A Nonpermeant Biotin Derivative Gains Access to the Parasitophorous Vacuole in Plasmodium falciparum-infected Erythrocytes Permeabilized with Streptolysin O*

In its host erythrocyte, the malaria parasitePlasmodium falciparum resides within a parasitophorous vacuole, the membrane of which forms a barrier between the host cell cytosol and the parasite surface. The vacuole is a unique compartment because it contains specific proteins that are believed to be involved in cell biological functions essential for parasite survival. As a prerequisite for the characterization of the vacuolar proteome, we have developed an experimental approach that allows the selective biotinylation of soluble vacuolar proteins. This approach utilizes nonpermeant biotin derivatives that can be introduced into infected erythrocytes after selective permeabilization of the erythrocyte membrane with the pore-forming protein streptolysin O. The derivatives gain access to the vacuolar lumen but not to the parasite cytosol, thus providing supportive evidence for the existence of nonselective pores within the vacuolar membrane that have been postulated based on electrophysiological studies. Soluble vacuolar proteins that are biotin-labeled can be isolated by affinity chromatography using streptavidin-agarose.

membrane presents a barrier between parasite and host cell cytosol, although electrophysiological studies in Plasmodiuminfected erythrocytes suggest the existence of nonselective pores within the PVM that allow passive bidirectional diffusion of small molecules (4,5). With respect to biogenesis and protein contents, the PV differs significantly from endocytic vacuoles (6), with the most apparent difference being the almost cytosolic pH of the vacuole (7,8). In recent years, many observations have indicated a number of cell biologically relevant processes within the vacuole that are important for parasite survival.
In the course of the infection, several parasite proteins are exported into the erythrocyte, and some of these proteins, such as the members of the PfEMP1 family, are key molecules involved in the pathogenesis of malaria (9). For at least some exported proteins, the PV is a transit compartment from which they are transported into the host cell cytosol (10 -12). Translocation across the vacuolar membrane must be a selective process because other proteins are retained within the PV (13,14). Recently, it has been suggested that proteins destined for the parasite apicoplast, a unique plastid-like intracellular organelle of apicomplexan parasites, also may be trafficked via the PV (15). Thus, the PV must contain a machinery involved in protein sorting. Upon completion of parasite development and multiplication, merozoites, the invasive stages, are released from the infected cell. Release of merozoites from the PV can be prevented by treatment of infected erythrocytes (IRBCs) with protease inhibitors (16). In addition, evasion of merozoites is accompanied by a specific sequential cleavage of the major merozoite surface protein 1 that is essential for the subsequent reinvasion of noninfected cells (17). These data suggest a role of vacuolar proteases in the release of parasites from the infected host cell. Collectively, the current observations underscore the notion that the PV is a unique intracellular compartment critical for parasite survival within the host cell and that it most likely contains novel proteins of elementary functions.
The volume of the vacuole compared with the volumes of the parasite cytosol and the erythrocyte cytosol, respectively, is very small. Estimates based on morphological data suggest a 1:10,000 ratio of vacuolar volume to erythrocyte cytosol in IRBCs infected with stages of the parasite that have completed ϳ30 h of the 48-h intraerythrocytic development (12). Cell fractionation experiments on IRBCs infected with the same developmental stages show that more than 70% of the total protein are erythrocyte cytosolic proteins, predominantly hemoglobin (18). Therefore, the isolation of vacuolar proteins is difficult; consequently, only a few vacuolar proteins have been identified thus far. In this report, we describe a novel strategy for the identification of vacuolar proteins as a prerequisite for a comprehensive proteome analysis of this compartment. * This work was supported by a grant from the Deutsche Forschungsgemeinschaft. 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.
§ Recipient of a scholarship from the German Academic Exchange Service.
ʈ Supported by the Alexander von Humboldt Foundation. ** Present address: Institute of Cell, Animal and Population Biology (ICAPB), University of Edinburgh, Edinburgh EH9 3JT, Scotland, United Kingdom.
Parasite Cultures and Permeabilization of Infected Erythrocytes-Parasites of the P. falciparum FCBR strain were cultivated in RPMI 1640 medium (Invitrogen) containing 10% human plasma and erythrocytes of blood group A ϩ (Marburg Blood Bank) under standard conditions (20). Trophozoite-infected erythrocytes (IRBCs) were enriched to a parasitemia of Ͼ90% by gel floatation (21). For metabolic labeling of newly synthesized proteins, enriched IRBCs were washed twice in methionine-free RPMI 1640 medium and cultivated for 1 h in plasmafree, methionine-free RPMI 1640 medium, in the presence of 50 Ci ml Ϫ1 L-[ 35 S]methionine. IRBCs were permeabilized with SLO as described previously (10). Briefly, 2 ϫ 10 8 IRBCs were incubated with 3-4 hemolytic units of SLO in RPMI 1640 medium at room temperature for 6 min. Samples were centrifuged at 10,000 ϫ g for 15 s. The pellet containing intact parasites, the vacuolar contents, and membranes was washed with 200 l of RPMI 1640 medium before biotinylation. Complete removal of the erythrocyte hemoglobin was monitored spectrophotometrically as described previously (10).
Biotin Labeling-For the biotinylation of intact erythrocytes, 10 8 parasitized or nonparasitized erythrocytes were washed three times in PBS containing 0.6 mM CaCl 2 and 1 mM MgCl 2 , pH 7.6 (PBS 2ϩ ), and then incubated in PBS 2ϩ containing 1 mg ml Ϫ1 sulfo-NHS-SS-biotin for 30 min on ice. Cells were sedimented by centrifugation at 10,000 ϫ g for 15 s at 4°C. The supernatant was analyzed photometrically at 412 nm for the release of hemoglobin. To block and remove unbound biotin, cells were washed three times in PBS 2ϩ containing 100 mM glycine. For cleavage of extracellularly exposed membrane proteins, an aliquot of the cells was treated with 1 mg ml Ϫ1 trypsin for 15 min at 37°C. The protease was inactivated using soybean trypsin inhibitor at a concentration of 1 mg ml Ϫ1 for 15 min on ice. Cells were lysed by repeated freezing and thawing in water supplemented with a protease inhibitor mixture containing antipain, chymostatin, aprotinin, trypsin inhibitor, Na-EDTA, pepstatin, leupeptin, and elastatinal, each at a concentration of 1 g ml Ϫ1 . A membrane fraction and a fraction of soluble proteins were prepared by centrifugation.
For the biotinylation of internal parasite proteins and vacuolar proteins, IRBCs permeabilized with SLO were washed with PBS 2ϩ and then resuspended in an optimal volume, routinely 800 l, of distilled water containing the above-mentioned protease inhibitors. Lysates were prepared by three cycles of freezing and thawing. Soluble proteins contained in the supernatant after centrifugation at 10,000 ϫ g for 15 min were biotin-labeled in PBS 2ϩ containing 1 mg ml Ϫ1 sulfo-NHS-LCbiotin for 30 min at 4°C with frequent shaking. The solution was centrifuged at 3,000 ϫ g using a microconcentrator (Millipore Corp.) with a size exclusion of 5 kDa to remove any free biotin. The sample was diluted with water containing the protease inhibitors. This step was repeated twice.
For the labeling of soluble vacuolar proteins, permeabilized cells were incubated with sulfo-NHS-LC-biotin as described above. The solution was centrifuged at 4°C at 1,300 ϫ g for 5 min, and the sediment of parasite-containing vacuoles was washed three times in 100 mM glycine in PBS 2ϩ to block the unreacted biotin derivative. After a final wash in PBS 2ϩ , cells were lysed and processed as above.
Affinity Purification of Biotin-labeled Proteins-Biotin-labeled proteins from 2 ϫ 10 8 IRBCs were incubated with streptavidin-conjugated agarose beads at 4°C for 2 h under constant shaking. The beads were sedimented by centrifugation at 10,000 ϫ g for 15 s. The supernatant was collected and stored at Ϫ20°C until further use. Beads were washed sequentially in buffer A (10 mM Tris-HCl, pH 7.5, 0.2% Nonidet P-40, 2 mM EDTA, and 150 mM NaCl), buffer B (10 mM Tris-HCl, pH 7.5, 0.2% Nonidet P-40, 2 mM EDTA, and 500 mM NaCl), buffer C (10 mM Tris-HCl, pH 7.5), and, finally, PBS 2ϩ . The beads were then washed with 10 mM HEPES buffer, pH 7.4, containing 20 mM MgCl 2 and 5 mM ATP. Bound proteins were eluted by boiling the beads in SDS-PAGE sample buffer before analysis.
Immunoprecipitation-The marker proteins SERP and GBP were precipitated from lysates of IRBCs as follows. After permeabilization of IRBCs and complete removal of the erythrocyte cytosol, parasite-containing vacuoles were lysed osmotically. Soluble proteins in a volume of 100 l, corresponding to 2 ϫ 10 7 IRBCs, were diluted with 400 l of distilled water and 500 l of solubilization buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, and 4 g ml Ϫ1 phenylmethylsulfonyl fluoride. The respective antisera were added, and the mixture was incubated at room temperature for 30 min. Subsequently, 15 l of protein A-Sepharose beads were added and incubated for 1 h at 4°C. The beads were sedimented by centrifugation, and the supernatant was collected. Proteins were precipitated from the supernatant with 10% trichloroacetic acid on ice for 10 min. The samples were centrifuged at 4°C, and the precipitate was washed with ice-cold acetone and dissolved in SDS-PAGE sample buffer. The protein A-Sepharose beads were washed sequentially with buffers A, B, and C and PBS 2ϩ as described above, and bound proteins were eluted by boiling the beads in SDS-PAGE sample buffer.
Gel Electrophoresis-Routinely, proteins were dissolved in SDS sample buffer and separated on 10% or 12% gels under reducing conditions. When proteins were labeled with the cleavable biotin derivative sulfo-NHS-SS-biotin, separation was on 5-20% gradient gels under nonreducing conditions. Protein bands were visualized by Coomassie Blue staining before exposure to x-ray film for the detection of radiolabeled proteins. For two-dimensional PAGE, soluble parasite and vacuolar proteins from 2 ϫ 10 9 permeabilized IRBCs were precipitated using 3 volumes of 10% trichloroacetic acid in acetone containing 20 mM DTT for 1 h at Ϫ20°C. The proteins were collected by centrifugation, washed with ice-cold acetone containing 20 mM DTT, and dried. Protein extracts were dissolved in 300 l of a rehydration solution (8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 4% DTT, 2% ASB (Calbiochem), and 2% immobilized pH gradient buffer pH 4 -7 (Amersham Biosciences)) and incubated at room temperature for 1 h. After rehydration for 24 h under low viscosity paraffin oil, 13-cm immobilized pH gradient strips (Amersham Biosciences) covering a pH range of 4 -7 were subjected to isoelectric focusing for 28,000 V-h. After isoelectric focusing, the individual strips were consecutively incubated in equilibration solution A and B (50 mM Tris-HCl, pH 6.8, 8 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, complemented with 3.5 mg ml Ϫ1 DTT (solution A) or 45 mg ml Ϫ1 iodoacetamide instead of DTT (solution B)), each for 15 min. The equilibrated strips were transferred to 12% slab gels and separated in the second dimension. After drying of the gels, radiolabeled bands were visualized by autoradiography.
Immunoblot Analysis-Proteins separated by SDS-PAGE or twodimensional PAGE were transferred to nitrocellulose membranes. For the detection of biotin-labeled proteins, membranes were blocked using 2% BSA in PBS, pH 7.4, for 1 h at room temperature. Filters were incubated for 20 min with alkaline phosphatase-conjugated streptavidin (Pharmingen) diluted 1:10,000 in 2% BSA in PBS, pH 7.4. The membranes were washed three times in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl for 5 min. Biotin-labeled proteins were stained with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate following standard procedures. The same membranes were then incubated with specific rabbit antisera at a dilution of 1:500 for 16 h at 4°C. Specific marker proteins were detected after incubation with horseradish peroxidase-conjugated anti-rabbit IgG for 1 h at room temperature and development of the filters using the ECL system (Amersham Biosciences) following standard procedures.
Indirect Immunofluorescence Assay-Permeabilized and biotintreated IRBCs were spread on glass slides and air-dried. Subsequently, they were fixed in acetone-methanol (1:1) for 10 min at Ϫ20°C. The slides were incubated with the primary antibodies for 2 h at room temperature. The monoclonal anti-band 3 IgG was diluted 1:20, and the polyclonal rabbit antisera specific for SERP and aldolase, respectively, were diluted 1:10 in PBS, pH 7.2. After three washes with PBS, pH 7.2, slides were probed with a mixture of the corresponding secondary antibody (1:100) and Cy3-conjugated streptavidin (1:200) for 30 min at room temperature in the dark. Autofluorescence and nonspecific fluorescence levels were determined by viewing control samples of either biotin-treated or untreated IRBCs not incubated with Cy3-conjugated streptavidin and secondary antibodies. The cells were mounted in glycerol containing 0.1% of the antifade reagent 1,4-diazobicyclo(2,2,2)octane. Fluorescence microscopy was performed using a Leica TCS SP2 laser scanning confocal microscope.

RESULTS AND DISCUSSION
A Membrane-impermeable Biotin Derivative Gains Access into Intact Infected Erythrocytes-The intracellular parasite depends on the acquisition of nutrients from the extracellular milieu and from the host cell cytosol. Extracellular nutrients must be taken up across three membranes, the erythrocyte plasma membrane, the PVM, and the parasite plasma membrane. In the course of infection, the erythrocyte plasma membrane undergoes major alterations. Most notably, the transport properties of the membrane change, increasing the influx rates for a number of low molecular weight solutes. The identification of the proteins that mediate these novel transport properties is being pursued intensely (for current reviews, see Refs. [22][23][24]. In preliminary experiments designed to study transport of small solutes across the erythrocyte membrane, we noticed that the membrane-impermeable sulfo-NHS-SS-biotin gained access to the interior of erythrocytes infected with trophozoite-stage parasites, but not to noninfected erythrocytes. Intact noninfected and infected erythrocytes were incubated with sulfo-NHS-SS-biotin. Cells were lysed in water by repeated freezing and thawing, and soluble proteins were separated from the membrane fraction by centrifugation. Whereas in noninfected cells, no biotin labeling of soluble proteins was detectable (Fig. 1, lanes 1 and 3), soluble proteins were found to be biotin-labeled in infected cells (Fig. 1, lanes 5 and 7). When intact erythrocytes were treated with trypsin after the biotin reaction, the pattern of biotin-labeled proteins in the membrane fraction changed, due to proteolytic cleavage of externally exposed protein domains (Fig. 1, lanes 2, 4, 6, and 8). More importantly, trypsin treatment had little effect on the pattern of biotin-labeled soluble proteins in infected cells (Fig.  1, lanes 5 and 7). This result demonstrates that the protease, unlike the biotin derivative, was excluded from the erythrocyte cytosol. Most likely, access of biotin into intact erythrocytes is due to the novel transport properties of the erythrocyte membrane, and we are currently investigating the pathways involved in this process. We never observed that biotin gained access to vacuolar proteins in infected erythrocytes using these experimental conditions (data not shown). We anticipate that internalization of biotin across the intact host cell plasma membrane is limited and that the excess of erythrocyte cytosolic proteins prevented efficient labeling of vacuolar proteins.
Biotin Accumulates in the Parasitophorous Vacuole in Permeabilized Infected Erythrocytes-Although the vacuolar membrane forms a barrier between the parasite surface and the host cell cytosol, it is most conceivable that the PVM allows the transport of nutrients essential for parasite survival. Using a patch-clamp technique, Desai et al. (4) measured high conductance channels that are permeable to organic and inorganic anions and cations. The channels are open 98% of the time, and they are highly abundant. In bilayers, this channel allows passage of molecules up to 1,400 Da (5), which is similar to the nonselective pores discovered in the PVM of the taxonomically related parasite Toxoplasma (25). The existence of these pores in Plasmodium-infected erythrocytes has not been verified biochemically, but if they exist, they should allow access of membrane-impermeable biotin derivatives that are ϳ600 Da in size.
To increase access of biotin to the interior of the infected erythrocyte, we utilized SLO to permeabilize the erythrocyte plasma membrane. In an initial assessment of our experimental strategy, infected erythrocytes were permeabilized and, after complete removal of the erythrocyte cytosol, incubated with the membrane-impermeable sulfo-NHS-LC-biotin, which was detected using a fluorescent streptavidin derivative (Fig. 2). The images show a distinct ring around the periphery of the intracellular parasite. The biotin co-localizes with the vacuolar marker protein SERP ( Fig. 2A). This location is distinct from the location of the erythrocyte membrane protein band 3 and from the location of the parasite cytosolic protein aldolase (Fig.  2, B and C). In some parts, the streptavidin and the antibody to band 3 co-localize; this is due to the fact that proteins of the erythrocyte plasma membrane also react with the biotin. Little if any biotin was detectable in the erythrocyte cytosol and in the parasite cytosol, respectively. A ring-like staining around the periphery of the parasite can be attributable to a reaction of the biotin with membrane proteins of the PVM and with soluble vacuolar proteins. Because the resolution of fluorescence microscopy does not allow discrimination between a staining of the PVM and a lumenal staining, a more detailed biochemical analysis was performed to assess the biotinylation of soluble vacuolar proteins.
Biotin-labeled Parasite Proteins Bind to SAv-agarose-Because mammalian erythrocytes have lost the ability to synthesize proteins de novo, in infected erythrocytes, radiolabeled amino acids are incorporated exclusively into parasite proteins. The following experiment was designed to optimize the biotinylation conditions and the selective isolation of biotinylated proteins by SAv-agarose. Infected cells were cultivated for 30 min in the presence of L-[ 35 S]methionine, and subsequently, the erythrocyte plasma membrane was permeabilized with SLO to release the soluble contents of the erythrocyte cytosol. In all experiments, the integrity of the PVM and the parasite plasma membrane was assessed as described previously (10). Parasite-containing vacuoles were lysed by repeated freezing and thawing, and the fraction of soluble proteins was collected after centrifugation. One aliquot of the lysate was treated with sulfo-NHS-LC-biotin, and another aliquot remained untreated. Both samples were incubated with SAv-agarose beads, and bound proteins were eluted by boiling the beads in SDS sample buffer. The fractions of unbound and bound proteins were analyzed by SDS-PAGE and autoradiography (Fig. 3A). No radiolabeled proteins from the nonbiotinylated sample bound to SAv-agarose (Fig. 3A, lane 2), whereas after biotinylation, most radiolabeled proteins were found in the fraction of bound proteins (Fig. 3A, lane 4). It is noteworthy that the pattern of FIG. 1. Infected erythrocytes are permeable for a nonpermeant biotin derivative. Noninfected (RBC) and infected (iRBC) erythrocytes were incubated with sulfo-NHS-SS-biotin, and a sample from each reaction was subsequently treated with trypsin (lanes 3, 4, 7, and 8) to cleave externally exposed proteins. Cells were lysed and separated into a fraction of soluble proteins (lanes 1, 3, 5, and 7) and into a membrane fraction (lanes 2, 4, 6, and 8). Proteins, corresponding to 2 ϫ 10 7 cells, were electrophoresed through a 5-15% nonreducing SDS-polyacrylamide gel, transferred to a nitrocellulose filter, and probed with alkaline phosphatase-conjugated SAv. biotinylated proteins differed from that of the nonbiotinylated proteins and that the bands appeared less focused. This observation is attributable to the variant degrees of biotin labeling of individual polypeptide chains. To assess the effects of biotin incorporation on the electrophoretic motility of proteins, BSA was incubated with different molar ratios of sulfo-NHS-LCbiotin and analyzed by SDS-PAGE. As shown in Fig. 3B, the electrophoretic motility of BSA is altered depending on the degree of biotinylation.
The subsequent experiments were devised to demonstrate compartment-specific biotinylation of soluble parasite proteins in permeabilized IRBCs. The so-called GBP, the SERP, and parasite aldolase (PfALD) are soluble marker proteins that are located in different compartments of the infected erythrocyte. Aldolase is restricted to the parasite cytosol (19), SERP is restricted to the vacuole (13), and GBP is found both inside the vacuole and within the erythrocyte cytosol (10). In the first step, it was confirmed that these proteins can be biotinylated and that they bind to SAv-agarose, provided that they are accessible to biotin. This was assessed using lysates of permeabilized infected erythrocytes; infected erythrocytes were permeabilized with SLO, and after complete removal of the erythrocyte cytosol, a lysate containing soluble proteins of the parasite cytosol and of the PV was prepared. One aliquot of soluble proteins was treated with sulfo-NHS-LC-biotin as described above. Another aliquot remained untreated. The samples were incubated with SAv-agarose beads, and the bound and unbound proteins were analyzed separately by immunoblotting using antisera to the respective marker proteins (Fig.  4A). In the sample of nonlabeled proteins, the three marker proteins were recovered in the fraction of unbound proteins (Fig. 4A, compare lanes 1 and 2). After biotin treatment, GBP, SERP, and PfALD were recovered in the fraction of bound proteins (Fig. 4A, compare lanes 3 and 4). In the case of GBP and SERP, recovery was almost quantitative. Although recovery was not entirely complete in the case of PfALD, these results demonstrate that the marker proteins are biotinylated and can be isolated on SAv-agarose.
After confirmation that all marker proteins are biotinylatable, IRBCs were permeabilized with SLO, washed to remove The remaining fraction consisted of intact parasites, including an intact PVM and parasite plasma membrane. The PVM and the parasite plasma membrane were then lysed, and membrane proteins and soluble proteins were separated. One aliquot of soluble proteins, corresponding to 2 ϫ 10 8 cells, was treated with sulfo-NHS-LC-biotin (lanes 3 and 4), and one aliquot remained untreated (lanes 1 and 2). Proteins that bound to SAv-agarose beads (lanes 2 and 4) and unbound proteins (lanes 1 and 3) were analyzed by SDS-PAGE and autoradiography. B, extensive biotin labeling increases the molecular size of proteins. BSA was treated with sulfo-NHS-LC-biotin at different molar ratios and analyzed by SDS-PAGE and Coomassie Blue staining (lanes 1-3) or by reaction with peroxidase-conjugated SAv on nitrocellulose filters (lanes  4 -6).
the contents of the erythrocyte cytosol, and incubated with sulfo-NHS-LC-biotin. Upon removal of free biotin and lysis of parasites, soluble proteins were immunoprecipitated using antibodies to SERP and GBP, respectively. Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose filters. Initially, the filters were probed with the SERP and GBP antibodies, and their binding was detected using an alkaline phosphatase-conjugated secondary antibody (Fig. 4B, lanes 1 and 3). Subsequently, the filters were reprobed with peroxidase-conjugated SAv and analyzed by chemiluminescence (Fig. 4B, lanes 2 and 4). The same analysis was carried out for PfALD, but because the antiserum against aldolase does not immunoprecipitate the protein, the total lysate was separated by SDS-PAGE and analyzed (Fig. 4B, lanes 5  and 6). The antisera specifically recognized the marker proteins. The bands that were detectable with SAv were of identical sizes as GBP and SERP, indicating that both proteins were biotin-labeled. No biotin-labeled band corresponding in size to aldolase was detectable; a protein band of smaller size reacted with SAv, but not with the aldolase-specific antibody.
Affinity isolation of biotin-labeled proteins on SAv-agarose beads and subsequent analysis by immunoblotting confirmed the compartment-specific labeling of the respective proteins (Fig. 4C). In permeabilized cells, PfALD was not labeled, and, consequently, it was not found in the fraction of proteins bound to SAv, whereas GBP was quantitatively labeled with biotin and recovered. Apparently, in permeabilized cells, the accessibility of the biotin to SERP is restricted; all of the biotin-labeled SERP protein is bound to SAv-agarose beads (Fig. 4C, lanes 1  and 2); however, a substantial proportion of the SERP molecules remained unbound, obviously because these molecules have not been labeled with biotin (Fig. 4C, compare lanes 3 and  4). A 5-fold increase of biotin in the biotinylation reaction did not result in more efficient labeling of SERP (data not shown). It is noteworthy that in lysates, i.e. when SERP is released, the protein is labeled and binds to SAv quantitatively (Fig. 4A).
Although it is possible that the unlabeled protein in Fig. 4C represents an intraparasite pool of SERP en route to the parasite plasma membrane, we consider this possibility unlikely.
In previous experiments, we were unable to detect levels of SERP within the parasite, unless secretion was actively inhibited by the drug brefeldin A (19,10). It is more likely that, within the vacuole, at least a proportion of the molecule assumes a tight conformation that severely compromises the access of biotin. In no experiments did we detect labeled or unlabeled hemoglobin, the most abundant soluble protein in trophozoite-infected erythrocytes, indicating that the soluble erythrocyte contents had been removed efficiently from permeabilized cells.
The Chaperone PfBiP Associates with Biotin-labeled Proteins-Although in none of our experiments using SLO-permeabilized erythrocytes were we able to detect biotin-labeled aldolase, we analyzed the fraction of SAv-agarose-bound proteins for the presence of several other intraparasite proteins. In the fraction of proteins that were isolated by affinity chromatography on SAv-agarose beads, we consistently detected PfBiP (26), the plasmodial homologue of BiP, a molecular chaperone and endoplasmic reticulum resident protein in eukaryotic cells (Fig.  5A). Because it is possible that some of this protein may not be retained within the parasite's secretory pathway and hence may be transported into the vacuole, we investigated whether PfBiP was biotin-labeled. Permeabilized cells were treated with sulfo-NHS-LC-biotin and subsequently lysed, and PfBiP was immunoprecipitated. The precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose filters. In the first reaction, binding of the anti-PfBiP antiserum was determined using an alkaline phosphatase-conjugated secondary antibody (Fig. 5B, lane 1). In a second reaction, biotin was detected by chemiluminescence using peroxidase-conjugated SAv (Fig. 5B, lane 2). Under these conditions, biotin was undetectable, suggesting that recovery of PfBiP in the fraction of proteins that bound to SAv-agarose was due to an interaction of the chaperone with other biotin-labeled proteins. PfBiP did not bind to SAv-agarose beads in the absence of the biotin (Fig. 5A), ruling out the possibility that the protein directly interacted with SAv or the resin. Most likely, when cell lysates are prepared, intraparasite chaperones such as PfBiP associate with biotinylated proteins bound to SAv-agarose beads and are coisolated. In the subsequent experiments, the protocol was revised to reduce the possibility of mistaking intraparasite chaperones as putative vacuolar resident proteins. Chaperones are released from their substrates in a process that requires the hydrolysis of ATP. Therefore, SAv-agarose beads were washed in the presence of 5 mM ATP before electrophoretic analysis. This step eliminated the co-isolation of PfBiP almost entirely,

FIG. 4. Selective biotin labeling of vacuolar marker proteins.
A, to confirm that the marker proteins can be biotinylated in solution, a soluble protein fraction was prepared and processed as described for Fig. 3A. The fraction of proteins bound to SAv-agarose beads (lanes 1 and 3) and the fraction of unbound proteins (lanes 2 and 4) were analyzed by immunoblotting for the presence of the marker proteins SERP, GBP, and PfALD. B and C, infected erythrocytes were permeabilized with SLO. After complete extraction of the erythrocyte cytosol, the cellular fraction consisting of intact parasites and the intact PV was incubated with sulfo-NHS-LC-biotin and lysed after removal of free biotin. B, proteins were immunoprecipitated using antisera against GBP and SERP, respectively, separated by SDS-PAGE, and transferred to nitrocellulose filters (lanes 1-4). The filters were first probed with antisera that were detected in a color reaction using alkaline phosphatase-conjugated secondary antibodies (lanes 1 and 3). The same filters were reprobed with peroxidase-conjugated SAv, which was subsequently detected by a chemiluminescence reaction (lanes 2 and 4). The incorporation of biotin into aldolase was analyzed in the same way (lanes 5 and 6), except that soluble proteins were separated electrophoretically without prior immunoprecipitation. C, the lysate was reacted with SAv-agarose beads, and the fractions of bound (lanes 1 and 3) and unbound (lanes 2 and 4) proteins were analyzed as described in B for the incorporation of biotin (lanes 1 and 2) and the presence of the respective marker proteins (lanes 3 and 4).
but it had no effect on the isolation of SERP (Fig. 5C) or of other biotin-labeled proteins.
Two-dimensional Gel Electrophoretic Analysis of Biotin-labeled Parasite Proteins-As a prerequisite for the identification of novel vacuolar proteins, IRBCs were labeled metabolically with L-[ 35 S]methionine, permeabilized with SLO, and treated with sulfo-NHS-LC-biotin. After cell lysis, soluble proteins were separated in triplicate on different two-dimensional gels using a pH gradient of 4 -7 in the first dimension. To estimate the relative abundance of proteins in permeabilized erythrocytes, one gel was silver-stained (Fig. 6A), and one gel was stained with Coomassie Blue (Fig. 6B). As expected, silver staining revealed the most complex protein pattern. The most abundant proteins were also detectable using the less sensitive Coomassie Blue stain. To compare biotinylated proteins versus total parasite proteins, proteins of a third gel were transferred to a nitrocellulose filter. The filter was exposed to x-ray films to analyze the pattern of radiolabeled proteins (Fig. 6C). Subsequently, biotin-labeled proteins were detected using alkaline phosphatase-conjugated SAv (Fig. 6D). A comparison of the biotin-labeled proteins and the radiolabeled proteins clearly demonstrates that the pattern of biotin-labeled proteins is less complex and that it does not simply reflect the pattern of the most abundant radiolabeled proteins. In contrast, the most intensely radiolabeled proteins appear not to be biotin-labeled. Considering that cytosolic housekeeping proteins of the parasite, such as enzymes of the glycolytic pathway, are likely to be highly abundant, this result underscores the notion that the incorporation of the biotin is selective. In fact, when a matrixassisted laser desorption ionization time-of-flight analysis was carried out using the two most prominent spots (I and II), they were identified unequivocally as P. falciparum enolase and as P. falciparum ornithine aminotransferase, respectively (data not shown). Biotin-labeled proteins form characteristic strings of individual spots. We attribute this pattern to various degrees of biotin incorporation into the same polypeptide chain. Be-cause each sulfo-NHS-LC-biotin adds a negative charge, molecules with a high incorporation of the derivative are expected to migrate toward the acidic end of the gradient. The shift to the acidic end of the gradient also correlates with a slight increase in molecular size. A detailed comparison reveals 12 prominent spots, circled in Fig. 6, AϪD, which represent highly abundant parasite proteins that are detectable by Coomassie Blue staining. Nine of these spots appear negatively stained in Fig. 6D because of the high local concentration of nonbiotinylated protein. Thirty-nine protein spots were identified that were both biotinylated and radiolabeled and that therefore fulfill the criteria expected for vacuolar proteins. Some of these spots may represent identical polypeptides that are biotinylated to various degrees. In conclusion, the accessibility of nonpermeant biotin derivatives to the vacuolar lumen in permeabilized IRBCs has allowed the identification of candidate vacuolar proteins. These proteins are restricted in number, and they segregate into a distinct two-dimensional pattern. The sequencing of the parasite's genome has almost been completed. This will allow the rapid identification of the genes encoding vacuolar proteins, as a prerequisite for a molecular understanding of the cell biological functions of this unusual compartment in the infected erythrocyte.  4) were analyzed for the presence of PfBiP using a specific antiserum. Lanes 1 and 2, untreated sample; lanes 3 and 4, biotinylated proteins. B, PfBip was immunoprecipitated from a sample of biotin-treated cells, and the precipitate was analyzed by immunoblotting using either the specific antiserum (lane 1) or peroxidase-conjugated SAv (lane 2). C, biotinylated proteins were bound to SAv beads, and the beads were washed with either HEPES buffer (lanes 1 and 2) or HEPES buffer containing 5 mM ATP (lanes 3 and 4). Proteins that remained bound to the beads (lanes 2 and 4) and unbound proteins (lanes 1 and 3) were analyzed for the presence of SERP and PfBiP.
FIG. 6. Protein pattern of putative vacuolar proteins. Proteins in intact infected erythrocytes were metabolically labeled, and cells were permeabilized and treated with biotin. The fraction of soluble proteins, each corresponding to 2 ϫ 10 9 infected erythrocytes, was subjected to two-dimensional gel electrophoresis using a pH gradient of 4 -7 in the first dimension and 12% SDS-PAGE in the second dimension. A, silver-stained gel. B, Coomassie Blue-stained gel. C and D, proteins from a third two-dimensional gel were transferred to a nitrocellulose filter. The filter was first exposed to x-ray film to visualize metabolically labeled parasite proteins (C) and subsequently reacted with alkaline phosphatase-conjugated SAv to visualize biotinylated proteins (D). The most prominent spots that were identified as parasite proteins are marked with circles. Proteins that are both biotinylated and radiolabeled are indicated by Arabic numerals; spots that may represent identical polypeptides at various degrees of biotinylation are marked with lowercase letters. I, P. falciparum enolase; II, P. falciparum ornithine aminotransferase.