled in Supplementary Table S1. Supplementary Figure S1 shows prediction of chaperone binding sites within STEVOR.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (La 941/7-1) and the European Commission (BioMalPar). We thank Catherine Braun-Breton for anti-PfSBP1 antibodies, Tim Gilberger for the transfection vector pARL1a⁺ and Klaus Lingelbach for anti-PfEXP1, anti-PfSERP, anti-human glycophorin B and streptolysin O. WR99210 was a gift from D Jacobus, Jacobus Pharmaceuticals, Princeton, NJ. We are grateful to Klaus Lingelbach and Stefan Charpian for helpful and stimulating discussions.

Adisa A, Albano FR, Reeder J, Foley M, Tilley L (2001) Evidence for a role for a Plasmodium falciparum homologue of Sec31p in the export of proteins to the surface of malaria parasite-infected erythrocytes. J Cell Sci 114: 3377–3386 | PubMed | ISI | ChemPort |

Albano FR, Berman A, La Greca N, Hibbs AR, Wickham M, Foley M, Tilley L (1999) A homologue of Sar1p localises to a novel trafficking pathway in malaria-infected erythrocytes. Eur J Cell Biol 78: 453–462 | PubMed | ISI | ChemPort |

Ansorge I, Benting J, Bhakdi S, Lingelbach K (1996) Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O. Biochem J 315: 307–314 | PubMed | ISI | ChemPort |

Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340: 783–795 | Article | PubMed | ISI | ChemPort |

Benting J, Mattei D, Lingelbach K (1994) Brefeldin A inhibits transport of the glycophorin-binding protein from Plasmodium falciparum into the host erythrocyte. Biochem J 300 (Part 3): 821–826

Blisnick T, Morales Betoulle ME, Barale JC, Uzureau P, Berry L, Desroses S, Fujioka H, Mattei D, Braun Breton C (2000) Pfsbp1, a Maurer's cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton. Mol Biochem Parasitol 111: 107–121 | Article | PubMed | ISI | ChemPort |

Blobel G, Dobberstein B (1975) Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 67: 835–851 | Article | PubMed | ISI | ChemPort |

Blythe JE, Surentheran T, Preiser PR (2004) STEVOR—a multifunctional protein? Mol Biochem Parasitol 134: 11–15 | Article | PubMed | ChemPort |

Burghaus PA, Lingelbach K (2001) Luciferase, when fused to an N-terminal signal peptide, is secreted from transfected Plasmodium falciparum and transported to the cytosol of infected erythrocytes. J Biol Chem 276: 26838–26845 | Article | PubMed | ChemPort |

Cheng Q, Cloonan N, Fischer K, Thompson J, Waine G, Lanzer M, Saul A (1998) stevor and rif are Plasmodium falciparum multicopy gene families which potentially encode variant antigens. Mol Biochem Parasitol 97: 161–176 | Article | PubMed | ISI | ChemPort |

Crabb BS, Cooke BM, Reeder JC, Waller RF, Caruana SR, Davern KM, Wickham ME, Brown GV, Coppel RL, Cowman AF (1997) Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress. Cell 89: 287–296 | Article | PubMed | ISI | ChemPort |

Crabb BS, Rug M, Gilberger TW, Thompson JK, Triglia T, Maier AG, Cowman AF (2004) Transfection of the human malaria parasite Plasmodium falciparum. Methods Mol Biol 270: 263–276 | PubMed | ChemPort |

Craig A, Scherf A (2001) Molecules on the surface of the Plasmodium falciparum infected erythrocyte and their role in malaria pathogenesis and immune evasion. Mol Biochem Parasitol 115: 129–143 | Article | PubMed | ISI | ChemPort |

Culvenor JG, Langford CJ, Crewther PE, Saint RB, Coppel RL, Kemp DJ, Anders RF, Brown GV (1987) Plasmodium falciparum: identification and localization of a knob protein antigen expressed by a cDNA clone. Exp Parasitol 63: 58–67 | Article | PubMed | ISI | ChemPort |

Elmendorf HG, Haldar K (1993) Identification and localization of ERD2 in the malaria parasite Plasmodium falciparum: separation from sites of sphingomyelin synthesis and implications for organization of the Golgi. EMBO J 12: 4763–4773 | PubMed | ISI | ChemPort |

Elmendorf HG, Haldar K (1994) Plasmodium falciparum exports the Golgi marker sphingomyelin synthase into a tubovesicular network in the cytoplasm of mature erythrocytes. J Cell Biol 124: 449–462 | Article | PubMed | ISI | ChemPort |

Fidock DA, Wellems TE (1997) Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil. Proc Natl Acad Sci USA 94: 10931–10936 | Article | PubMed | ChemPort |

Foth BJ, Ralph SA, Tonkin CJ, Struck NS, Fraunholz M, Roos DS, Cowman AF, McFadden GI (2003) Dissecting apicoplast targeting in the malaria parasite Plasmodium falciparum. Science 299: 705–708 | Article | PubMed | ISI | ChemPort |

Gunther K, Tummler M, Arnold HH, Ridley R, Goman M, Scaife JG, Lingelbach K (1991) An exported protein of Plasmodium falciparum is synthesized as an integral membrane protein. Mol Biochem Parasitol 46: 149–157 | PubMed | ChemPort |

Haeggstrom M, Kironde F, Berzins K, Chen Q, Wahlgren M, Fernandez V (2004) Common trafficking pathway for variant antigens destined for the surface of the Plasmodium falciparum-infected erythrocyte. Mol Biochem Parasitol 133: 1–14 | Article | PubMed | ChemPort |

Haldar K, Mohandas N, Samuel BU, Harrison T, Hiller NL, Akompong T, Cheresh P (2002) Protein and lipid trafficking induced in erythrocytes infected by malaria parasites. Cell Microbiol 4: 383–395 | Article | PubMed | ISI | ChemPort |

Hiller NL, Bhattacharjee S, van Ooij C, Liolios K, Harrison T, Lopez-Estrano C, Haldar K (2004) A host-targeting signal in virulence proteins reveals a secretome in malarial infection. Science 306: 1934–1937 | Article | PubMed | ISI | ChemPort |

Kara UA, Stenzel DJ, Ingram LT, Bushell GR, Lopez JA, Kidson C (1988) Inhibitory monoclonal antibody against a (myristylated) small-molecular-weight antigen from Plasmodium falciparum associated with the parasitophorous vacuole membrane. Infect Immun 56: 903–909 | PubMed | ISI | ChemPort |

Kaviratne M, Khan SM, Jarra W, Preiser PR (2002) Small variant STEVOR antigen is uniquely located within Maurer's clefts in Plasmodium falciparum-infected red blood cells. Eukaryot Cell 1: 926–935 | Article | PubMed | ChemPort |

Kilejian A (1979) Characterization of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum. Proc Natl Acad Sci USA 76: 4650–4653 | PubMed | ChemPort |

Knuepfer E, Rug M, Klonis N, Tilley L, Cowman AF (2005) Trafficking of the major virulence factor to the surface of transfected P. falciparum-infected erythrocytes. Blood 105: 4078–4087 | Article | PubMed | ChemPort |

Kriek N, Tilley L, Horrocks P, Pinches R, Elford BC, Ferguson DJ, Lingelbach K, Newbold CI (2003) Characterization of the pathway for transport of the cytoadherence-mediating protein, PfEMP1, to the host cell surface in malaria parasite-infected erythrocytes. Mol Microbiol 50: 1215–1227 | Article | PubMed | ChemPort |

Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65: 418–420 | Article | PubMed | ISI | ChemPort |

Limpaiboon T, Shirley MW, Kemp DJ, Saul A (1991) 7H8/6, a multicopy DNA probe for distinguishing isolates of Plasmodium falciparum. Mol Biochem Parasitol 47: 197–206 | Article | PubMed | ChemPort |

Lopez-Estrano C, Bhattacharjee S, Harrison T, Haldar K (2003) Cooperative domains define a unique host cell-targeting signal in Plasmodium falciparum-infected erythrocytes. Proc Natl Acad Sci USA 100: 12402–12407 | Article | PubMed | ChemPort |

Marti M, Good RT, Rug M, Knuepfer E, Cowman AF (2004) Targeting malaria virulence and remodeling proteins to the host erythrocyte. Science 306: 1930–1933 | Article | PubMed | ISI | ChemPort |

McRobert L, Preiser P, Sharp S, Jarra W, Kaviratne M, Taylor MC, Renia L, Sutherland CJ (2004) Distinct trafficking and localization of STEVOR proteins in three stages of the Plasmodium falciparum life cycle. Infect Immun 72: 6597–6602 | Article | PubMed | ChemPort |

Miller LH, Baruch DI, Marsh K, Doumbo OK (2002) The pathogenic basis of malaria. Nature 415: 673–679 | Article | PubMed | ISI | ChemPort |

Moller S, Croning MD, Apweiler R (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17: 646–653 | Article | PubMed | ISI | ChemPort |

Neupert W, Brunner M (2002) The protein import motor of mitochondria. Nat Rev Mol Cell Biol 3: 555–565 | Article | PubMed | ISI | ChemPort |

Papakrivos J, Newbold CI, Lingelbach K (2005) A potential novel mechanism for the insertion of a membrane protein revealed by a biochemical analysis of the Plasmodium falciparum cytoadherence molecule PfEMP-1. Mol Microbiol 55: 1272–1284 | Article | PubMed | ChemPort |

Pologe LG, Pavlovec A, Shio H, Ravetch JV (1987) Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum. Proc Natl Acad Sci USA 84: 7139–7143 | PubMed | ChemPort |

Przyborski J, Lanzer M (2004) Parasitology. The malarial secretome. Science 306: 1897–1898 | Article | PubMed | ChemPort |

Rudiger S, Germeroth L, Schneider-Mergener J, Bukau B (1997) Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J 16: 1501–1507 | Article | PubMed | ISI | ChemPort |

Sam-Yellowe TY, Florens L, Johnson JR, Wang T, Drazba JA, Le Roch KG, Zhou Y, Batalov S, Carucci DJ, Winzeler EA, Yates III JR (2004) A Plasmodium gene family encoding Maurer's cleft membrane proteins: Structural properties and expression profiling. Genome Res 14: 1052–1059 | Article | PubMed | ChemPort |

Soll J, Schleiff E (2004) Protein import into chloroplasts. Nat Rev Mol Cell Biol 5: 198–208 | Article | PubMed | ISI | ChemPort |

Staalsoe T, Giha HA, Dodoo D, Theander TG, Hviid L (1999) Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry. Cytometry 35: 329–336 | Article | PubMed | ISI | ChemPort |

Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680 | Article | PubMed | ISI | ChemPort |

Trager W, Jensen JB (1976) Human malaria parasites in continuous culture. Science 193: 673–675 | Article | PubMed | ISI | ChemPort |

Vincensini L, Richert S, Blisnick T, Van Dorsselaer A, Leize-Wagner E, Rabilloud T, Braun Breton C (2005) Proteomic analysis identifies novel proteins of the Maurer's clefts, a secretory compartment delivering Plasmodium falciparum proteins to the surface of its host cell. Mol Cell Proteomics 4: 582–593 | Article | PubMed | ChemPort |

Waller KL, Cooke BM, Nunomura W, Mohandas N, Coppel RL (1999) 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). J Biol Chem 274: 23808–23813 | Article | PubMed | ISI | ChemPort |

Waller KL, Nunomura W, Cooke BM, Mohandas N, Coppel RL (2002) Mapping the domains of the cytoadherence ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) that bind to the knob-associated histidine-rich protein (KAHRP). Mol Biochem Parasitol 119: 125–129 | Article | PubMed | ChemPort |

Waller RF, Reed MB, Cowman AF, McFadden GI (2000) Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway. EMBO J 19: 1794–1802 | Article | PubMed | ISI | ChemPort |

Wickert H, Gottler W, Krohne G, Lanzer M (2004) Maurer's cleft organization in the cytoplasm of Plasmodium falciparum-infected erythrocytes: new insights from three-dimensional reconstruction of serial ultrathin sections. Eur J Cell Biol 83: 567–582 | Article | PubMed

Wickert H, Rohrbach P, Scherer S, Krohne G, Lanzer M (2003a) A putative Sec23 homologue of Plasmodium falciparum is located in Maurer's clefts. Mol Biochem Parasitol 129: 209–213 | Article | ChemPort |

Wickert H, Wissing F, Andrews KT, Stich A, Krohne G, Lanzer M (2003b) Evidence for trafficking of PfEMP1 to the surface of P. falciparum-infected erythrocytes via a complex membrane network. Eur J Cell Biol 82: 271–284 | ChemPort |

Wickham ME, Rug M, Ralph SA, Klonis N, McFadden GI, Tilley L, Cowman AF (2001) Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes. EMBO J 20: 5636–5649 | Article | PubMed | ChemPort |

Wiser MF, Lanners HN, Bafford RA, Favaloro JM (1997) A novel alternate secretory pathway for the export of Plasmodium proteins into the host erythrocyte. Proc Natl Acad Sci USA 94: 9108–9113 | Article | PubMed | ChemPort |

World Health Organization (2000) WHO Expert Committee on Malaria. World Health Organ Tech Rep Ser 892: i–v, 1–74

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