Proteomic Analysis Identifies Novel Proteins of the Maurer’s Clefts, a Secretory Compartment Delivering Plasmodium falciparum Proteins to the Surface of Its Host Cell*,S

Laetitia Vincensini{ddagger},§, Sophie Richert,||, Thierry Blisnick{ddagger}, Alain Van Dorsselaer, Emmanuelle Leize-Wagner, Thierry Rabilloud** and Catherine Braun Breton{ddagger},{ddagger}{ddagger},§§

From the {ddagger} Unité de Biologie des Interactions Hôte-Parasite, CNRS URA 2581, Institut Pasteur, 25–28 Rue du Dr Roux, 75724 Paris Cedex 15, the Laboratoire de Spectrométrie de Masse Bio-Organique, 25 rue Becquerel, 67087 Strasbourg Cedex 2, the ** Laboratoire de Bioénergétique Cellulaire et Pathologique, Commissariat à l’Energie Atomique Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 9, and {ddagger}{ddagger} UMR 5539 CNRS-Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A novel method was validated for the efficient distinction between malaria parasite-derived and host cell proteins in mass spectrometry analyses. This method was applied to a ghost fraction from Plasmodium falciparum-infected erythrocytes containing the red blood cell plasma membrane, the erythrocyte submembrane skeleton, and the Maurer’s clefts, a Golgi-like apparatus linked to and addressing parasite proteins to the host cell surface. This method allowed the identification of 78 parasite proteins. Among these we identified seven novel proteins of the Maurer’s clefts based on immunofluorescence studies and proteinase K digestion assays. The products of six contiguous genes located on chromosome 5 were identified, and the location within the Maurer’s clefts was established for two of them. This suggests a clustering of genes encoding Maurer’s cleft proteins. Our study sheds new light on the biological function of the Maurer’s clefts, which are central to the pathogenesis and to the intraerythrocytic development of P. falciparum.


Upon invasion of the red blood cell, Plasmodium falciparum, the most life-threatening species of parasites causing human malaria, establishes a parasitophorous vacuole inside which it develops. The parasite nevertheless interacts with its host cell and its environment by exporting a membrane network into the cytoplasm of its host cell (1). Maurer’s clefts are part of this network extending or budding from the parasitophorous vacuole membrane surrounding the growing parasite; electron microscopy studies detect them as flattened elongated vesicles close to the red cell plasma membrane (2). Since the characterization of PfSBP1,1 a Maurer’s cleft transmembrane protein, which allowed precise identification of these structures (3), Maurer’s clefts have been the subject of several studies revealing very interesting features. Maurer’s clefts have characteristics of a Golgi-like secretory compartment exported into the host cell cytoplasm and trafficking parasite proteins to the red cell membrane (48). One of their important roles is in the assembly of the cytoadherence complex, a function particularly significant to the pathogenesis of severe malaria. Interestingly we have shown recently that Maurer’s clefts are also implicated in merozoite release, which depends on the phosphorylation status of the Maurer’s cleft transmembrane protein PfSBP1, which is modulated by a parasite type 1 phosphatase located in the lumen of the clefts.2

Because the Maurer’s clefts are linked to the red cell plasma membrane throughout the intraerythrocytic parasite development, they can be recovered together with a ghost preparation containing the erythrocyte plasma membrane and submembrane skeleton (3). To better understand the biological role of the Maurer’s clefts and possibly identify novel putative drug targets, we performed a global MS proteomic analysis of P. falciparum-infected ghost preparations. Because P. falciparum is an obligate intracellular parasite, it is not possible to fractionate parasite-derived proteins away from erythrocyte proteins, and this is a drawback for systematic MS analyses. In this study, we showed that isotope metabolic labeling is a quick and efficient method for differentiation of parasite- versus host cell-derived proteins. Our proteomic analysis confirmed that ghost preparations are valuable to study Maurer’s clefts and enabled us, together with localization and topological studies, to identify new Maurer’s clefts proteins, providing insight into the biology of these interesting organelles.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Parasite Culture and Deuterium Labeling—
P. falciparum 3D7 strain was grown in vitro under standard culture conditions (9). For the deuterium labeling experiment, parasites at the ring stage were grown for 24 h at 37 °C in standard culture medium supplemented with 0.875 mM DL-lysine 4,4,5,5-d4 (Isotec, Miamisburg, OH) (10). Mature trophozoites and schizonts were then harvested by Plasmion floatation (Fresenius Kabi, Bad Homburg, Germany) (11).

Preparations of Ghosts from Infected Erythrocytes—
Infected erythrocytes were washed extensively in RPMI 1640 medium and lysed in hypotonic buffer as described previously (3). The lysate was then separated by centrifugation into a cytosolic fraction and a pellet containing ghosts and free parasites; ghosts were recovered from the pellet as described previously (3) and stored as a pellet at –20 °C or incubated in PBS for 30 min at 37 °C and further handled for indirect immunofluorescence assays.

Sample Preparation for Mass Spectrometry Analyses—
Deuterium-labeled infected red blood cell ghost proteins were extracted by incubation in Laemmli sample buffer (12) for 10 min and extensive vortexing. Proteins were then separated by SDS-PAGE in a 10% polyacrylamide gel and stained with Biosafe Coomassie (Bio-Rad). Sixty-seven bands were excised from the gel and frozen for further MS analyses.

Mass Spectrometry Analyses—
Separated gel bands were subjected to tryptic digestion overnight at room temperature in 25 mM ammonium bicarbonate buffer. The resulting peptides were extracted from the gel and analyzed by nanoscale capillary liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Purification and analysis were performed on a C18 capillary column (PepMap, LC Packings, Sunnyvale, CA) using a CapLC capillary LC system (Waters, Milford, MA) coupled to a hybrid quadrupole orthogonal acceleration time-of-flight tandem mass spectrometer (Q-TOF II, Micromass, Manchester, UK). The LC-MS union was made with a PicoTip (New Objective, Woburn, MA) fitted on a ZSPRAY (Micromass, Beverly, MA) interface. Chromatographic separations were conducted on a reversed-phase capillary column (PepMap C18, 75-µm inner diameter, 15-cm length; LC Packings, Voisins le Bretonneux, France) with a 200 nl min–1 flow. The gradient profile used consisted of a linear gradient from 95% A (H2O, 0.05% HCOOH) to 45% B (acetonitrile, 0.05% HCOOH) in 35 min followed by a linear gradient to 95% B in 1 min. Mass data acquisitions were piloted by MassLynx software using automatic switching between MS and MS/MS modes. The internal parameters of Q-TOF II were set as follows. The electrospray capillary voltage was set to 3.0 kV, the cone voltage was set to 30 V, and the source temperature was set to 80 °C. The MS survey scan was m/z 300–1500 with a scan time of 1 s and an interscan time of 0.1 s. When the intensity of a peak rose above a threshold of 8 counts, tandem mass spectra were acquired. Normalized collision energies for peptide fragmentation were set using the charge-state recognition files for +1, +2, and +3 peptide ions. The scan range for MS/MS acquisition was from m/z 50 to 1500 with a scan time of 3 s and an interscan time of 0.1 s. Fragmentation was performed using argon as the collision gas and with a collision energy profile optimized for various mass ranges of precursor ions. Mass data collected during a nano LC-MS/MS analysis were processed and converted into a Pocket Builder Library file and then fed into the search engine Mascot (Matrix Science, London, UK) (13). The data were searched against Swiss-Prot and TrEMBL data bases with trypsin plus potentially one missed cleavage. All proteins present in the data base were taken into account without any pHi and molecular mass restriction. In addition to substitution with deuterated lysine, other putative modifications were taken into account, such as carbamidomethylation of cysteine and oxidation of methionine. The peptide mass error was limited to 0.25 Da in MS mode and 0.5 Da in MS/MS mode for nano LC-MS/MS data.

Expression of Recombinant Proteins and Production of Specific Antibodies—
Different fragments of the selected proteins were amplified from P. falciparum 3D7 genomic DNA using the corresponding primers (Table I). The resulting PCR products were ligated into the pGEX-B vector and expressed in Escherichia coli BL21 cells (Stratagene, La Jolla, CA) as glutathione S-transferase (GST) fusion proteins (14), which were purified on glutathione-agarose beads as described previously (15). Specific antibodies were obtained by immunization of BALB/c mice as described previously (3).


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TABLE I Sequence of the primers used for the PCR amplification of selected gene fragments to express GST-recombinant proteins

The proteins have been named according to the locus annotated in the Plasmodium data base. The added EcoRI and BamHI restriction sites are underlined in the primer sequence. The protein region expressed as a GST-recombinant polypeptide is indicated by its amino- and carboxyl-terminal amino acids.

 
Protein Immunodetection—
Immunoblotting assays were performed as described previously (3). The new specific sera were used at a 1:50 dilution. Indirect immunofluorescence assays were performed on air-dried samples as described previously (3). Sera were used at a 1:200 dilution.

Proteinase K Digestion of Ghost Preparations—
Ghost preparations from P. falciparum-infected erythrocytes were subjected to proteinase K digestion as described previously (3). The resulting samples were analyzed by Western blotting using specific sera as indicated.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mass Spectrometry Analysis of Deuterium-labeled P. falciparum-infected Red Blood Cell Ghosts—
Ring stage P. falciparum-infected erythrocytes were matured for 24 h to late trophozoites and young schizonts in the presence of deuterated lysine (see "Experimental Procedures"). Because erythrocytes do not synthesize proteins, parasite-derived proteins were specifically labeled. Deuterated lysine, DL-lysine 4,4,5,5-d4, was added at twice the concentration of cold lysine used in the cell culture medium: 0.876 mM for an equivalent of 0.438 mM L-lysine 4,4,5,5-d4. With two-thirds of the lysine in the medium being labeled and taking into consideration that hemoglobin contains nearly 10% lysine, we estimated that ~50% of the lysines that were incorporated into parasite proteins during the labeling were deuterated. To enrich infected erythrocyte preparations with Maurer’s clefts proteins, deuterium-labeled mature trophozoites and young schizonts (33 to 40 h postinvasion) were harvested by Plasmion floatation, and ghosts were recovered following cell hypotonic lysis (see "Experimental Procedures"). Previous work has shown that infected erythrocyte ghosts are devoid of detectable contamination with proteins of the parasite cytoplasm and the parasite membrane, but they do contain Maurer’s clefts linked to the erythrocyte membrane (3). PfSBP1 was used as a positive control to confirm the presence of the Maurer’s clefts. Ghost proteins were then extracted in sample buffer and processed by SDS-PAGE. Sixty-seven bands were cut from the gel and trypsin-digested, and the resulting peptides were analyzed by MS.

Because trypsin specifically cleaves after lysine and arginine residues, resulting peptides cleaved after a lysine residue contain usually one and no more than two lysine residues in case of a missed cleavage. Moreover, as only 50% of the lysines incorporated into parasite proteins during the labeling are deuterated, such peptides that are derived from parasite proteins may exist in two forms, deuterated or not deuterated, and have a molecular mass difference of 4 Da (if containing one lysine) or 8 Da (two lysines). These peptides thus appear in nano LC-MS/MS analyses as doublets with a mass shift of 4 or 8 Da and an intensity ratio of the two peaks characteristic of the labeling conditions (close to 1; Fig. 1). MS/MS analysis focused solely on such differentially labeled peptides. The Mascot software package (13) was used to take into account modifications such as deuterated lysine, thus excluding from the analysis major proteins devoid of this modification.



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FIG. 1. An example of a nano LC Q-TOF mass spectrum. A, the spectrum of the tryptic digest of band 17 containing the RESA-like protein is presented. B, enlargement of the peaks corresponding to a selected peptide of this protein; the two peaks with a 4-Da mass difference (m/z +2) and a peak intensity ratio close to 1 are identified as P1 and P2 for the unlabeled and labeled peptides, respectively. Only such doublets were taken into account for the Mascot search.

 
In total, 78 parasite proteins were identified including 28 proteins from the protein translation machinery (ribosomal proteins and translation initiation and elongation factors) that are classical contaminants in global proteomic studies due to their high abundance (16) (Supplemental Table 1). The remaining 50 identified proteins, listed in Table II, are divided into four classes (A–D) based on the presence or not of a signal peptide and/or transmembrane domains. Among these proteins, PfSBP1 is known to be a transmembrane protein of the Maurer’s clefts (3). Interestingly, as illustrated by PfSBP1, a classical amino-terminal signal peptide (17) is not required to target a protein outside the parasite. Thus, to validate our approach and identify novel Maurer’s cleft proteins, we selected seven hypothetical proteins of unknown function with high coverage in the MS analysis (20–49%) and from classes B–D corresponding to proteins harboring a signal peptide and/or a transmembrane domain. The selected proteins, PfA680, PfD80, PfE60, PfG174, PfJ13, PfJ23, and PfJ323, correspond to the PlasmoDB accession numbers PFA0680c, PFD0080c, PFE0060w, MAL7P1.174, PF10_0013, PF10_0023, and PF10_0323, respectively (Table II).


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TABLE II Proteins identified from P. falciparum-infected red blood cell ghosts by tandem mass spectroscopy

The peptides were assigned by Mascot analysis, and the corresponding proteins were categorized into four classes depending on predicted signal peptide (SP) and transmembrane domains (TMD). The gene locus as annotated in the Plasmodium data base (www.plasmodb.org) is indicated for each identified protein. The protein names or putative function (protein description), their predicted molecular mass (MM), and calculated pHi as determined using DNAStriderTM1.3 (Commissariat à l’Energie Atomique, Saclay, France) are indicated. The final two columns give the number of peptides identified in the mass spectrum and the percentage of coverage of the total protein sequence of those peptides.

 
Detection of Proteins within the Infected Erythrocyte Ghost Fraction by Western Blotting—
To confirm that the selected proteins are associated with the red blood cell ghost fraction we raised mice antibodies against GST fusion recombinant proteins (Table I). The corresponding sera were used in Western blotting experiments (Fig. 2). Preimmune sera did not react with erythrocyte or parasite proteins (not shown). Taking into account the cleavage of predicted signal peptides (Table III), proteins of approximately the expected size were detected in whole cell lysates of parasite-infected erythrocytes (Fig. 2, lanes 1) but not in uninfected erythrocyte lysates (Fig. 2, lanes 3). The apparent molecular masses of PfJ13 (20 kDa) and PfJ23 (25 kDa) are slightly lower than expected, and that of PfD80 (70 kDa) is slightly larger. Two polypeptides of 37 and 48 kDa were specifically detected for PfJ323, and the 37-kDa polypeptide was more abundant in the ghost fraction; this suggests a maturation of the PfJ323 protein correlated with its export outside the parasite. Importantly and concordant with their identification within the labeled ghost preparation, all these proteins were detected by Western blot in ghost preparations from P. falciparum-infected erythrocytes (Fig. 2, lanes 2). The same analysis was performed for the protein-disulfide isomerase (PfPDI). PfPDI-specific antibodies (a gift from P. Grellier) reacted with a protein of the expected molecular mass (50 kDa) in both the total extract and the ghost fraction from P. falciparum-infected red blood cells. The intensities of labeling specific for PfA680, PfJ23, and PfPDI on ghost versus total extracts from P. falciparum-infected cells suggest that these proteins are mainly not associated with the ghost fraction.



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FIG. 2. Subcellular allocation of the selected proteins. Western blot analyses were performed using mouse antibodies raised against GST fusion proteins. Lanes 1, whole cell lysate of P. falciparum-infected erythrocytes; lanes 2, infected erythrocyte ghosts; lanes 3, whole cell lysate of uninfected erythrocytes. The samples correspond to 107 (total infected red blood cells), 3 x 107 (infected red blood cell ghosts) and 107 (red blood cells) erythrocytes, respectively. The specifically detected polypeptides are designated with arrows. Molecular mass markers are indicated (kDa).

 

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TABLE III Summary of the data obtained for PfPDI and the seven selected hypothetical proteins

The predicted (taking into account cleavage of a predicted signal peptide) and observed (Western blot) molecular masses are indicated as well as the predicted VTS/Pexel motifs (38, 39). The association of the proteins with the infected red blood cell ghost fraction was determined by a Western blot comparative analysis of this fraction with a total infected red blood cell extract (+, detected in; ++, mainly associated with; +++, enriched in the infected red blood cell ghost fraction). The specific sera were used in immunofluorescence experiments (–, no labeling; +, specific labeling; ND, not done), and the observed pattern is indicated (MC, Maurer’s clefts; PV, parasitophorous vacuole). Whether the domain of the protein reacting with the corresponding specific serum was (+) or was not (–) protected from proteinase K digestion is indicated.

 
Localization of the Selected Proteins by Immunofluorescence Microscopy—
To determine the exact subcellular localization of the selected proteins, immunofluorescence studies were performed on air-dried P. falciparum-infected red blood cells and resealed ghosts (Fig. 3) (see "Experimental Procedures"). None of the sera reacted with uninfected erythrocytes (not shown). The sera specific for PfJ13, PfD80, and PfG174 did not label infected red blood cells or infected erythrocyte ghosts using these methods (not shown). A labeling of both intact infected erythrocytes and ghosts was obtained with the sera directed against PfJ23, PfE60, PfJ323, and PfA680 (Fig. 3). Most interestingly, the pattern characteristic of the Maurer’s clefts and observed for PfSBP1 was detected for PfJ23 and PfE60 in both intact erythrocytes and ghosts. Using PfA680-specific antibodies, a similar pattern was obtained for ghost preparations, whereas the vesicular-like and diffuse labeling of intact infected red cells is indicative of a dual location. PfJ323-specific antibodies also labeled vesicular-like structures in infected erythrocyte ghosts; however, this pattern seems slightly different from the characteristic pattern of Maurer’s clefts.



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FIG. 3. Indirect immunofluorescence of P. falciparum-infected erythrocytes and infected red blood cell (IRBC) ghosts. Air-dried infected red blood cells and infected red blood cell ghosts were incubated with mouse antibodies raised against GST fusion proteins as indicated. The nuclei were stained with 4',6-diamidino-2-phenylindole (blue). Negative controls were performed using preimmune sera and anti-GST antibodies (not shown).

 
Localization and Orientation of the Selected Proteins within the Erythrocyte Ghost Preparations—
To further characterize the association of PfJ23, PfE60, PfJ323, and PfA680 with vesicles present in ghost preparations and to specify the location of PfJ13, PfD80, and PfG174, proteinase K digestion experiments were performed on intact (Fig. 4, lanes 3) and Triton X-100-ruptured (Fig. 4, lanes 4) ghosts (see "Experimental Procedures"). The selected proteins were detected by Western blot using specific antibodies. When Triton X-100 was added together with proteinase K, none of the proteins were detected, indicating their sensitivity to proteolysis in detergent-lysed ghost preparations. PfJ323 and PfG174 were not detected following addition of proteinase K in the absence of detergent. However, full-length PfJ13, PfPDI, PfA680, PfJ23, and PfE60 and a degradation fragment of PfD80 were detected under the same conditions, establishing their association with closed vesicles. The degradation of PfG174 indicates that the hypotonic buffer prevents the resealing of erythrocyte plasma membrane vesicles; the protection of others, including PfSBP1 (Supplemental Fig. 1), indicates that Maurer’s cleft closed vesicles are recovered with the ghost fraction (3).



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FIG. 4. Topology analysis of the selected proteins within infected erythrocyte ghost preparations. Ghosts were prepared from Plasmion-enriched trophozoites and schizonts and incubated in RPMI 1640 medium (lanes 1) or RPMI 1640 medium supplemented with 0.5% Triton X-100 (lanes 2), 5 mg ml–1 proteinase K (lanes 3), and 0.5% Triton X-100 and 5 mg ml–1 proteinase K (lanes 4). The digest products were analyzed by SDS-PAGE and Western blotting using mouse antibodies specific for the selected proteins as indicated. Molecular mass markers are indicated (kDa). Schematic drawings of the protein sequences are presented with predicted signal peptide and transmembrane domains (hatched boxes). Only transmembrane domains predicted by at least two of three prediction softwares (TMAP, bioweb.pasteur.fr/seqanal/interfaces/tmap.html; TMPRED, www.ch.embnet.org/software/TMPRED_form.html; TOPRED, bioweb.pasteur.fr/seqanal/interfaces/toppred.html) were considered. The protein region used to raise specific antibodies is underlined by dotted lines, and the protein regions protected from proteinase K digestion are indicated in brackets. Full-length proteins are indicated by an arrow, and degradation products are indicated by an asterisk.

 
Consequently we propose a topology model for the selected proteins based on these results, on transmembrane predictions, and the specificity of the antibodies (Fig. 5). PfG174, PfJ13, and PfPDI have a predicted signal sequence but no putative transmembrane domain. Because PfG174 was fully degraded in the absence of Triton X-100, it is likely located in the red cell cytoplasm, while its recovery together with the ghost fraction suggests that PfG174 is at least transiently associated with the Maurer’s clefts or the erythrocyte plasma membrane. PfJ13 and PfPDI were protected from proteolysis indicating their localization in the lumen of closed vesicles.



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FIG. 5. Schematic representation of the topology of the selected proteins within ghost preparations. The selected proteins and PfSBP1 have been assigned to the Maurer’s cleft membrane or lumen or to the red cell cytosol according to their behavior in proteinase K digestion experiments and the location of predicted transmembrane domains. The location of the amino and carboxyl termini is presented for the transmembrane proteins. Proteins with a predicted signal peptide are represented in blue, and proteins without a signal peptide are in red.

 
PfJ323, PfD80, PfE60, and PfA680 have a predicted signal peptide and transmembrane domain(s). Based on the specificity of the PfJ323 antibodies and the lack of detection of a degradation product in the absence of detergent, the carboxyl-terminal region of PfJ323 is likely to be located in the red cell cytoplasm. For PfD80, a degradation product of 30-kDa was detected, concordant with the location of the Pro235–Lys511 region of the protein in the lumen of closed vesicles. The size of this degradation product also strengthens the TMPRED prediction of a His512–Tyr532 transmembrane domain. Finally full-length PfE60 and PfA680 were observed following proteinase K treatment, indicating that the amino-terminal region of these proteins, which is detected by the specific antibodies, is located in the lumen of closed vesicles. For both proteins, two transmembrane domains separated by only two to six residues are predicted close to their carboxyl terminus. These very short stretches are not likely to be accessible to proteolysis, thus explaining the detection of full-length proteins. Based on the same rationale, we propose that the central loop of PfJ23 (Asn75–Lys221) is located in the lumen of closed vesicles.


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mass Spectrometry Identification of Deuterium-labeled P. falciparum Maurer’s Cleft Proteins—
Our proteomic study focused on Maurer’s clefts, a Golgi-like compartment linked to the erythrocyte plasma membrane. They are recovered together with the red cell plasma membrane and submembrane skeleton as infected erythrocyte ghost preparations. We studied these preparations using a method based on metabolic labeling with deuterated lysine that allows distinction between host cell- and parasite-derived proteins in MS analyses. Because only 50% of the lysine residues incorporated into newly synthesized proteins were deuterated, peptides originating from parasite-derived proteins cleaved after a lysine residue appeared as doublets with a mass difference of 4 Da (or 8 Da in the case of a missed cleavage). When the Mascot search was restricted to such peptides, only parasite proteins were identified, validating our approach for an efficient and quick identification of parasite proteins. This approach has the advantage of limiting the number of Mascot searches, therefore increasing the efficiency and precision of protein identification. Nevertheless this approach was not too restrictive as the access to other species was conserved, allowing the identification of proteins by homology in case the P. falciparum genome data base missed some coding regions.

From 67 polyacrylamide gel slices, 78 parasite proteins were identified from ghost preparations. Twenty-eight ribosomal proteins or translation factors were identified as well as five P. falciparum glycolytic enzymes (Supplemental Table 1). Parasite protein synthesis and the entire glycolytic pathway take place in the parasite cytosol, and the identification of these proteins in the ghost preparation suggests that a small fraction of infected erythrocytes is completely lysed when ghosts are being prepared. Nevertheless ribosomal proteins are known to be very abundant in eukaryotic cells, and they are notorious contaminants of global proteomic studies (except following two-dimensional gel separation because of their basic pHi). Similarly glycolytic enzymes are particularly abundant in P. falciparum extracts. Indeed microarray studies showed that the lactate dehydrogenase gene transcript is 10 times more abundant than the transcript of HSP70 and 100 times more abundant than that of actin (16). This is consistent with the observed increased glucose consumption by 30–50-fold following erythrocyte infection by P. falciparum (18). However and concordant with its recovery in the ghost fraction, it has been recently proposed that, in mammals, the glyceraldehyde-3-phosphate dehydrogenase is also involved in vesicular transport (19, 20).

Our study identified proteins that have been described as Maurer’s cleft proteins (PfSBP1 (3)) or as transiently associated with Maurer’s clefts (HRP1 (7, 21)). On the other hand, several proteins known to be associated with the Maurer’s clefts were not detected within the ghost preparations: PfEMP1 (7), PfEMP3 (7, 22), and the PfSEP (23) and STEVOR proteins (24). As no protein over 187 kDa and under 12 kDa was identified, PfEMP1 (200–400 kDa) and PfEMP3 (273 kDa) should not have entered the 10% polyacrylamide gel, while the SEP proteins (12–16 kDa) might have run out of the gel. The lack of detection of the STEVOR proteins may be related to the timing of their translocation, although they have been described to be located in the Maurer’s clefts in late trophozoites and early schizonts (24). Alternatively it can illustrate the inefficient recovery of peptides from STEVOR proteins for the MS analysis.

Five proteins of the parasitophorous vacuole were identified: SERA3, SERA4 (25), ABRA (26), and EXP1 and EXP2 (27). It is unclear whether the Maurer’s clefts and the parasitophorous vacuole form a continuous membrane network in the erythrocyte cytoplasm, joining the parasite cytoplasm and the red blood cell membrane together, or whether proteins are transferred across these two compartments by vesicular transport (28, 29). While the latter hypothesis is supported by the presence of secretory vesicle components in the Maurer’s clefts (57, 30), some data also suggest that the Maurer’s clefts constitute specialized domains of the parasitophorous vacuole membrane (31). In any case, only a limited number of proteins are trafficked between these two compartments. Indeed only 10 proteins are common to our study and a proteomic study of the parasitophorous vacuole3: two glycolytic enzymes (triose-phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase), SERA4, three heat shock proteins (PfHsp86 and proteins encoded by the PF11_0351 and PF08_0054 loci), GBP130, the merozoite surface protein 7, an endoplasmin homologue, and the protein-disulfide isomerase. In addition, two proteins of the parasitophorous vacuole membrane identified in our study, EXP1 and EXP2, have been shown to be exported to a parasite-derived compartment within the erythrocyte cytosol (27, 32). These observations strengthen the validity of this proteomic study.

Characterization of Seven Novel Maurer’s Cleft Proteins (Table III)—
Our proteomic study identified 13 hypothetical proteins, putatively located in the Maurer’s clefts and expressed in P. falciparum blood stages (16, 33). Sera were raised against seven of these hypothetical proteins and specifically detected each of the relevant proteins by Western blot in infected erythrocyte ghosts. Indirect immunofluorescence studies were also performed. The pattern observed for PfE60 and PfJ23 clearly indicates that the proteins are associated with Maurer’s clefts. Alternatively PfA680 and PfJ323 were detected outside the parasite both with a Maurer’s cleft pattern and a more diffuse labeling of the parasitophorous vacuole, suggesting a dual localization. Immunoelectron microscopy studies should be performed to address this matter. The location of the hypothetical proteins and/or their orientation within closed vesicles of the ghost preparations was then investigated with proteinase K digestion experiments. The predicted soluble protein PfG174 was fully degraded by proteinase K in the absence of detergent. Thus PfG174 is likely to be located in the red cell cytosol. Whether PfG174 is soluble in the erythrocyte cytosol or associated with a membrane or the submembrane skeleton requires further investigation. Five of the selected proteins were at least partially protected from proteinase K digestion, concordant with their association with closed vesicles as presented Fig. 5. Maurer’s clefts are the only parasite-derived membrane structures identified in ghost preparations, and they form closed vesicles following the hypotonic lysis of infected erythrocytes as shown by the inaccessibility of the amino-terminal domain of PfSBP1 to proteinase K in the absence of detergent (Supplemental Fig. 1 and Ref. 3). Taken together, our results suggest that PfA680, PfD80, PfE60, and PfJ23 are transmembrane proteins of the Maurer’s clefts, while PfJ13 and PfPDI are soluble proteins in the lumen of the Maurer’s clefts. PfJ323 was not detected following proteinase K treatment indicating that its carboxyl-terminal domain, against which the antibodies were raised, is in the red cell cytoplasm. Because immunofluorescence studies located PfJ323 in vesicular structures, we propose that PfJ323 is a transmembrane protein of the Maurer’s clefts.

A complementary study has been published recently using a multidimensional protein identification technology (MudPIT) to identify surface proteins from P. falciparum-infected erythrocytes (34). Following surface biotinylation of mature schizonts or trophozoites, subcellular fractions were prepared by sedimentation following hypotonic lysis. These fractions should contain the red cell plasma membrane and submembrane skeleton as well as Maurer’s clefts. The biotinylated proteins were recovered by affinity chromatography on a streptavidin column and processed for MS analysis. Among the 36 identified proteins considered by the authors as potential parasite-infected erythrocyte surface proteins, four were also identified in our study: PfE60/PIESP2, PfJ323, PfG174, and PfE50 (product of gene PFE00050w). We showed that PfE60/PIESP2 and PfJ323 are transmembrane proteins of the Maurer’s clefts; Florens and colleagues (34) also determined that PfE60/PIESP2 may be associated with the red cell membrane. Consequently, like PfEMP1, PfE60/PIESP2 might be transiently associated with the membrane of the Maurer’s clefts and then translocated to the red cell surface. Alternatively, because schizonts might be slightly permeable to biotin (35), submembrane proteins of the infected erythrocyte, including transmembrane proteins of the Maurer’s clefts, might have been labeled. Concordant with this permeability of the erythrocyte membrane is the accessibility of HRP1 to specific antibodies in late schizonts (36). This would also explain the labeling of PfG174 that we located in the red cell cytosol.

Trafficking of Proteins to the Maurer’s Clefts—
Overall the Maurer’s cleft proteins we identified have varied topologies (Fig. 5). Because PfA680, PfE60, and PfJ323 display a signal peptide and have the same topology within the clefts, they should be trafficked to this compartment via the same pathway. However, while PfD80 has a signal peptide, its topology is different. Similarly PfSBP1 and PfJ23 lack a signal peptide but have an odd number of transmembrane domains, yet their orientation in the Maurer’s clefts is different. Because a recent study showed that the trafficking of parasite-derived proteins in the host erythrocyte depends on the timing of expression (37), we investigated whether this hypothesis could account for the observed variability. This was not the case. Based on microarray data (33), PfA680, PfE60, and PfD80 have the same pattern of transcription, but their topologies differ, while PfJ323, with the same topology as PfA680 and PfE60, has a different pattern of expression.

The pathway addressing parasite proteins to the Maurer’s clefts has not been characterized nor have the amino acid sequences signaling them to this machinery been identified. However, recent studies identified a conserved motif in parasite proteins trafficking outside the parasite (38, 39). This VTS or Pexel motif is present in the sequence of the seven Maurer’s clefts proteins newly identified in this study (Table III) and in PfPDI but not in the PfSBP1 sequence. Moreover some of the known Maurer’s cleft membrane proteins like PfSBP1 have no signal peptide, while others including members of a recently identified family of putative Maurer’s cleft proteins do (3, 40). Interestingly Sam-Yellowe and colleagues (40) defined PfA680 as belonging to this family. Here we established its location in the Maurer’s clefts. Members of this family have two predicted transmembrane domains close to their carboxyl terminus and separated by a three to nine-residue loop. Although not members of this family, PfE60 and PfJ23, unambiguously located in Maurer’s clefts, display the same characteristic. However, if this property participates in the targeting of proteins to the Maurer’s clefts, it is again not a ubiquitous feature of Maurer’s cleft transmembrane proteins as illustrated by PfSBP1.

Taken together, our results strongly support that several pathways based on various signal motifs are addressing parasite proteins to the Maurer’s clefts. As proposed by Cooke and colleagues (29), a distinctive class of double membrane vesicles exported from the parasite endoplasmic reticulum to the parasitophorous vacuole membrane may participate in the relocation of some parasite proteins to the erythrocyte cytoplasm.

A Study Shedding New Light on the Biological Roles of Maurer’s Clefts—
Some of the proteins that we detected in the ghost preparations were not precisely located but have a predicted function consistent with their localization in a secretory compartment. These include several chaperones (five heat shock proteins), endoplasmin, and PfPDI, involved in protein folding. Endoplasmin and PfPDI are located in the endoplasmic reticulum in mammalian cells, but confocal microscopy studies suggest that PfPDI is also partly exported to the Maurer’s clefts.4 This is consistent with our study in which PfPDI was detected by Western blotting and protected from proteinase K digestion in ghost preparations. Taken together, our results support that Maurer’s clefts are a secretory compartment transposed in the cytoplasm of the parasite host cell.

Proteins of known function were also identified in ghost preparations that may suggest that the Maurer’s clefts are not only involved in protein trafficking. One such protein, hypoxanthine phosphoribosyltransferase, is involved in purine metabolism and has been located in vesicle-like structures within the cytoplasm of infected erythrocytes (41). Phosphoethanolamine N-methyltransferase is required for phosphatidylcholine synthesis (42) and may participate in the extensive synthesis of membrane as the parasite develops inside its host cell. The 14-3-3 protein is evolutionarily conserved and has been described as a serine/threonine-binding protein involved in signal transduction events (43). In addition, two small GTP-binding proteins from the Ran and Rack signal transduction families were identified. Although their location within Maurer’s clefts requires further investigation, signal transduction events controlling the biological function of Maurer’s clefts have been suggested by the presence of the PfPP1 phosphatase in the lumen of this compartment and its central role for merozoite release.2

Our study also identified two rhoptry-associated proteins, RhopH2 and PfE75, a protein similar to RAP2 encoded by gene PFE0075c. These findings are consistent with results from Sam-Yellowe and colleagues (44) showing that RhopH3, a rhoptry-associated protein and member of the rhoptry high molecular weight complex including three proteins (RhopH1–3), might transit through Maurer’s cleft-like vesicles in early parasite stages when the rhoptries have not formed.

Interestingly the product of adjacent genes on chromosome 5, PFE0050w, PFE0055c, PFE0060w, PFE0065w (encoding PfSBP1), and PFE0075c were all identified by MS analyses of infected erythrocyte ghosts. Among them, PfSBP1 and PFE60 are transmembrane proteins of the Maurer’s clefts. Whether the proteins encoded by this cluster of genes are all located in the Maurer’s clefts is currently being investigated. PFE0055c is annotated as encoding a putative chaperone protein, and PfE75 is annotated as a rhoptry-associated protein. PFE0070w encodes the PfE70 protein with basic amino acid repeats similar to those of PfSBP1 that have been shown to mediate an interaction with the erythrocyte protein LANCL1 (lantibiotic synthetase component c-like protein).5 This interaction has been proposed to participate in the binding of the Maurer’s clefts to the red cell membrane in late parasite stages. Because Maurer’s clefts are linked to the red cell membrane throughout the intraerythrocytic parasite development, other interactions have to be involved. Whether PfE70 is mediating such an interaction is currently being investigated.

In conclusion, our study was validated by the identification of seven new Maurer’s cleft proteins and sheds new light on the important biological functions of this parasite-derived compartment. Our results confirmed that Maurer’s clefts have characteristics of a secretory compartment addressing parasite proteins to the red cell surface. They also support the hypothesis that rhoptry proteins may transit through the Maurer’s clefts and suggest the occurrence of signal transduction events and the presence of enzymes. Some of these proteins may be essential for the parasite development, and because they are likely parasite-specific, they could constitute new attractive drug targets.


    ACKNOWLEDGMENTS
 
We are grateful to H. Vial for support. We thank M. Guillotte for help in animal handling, P. Grellier for the anti-PfPDI antibody, K. Lingelbach and J. Nyalwidhe for sharing unpublished data, and S. Ralph for critical reading of the manuscript. The Laboratoire de Spectrométrie de Masse Bio-Organique thanks Bruker Daltonics for technical support.


    FOOTNOTES
 
Received, November 10, 2004, and in revised form, January 21, 2005.

Published, MCP Papers in Press, January 24, 2005, DOI 10.1074/mcp.M400176-MCP200

1 The abbreviations used are: PfSBP1, P. falciparum skeleton-binding protein 1; MS/MS, tandem mass spectrometry; nano LC, nanoscale capillary liquid chromatography; GST, glutathione S-transferase; PfPDI, P. falciparum protein-disulfide isomerase; STEVOR, sublelomeric variable open reading frame; HRP1, histidine rich protein 1; VTS, vacuolar transport signal; SERA, serine rich antigen; RESA, ring-infected erythrocyte surface antigen; EXP, exported protein. Back

2 Blisnick, T., Vincensini, L., Fall, G., and Braun Breton, C. (2005) LANCL1, an erythrocyte protein recruited to the Maurer’s clefts during Plasmodium falciparum development. Mol. Biochem. Parasitol., in press. Back

3 K. Lingelbach and J. Nyalwidhe, personal communication. Back

4 P. Grellier, personal communication. Back

5 T. Blisnick, L. Vincensini, J. C. Barale, A. Namane, and C. Braun Breton, submitted for publication. Back

* This work was supported by the Pasteur Institute, the CNRS, and the "Programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires" from the Ministère Français de l’Éducation Nationale, de la Recherche et de la Technologie. Bruker Daltonics also provided financial support. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

§ Supported by fellowships from the Ministère Français de l’Éducation Nationale de la Recherche et de la Technologie and from the Fonds Inkerman de la Fondation de France. Back

|| Supported by a fellowship from the Ministère Français de l’Éducation Nationale, de la Recherche et de la Technologie and the Internationales Graduiertenkolleg/International Research Training Group 532. Back

§§ To whom correspondence should be addressed. Tel.: 33-4-67-14-33-81; Fax: 33-4-67-14-42-86; E-mail: cbb{at}univ-montp2.fr


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