Luciferase, When Fused to an N-terminal Signal Peptide, Is Secreted from Transfected Plasmodium falciparum and Transported to the Cytosol of Infected Erythrocytes
2001; Elsevier BV; Volume: 276; Issue: 29 Linguagem: Inglês
10.1074/jbc.m100111200
ISSN1083-351X
AutoresPetra A. Burghaus, Klaus Lingelbach,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoPlasmodium falciparum, a unicellular parasite that causes human malaria, infects erythrocytes where it develops within a vacuole. The vacuolar membrane separates the parasite from the erythrocyte cytosol. Some secreted parasite proteins remain inside the vacuole, and others are transported across the vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent protein and luciferase as reporters in transiently transfected parasites. Because of the higher sensitivity of the enzymatic assay, luciferase was quantified 3 days after transfection, whereas reliable detection of green fluorescent protein required prolonged drug selection. Luciferase was confined to the parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-terminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar content and the erythrocyte cytosol. The results suggest that no specific protein sequences are required for translocation across the vacuolar membrane. The high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involve a rate-limiting step. Plasmodium falciparum, a unicellular parasite that causes human malaria, infects erythrocytes where it develops within a vacuole. The vacuolar membrane separates the parasite from the erythrocyte cytosol. Some secreted parasite proteins remain inside the vacuole, and others are transported across the vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent protein and luciferase as reporters in transiently transfected parasites. Because of the higher sensitivity of the enzymatic assay, luciferase was quantified 3 days after transfection, whereas reliable detection of green fluorescent protein required prolonged drug selection. Luciferase was confined to the parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-terminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar content and the erythrocyte cytosol. The results suggest that no specific protein sequences are required for translocation across the vacuolar membrane. The high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involve a rate-limiting step. parasitophorous vacuolar membrane infected red blood cell green fluorescent protein streptolysin O exported protein 1 of P. falciparum serine-rich protein of P. falciparum relative light units Plasmodium falciparum, the parasite that causes the most severe form of malaria, spends part of its life cycle in human erythrocytes. Here it resides within the so-called parasitophorous vacuole, which is bound by the parasitophorous vacuolar membrane (PVM).1 The vacuole constitutes a separate compartment in the infected red blood cell (iRBC) that is distinct from the cytosol of the parasite and from the cytosol of the erythrocyte, respectively (1Lingelbach K. Joiner K.A. J. Cell Sci. 1998; 111: 1467-1475PubMed Google Scholar). Most proteins secreted from P. falciparum are not released into an extracellular space but are transported to various destinations within the iRBC: the parasitophorous vacuole or the erythrocyte cytosol. Membrane-bound proteins are found in the PVM and erythrocyte plasma membrane (2Foley M. Tilley L. Int. J. Parasitol. 1998; 28: 1671-1680Crossref PubMed Scopus (38) Google Scholar, 3Cooke B.M. Wahlgren M. Coppel R.L. Parasitol. Today. 2000; 16: 416-420Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). It is completely unknown which information within a polypeptide chain determines whether a protein is transported across the PVM. Recently we described experimental evidence for a transport pathway that involves the release of secreted parasite proteins into the vacuolar space and translocation across the PVM in a subsequent step (4Ansorge I. Benting J. Bhakdi S. Lingelbach K. Biochem. J. 1996; 315: 307-314Crossref PubMed Scopus (162) Google Scholar). This model infers that a sorting mechanism must operate within the vacuole that discriminates between vacuolar resident proteins and proteins destined for a location beyond the PVM (5Lingelbach K. Ann. Trop. Med. Parasitol. 1997; 91: 543-549Crossref PubMed Scopus (22) Google Scholar). Sorting could involve two different principles: the retention of vacuolar proteins or, alternatively, the recognition of protein signals that mediate translocation across the PVM. A widely used experimental approach for the identification of protein targeting and sorting signals in eukaryotic cells is the fusion of putative signal sequences to reporter proteins and their subsequent localization in the transfected cell. Although transfection of P. falciparum blood stages has been established during the last 5 years (6Wu Y. Sifri C.D. Lei H.H. Su X.Z. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 973-977Crossref PubMed Scopus (320) Google Scholar, 7Waters A.P. Thomas A.W. van Dijk M.R. Janse C.J. Methods (San Diego, Calif.). 1997; 13: 134-147Crossref PubMed Scopus (111) Google Scholar), the extremely low transfection efficiency has remained a major obstacle to a systematic analysis of putative signal sequences in this organism. Because the amount of a recombinant protein in a culture of transfected parasites is minute, either a very sensitive detection assay is required or parasite lines have to be selected for several months that stably express the reporter protein (8Wu Y. Kirkman L.A. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1130-1134Crossref PubMed Scopus (339) Google Scholar, 9Crabb B.S. Cowman A.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7289-7294Crossref PubMed Scopus (237) Google Scholar). Furthermore, it is essential that the reporter protein can be localized reliably and reproducibly in each compartment of the iRBC and that its concentration can be precisely quantified therein. In this study we compare the green fluorescent protein (GFP) of Aequoria victoria, the luciferase of Photinus pyralis, and an epitope of the human c-myc oncogene (10Chalfie M. Yuan T. Euskirchen G. Ward W.W. Prasher D.C. Science. 1991; 263: 802-805Crossref Scopus (5540) Google Scholar, 11de Wet J.R. Wood K.V. Deluca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2482) Google Scholar, 12Chang H. Bush D.R. J. Biol. Chem. 1997; 272: 30552-30557Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) for their suitability as reporters for protein transport in P. falciparum. GFP and the c-Myc epitope are detected by fluorescence microscopy of transfected cells. The enzymatic activity of luciferase is determined in cell lysates; hence, a selective fractionation of iRBCs is essential. Previously we used the treatment of iRBCs with saponin and the bacterial pore-forming protein streptolysin O (SLO) to investigate the distribution of parasite proteins within the erythrocyte cytosol and vacuolar space (4Ansorge I. Benting J. Bhakdi S. Lingelbach K. Biochem. J. 1996; 315: 307-314Crossref PubMed Scopus (162) Google Scholar). Thus the use of an enzymatically active reporter in combination with the fractionation of iRBCs should allow for the quantification of luciferase activity in different compartments of the iRBC, namely the parasite, vacuolar space, and erythrocyte cytosol. When luciferase was fused to the previously characterized N-terminal secretory signal sequence of the plasmodial exported protein 1 (EXP1), it was secreted from the parasite and recovered in the vacuolar space and erythrocyte cytosol. This suggests that translocation across the PVM does not involve protein sequences specific for secreted parasite proteins. Luciferase and GFP were expressed from plasmids pHLH1 or pHRP-GFPM2, kindly provided by T. E. Wellems and K. Haldar, respectively (6Wu Y. Sifri C.D. Lei H.H. Su X.Z. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 973-977Crossref PubMed Scopus (320) Google Scholar, 13van Wye J.D. Haldar K. Mol. Biochem. Parasitol. 1997; 87: 225-229Crossref PubMed Scopus (42) Google Scholar). The plasmid pDTTg23 coding for dihydrofolate reductase-thymidylate synthase of Toxoplasma gondii that confers pyrimethamine resistance to P. falciparum was provided by Y. Wu and T. E. Wellems. All these plasmids contain the same control elements for transcription: the 5′-flanking regions of the gene encoding the histidine-rich protein 3 and the 3′-flanking region of the gene encoding the histidine-rich protein 2, respectively. Plasmid pHluc-3 m, which encodes the c-Myc epitope fused to the C terminus of luciferase in plasmid pHLH1, was constructed by polymerase chain reaction as follows: using the primers 5′-GCT GAA TTG GAA TCG ATA and 5′-GGC CCA AGC TTA TAA ATC TTC CTC ACT TAT TAA TTT CTG TTC AGA TCT CAA TTT GGA CTT TCC GCC CTT GG and pHLH1 as template, a 345-base pair DNA fragment was synthesized that comprises the 3′ region of theluciferase gene downstream of the endogenousClaI restriction site, the sequence coding for the epitope EQKLISEEDL, and a HindIII site. Restriction sites are underlined. The ClaI-HindIII fragment was excised from plasmid pHLH1 and replaced by the polymerase chain reaction product. Plasmid pHx1luc, which codes for the 26 N-terminal amino acids of EXP1 fused to the N terminus of luciferase, was constructed as follows: the 5′ sequence of the exp1 gene was amplified from plasmid pGEM3/exp1 (14Guenther K. Tuemmler M. Arnold H.H. Ridley R. Goman M. Scaife J.G. Lingelbach K. Mol. Biochem. Parasitol. 1991; 46: 149-158Crossref PubMed Scopus (102) Google Scholar) using the primers 5′-GG CTG CAG AAA ATC TTA TCA GTA TTT TTT C and 5′-GG CGC ATG CAT TTT GTT TGT TTT TTC GGC, which contain a PstI or NsiI site (underlined), respectively. After digestion with PstI andNsiI the polymerase chain reaction product was ligated into the NsiI site of pHLH1. The P. falciparum isolate FCBR was cultured in RPMI 1640 medium (Life Technologies, Inc.) containing 10% human plasma and human erythrocytes of blood group A+ (Marburg Blood Bank) following standard procedures (15Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6219) Google Scholar). Ring-stage parasites of a parasitemia of 10–15% were electroporated with 30–100 µg of plasmid DNA as described by Wuet al. (6Wu Y. Sifri C.D. Lei H.H. Su X.Z. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 973-977Crossref PubMed Scopus (320) Google Scholar). For the assessment of transient expression, trophozoite-stage parasites were harvested 3 days after transfection. To generate stable recombinant parasites, co-transfection was carried out with either pHLH1 or pHRP-GFPM2 together with pDTTg23 in a molar ratio of 9:1. Selection was carried out essentially as described in Ref. 8Wu Y. Kirkman L.A. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1130-1134Crossref PubMed Scopus (339) Google Scholar. 100 ng/ml pyrimethamine was added to the culture medium from days 3 to 8 and then reduced to 15 ng/ml. Daily blood smears were screened for parasitized erythrocytes. iRBCs were enriched to parasitemias of >60% by gel flotation (16Pasvol G. Wilson R.J.M. Smally M.E. Brown J. Ann. Trop. Med. Parasitol. 1978; 72: 87-88Crossref PubMed Scopus (302) Google Scholar). Saponin treatment was carried out as described previously (4Ansorge I. Benting J. Bhakdi S. Lingelbach K. Biochem. J. 1996; 315: 307-314Crossref PubMed Scopus (162) Google Scholar). Briefly, parasites were released from host cells by treatment with 0.1% saponin (grade pure, Serva) in phosphate-buffered saline for 10 min on ice and separated by centrifugation in a microcentrifuge at 16,000 ×g for 2 min. The pellet contained intact parasites and membranes. The supernatant was centrifuged again for 25 min at 16,000 × g, and the small white pellet was discarded. The supernatant consisted of soluble proteins of the parasitophorous vacuole and erythrocyte cytosol. For the localization of proteins inside the parasite, fractionation of iRBCs was carried out with higher concentrations of saponin ranging from 0.2 to 2%. Samples were processed as described above and analyzed for the distribution of two cytosolic parasite proteins, namely aldolase and lactate dehydrogenase, for the endoplasmic reticulum resident protein PfBiP and for luciferase as detailed below. For selective permeabilization of red blood cell membranes with SLO, 1–2 × 108 iRBCs were incubated with SLO (kindly provided by S. Bhakdi) in RPMI 1640 medium at room temperature for 6 min at a concentration of 3–4 hemolytic units for 1 × 108 iRBCs (4Ansorge I. Benting J. Bhakdi S. Lingelbach K. Biochem. J. 1996; 315: 307-314Crossref PubMed Scopus (162) Google Scholar). Samples were centrifuged at 4000 ×g for 5 min. After separation the supernatant was recentrifuged as described above. The pellet contained intact parasites, the vacuolar content, and membranes. The supernatant contained the erythrocyte cytosol. All supernatants were kept on ice, and pellets were washed twice with phosphate-buffered saline, frozen, and then thawed in 2 volumes of parasite lysis buffer (25 mm Tris-phosphate, pH 7.8, 2 mm dithiothreitol, 2 mm EDTA, 10% glycerol, and 1% Triton X-100) containing Complete™ protease inhibitor mixture prepared according to manufacturer instructions (Sigma). Antibodies to plasmodial aldolase and the vacuolar resident parasite-encoded serine-rich protein (SERP) have been described earlier (4Ansorge I. Benting J. Bhakdi S. Lingelbach K. Biochem. J. 1996; 315: 307-314Crossref PubMed Scopus (162) Google Scholar). A recombinant protein comprising 125 amino acids (Leu-259 to Phe-375) of P. falciparum PfBiP (35The Plasmodium Genome Database Collaborative Nucleic Acids Res. 2001; 29: 66-69Crossref PubMed Google Scholar) was produced in Escherichia coli from the plasmid vector pJC45 (36Schluter A. Wiesgigl M. Hoyer C. Fleischer S. Klaholz L. Schmetz C. Clos J. Biochim. Biophys. Acta. 2000; 1491: 65-74Crossref PubMed Scopus (42) Google Scholar) and used for the immunization of a rabbit. The specificity of the serum was tested by immunoblotting. A protein of the expected molecular mass of 75 kDa was recognized in lysates of P. falciparumiRBCs but not in noninfected cells. iRBCs were concentrated by gel flotation (16Pasvol G. Wilson R.J.M. Smally M.E. Brown J. Ann. Trop. Med. Parasitol. 1978; 72: 87-88Crossref PubMed Scopus (302) Google Scholar). For the detection of GFP fluorescence, iRBCs were spread onto microscope slides and analyzed immediately using a wavelength of 488 nm for excitation and 530 nm for emission. For the detection of the c-Myc tag iRBCs were fixed with methanol/acetone (1:9 (v/v)), incubated first with anti-c-Myc monoclonal antibody 9E10 (17Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2166) Google Scholar), and then incubated with a fluorescein-labeled anti-mouse IgG (Jackson ImmunoResearch Laboratories) following standard procedures (18Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). For the detection of luciferase activity, iRBCs were fractionated with either SLO or saponin and separated into a pellet and a supernatant fraction. The hemoglobin content of each fraction was assessed photometrically at 412 nm after 500-fold dilution. Reagents for the luciferase assay were purchased from Promega, and a Lumat type LB9501 (Berthold) was used to measure the enzyme activity as relative light units (RLU) in pellets and in supernatants of fractionated iRBCs. 100 µl of substrate solution was automatically injected into a 10-µl aliquot of the sample and chemiluminescence-measured for 60 s at room temperature. Background chemiluminescence was measured (ranging from 50 to 100 RLU) before luciferase activity in the samples was determined. Background values were subsequently subtracted from the reading obtained for each sample. Recombinant P. pyralisluciferase produced in E. coli was purchased from Promega, diluted to 10−8–10−10 g/liter in luciferase storage buffer (Promega), and used to generate a standard curve of relative light units versus luciferase concentration in parasite lysis buffer. To assess the effect of hemoglobin and possible other components contained within iRBCs on the determination of luciferase activity, different numbers of nontransfected iRBCs were treated with either SLO or saponin. The hemoglobin content of the supernatant that contained the released proteins was determined photometrically and recorded. The defined amounts of recombinant luciferase were added to each supernatant, and its activity was determined in RLU. Standard curves were plotted for each hemoglobin concentration as for lysis buffer and used to calculate the amount of luciferase present in samples prepared from transfected iRBCs. The total protein content of samples was determined by Bradford assay (19Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar) following manufacturer protocols (Bio-Rad). RLU were either normalized to total protein content of isolated parasites or to total volume of a sample that contained a fraction of iRBCs. Cytosolic plasmodial lactate dehydrogenase was monitored as described in Ref. 20Oduola A.M.J. Omitowoju G.O. Sowunmi A. Makler M.T. Falade C.O. Kyle D.E. Fehintola F.A. Ogundahunsi O.A.T. Piper R.C. Schuster B.G. Milhous W.K. Exp. Parasitol. 1997; 87: 283-289Crossref PubMed Scopus (58) Google Scholarusing the MalStat™ assay (Flow, Inc.). For the detection of the vacuolar resident SERP, the endoplasmic reticulum resident PfBiP, and cytosolic parasite aldolase in different fractions obtained after SLO or saponin treatment of iRBCs, aliquots of the supernatant and pellet fraction, each corresponding to 1 × 106 iRBCs for aldolase, 3 × 106 iRBCs for PfBiP, or 1 × 107 iRBCs for SERP, were dissolved in SDS sample buffer, electrophoresed through 10% SDS-polyacrylamide gels, and transferred to nitrocellulose. Blots were probed with specific rabbit antisera at a dilution of 1:500 and then incubated with alkaline phosphatase-conjugated anti-rabbit IgG adsorbed with human serum proteins (Sigma). Blots were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphoshate (Sigma) following standard procedures (18Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). The DNA constructs analyzed in this study are shown schematically in Fig.1. In initial experiments we compared the detectability of GFP and luciferase as reporters in transiently transfected parasites. P. falciparum was transfected with the plasmid pHRP-GFPM2 or pHLH1, and parasites were analyzed for the expression of the recombinant genes 24–72 h after transfection. Analysis of iRBCs transfected with the plasmid pHRP-GFPM2 revealed no fluorescent parasites. Consequently, the following transfection conditions varied by: (i) electroporation settings (21Fidock D.A. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10931-10936Crossref PubMed Scopus (414) Google Scholar), (ii) the amount of plasmid DNA used in a single transfection, ranging from 30 to 100 µg, and (iii) the numbers of iRBCs, ranging from 1 × 108 to 3 × 108 iRBCs. None of these conditions resulted in detectable levels of GFP. Transfections were conducted in parallel with the plasmid pHLH1, and luciferase activity was determined in parasite lysates, which were obtained after treatment of iRBCs with saponin. Reproducibly, luciferase activity was detectable in transfected but not in mock-transfected parasites. When transfections were carried out using either larger amounts of DNA or larger numbers of iRBCs, chemiluminescence consistently increased, at most 5-fold. In subsequent experiments we routinely used 75 µg of DNA and 2 × 108 iRBCs. It is noteworthy that both genes contained an identical 5′- and 3′-flanking region including the transcriptional control elements. To elucidate the reason for the failure to detect GFP in transiently transfected parasites, we intended to produce stable transfectants by co-transfection of iRBCs with either pHRP-GFPM2 or pHLH1 and pDTTg23, a plasmid that encodes T. gondiidihydrofolate reductase-thymidylate synthase and thus confers resistance to pyrimethamine (8Wu Y. Kirkman L.A. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1130-1134Crossref PubMed Scopus (339) Google Scholar). From day 5, pyrimethamine was added to the cultures, and daily blood smears confirmed elimination of the parasites by the drug. 20 days after infection, parasitemia began to rise again, and cultures were maintained like wild-type parasites. As shown in Fig. 2, 35 days after transfection, luciferase activity in the parasite lysate was three orders of magnitude higher than after 3 days. After this prolonged period of selection, GFP was also detectable by fluorescence microscopy in parasites transfected with pHRP-GFPM2. Bright green parasites of all developmental stages were clearly visible, amounting to a rate of 24% of the infected erythrocytes at day 30. When pyrimethamine pressure was applied further, the expression of both reporter genes declined. Most likely, the episomal replication of pDTTg23, the plasmid that carries the selectable marker, was favored. Alternatively, it is possible that spontaneous mutations in the endogenous plasmodial dihydrofolate reductase gene may have occurred, thus conferring resistance to pyrimethamine (22Peterson D.S. Walliker D. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9114-9118Crossref PubMed Scopus (541) Google Scholar). For about 3 weeks, the amount of recombinant protein produced was sufficient for analytical purposes. Considering that the regions flanking the luciferase and gfpgenes are identical and that transfections were carried out in parallel, the most plausible explanation for the early detection of recombinant luciferase remains that the enzymatic assay is far more sensitive than the visual analysis of GFP by fluorescence microscopy. This interpretation was corroborated using a chimeric construct in which the c-Myc epitope was fused to the C-terminal end of luciferase. Although luciferase activity was detectable in transfected parasites after 3 days, no signal was obtained in the immunofluorescence analysis using a specific monoclonal antibody to the c-Myc tag (data not shown). The results described above clearly demonstrate that luciferase is the preferable reporter when it is required to analyze a large number of different DNA constructs, which is necessary for the identification of protein targeting and sorting signals. Apart from being detectable at low concentrations, the enzymatic activity can be quantified precisely. Luciferase carries at its C terminus the sequence SKL, which functions as a peroxisomal import sequence in higher eukaryotes. Therefore we first investigated whether the recombinant protein in transfected parasites segregated from a characterized cytosolic plasmodial protein, parasite lactate dehydrogenase, that can also be quantified by an enzymatic assay. Infected erythrocytes, 3 days after transfection, were treated with increasing concentrations of saponin and separated into a pellet fraction and a supernatant. Both fractions were analyzed for the activity of parasite lactate dehydrogenase and luciferase (Fig.3). Consistent with previous reports (4Ansorge I. Benting J. Bhakdi S. Lingelbach K. Biochem. J. 1996; 315: 307-314Crossref PubMed Scopus (162) Google Scholar,23Saliba K.J. Kirk K. J. Biol. Chem. 1999; 274: 33213-33219Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), treatment of iRBCs with low saponin concentrations resulted in almost complete hemolysis, but up to a concentration of 0.15% saponin less than a 10% release of parasite lactate dehydrogenase and luciferase from the parasite was observed. At these concentrations more than 95% of the erythrocyte hemoglobins were released, demonstrating almost complete lysis of the infected host cell. At higher concentrations of saponin, the release of enzymatic activity shows essentially the same profile for both proteins, suggesting that the majority of luciferase was localized cytosolically (Fig. 3). In the following experiments, the distribution of luciferase in transfected parasites that had been selected for high expression by prolonged treatment with pyrimethamine (Fig. 2) was determined. In these analyses an independent cytosolic protein, parasite aldolase, and a marker for a compartmentalized protein, PfBiP, were included. Infected erythrocytes were treated with different saponin concentrations applying a more narrow range of 0.2–0.8% saponin. Parasite aldolase and PfBiP were detected by immunoblotting, and recombinant luciferase was quantified by enzymatic activity (Fig. 4). Release of aldolase was detectable after the treatment of iRBCs with 0.2% saponin, and complete release was observed at 0.5% saponin. In contrast, release of low amounts of PfBiP required saponin concentrations of >0.5%. The amounts of the respective proteins in the pellet fractions decreased accordingly (data not shown). The segregation of luciferase resembled that of aldolase. After lysis with 0.2% saponin, a significant amount of luciferase activity was released, and a steep increase of activity was detected at the next higher concentrations of saponin. Activity in the pellets showed a sharp decline from 0.2 to 0.6% saponin. In conclusion, these results argue against a localization of the recombinant protein within a subcellular compartment of the parasite.Figure 4Differential solubility of recombinant luciferase and the endoplasmic reticulum resident protein PfBiP.Erythrocytes infected with parasites, co-transfected with pHLH1 and pDTTg23, and selected with pyrimethamine were treated with increasing concentrations of saponin. The samples were centrifuged, and the supernatants containing released proteins were analyzed by immunoblotting for the distribution of parasite aldolase (A) and PfBiP (B). C, luciferase activity was determined in the pellet fraction (closed circles) and the supernatant (open circles) and plotted as RLU against saponin concentration.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The detection of luciferase as a reporter protein in various compartments of the infected erythrocyte, namely within the parasite, the vacuolar space and the erythrocyte cytosol depends on (i) an accurate subcellular fractionation and (ii) an accurate quantification of luciferase activity that takes into account possible quenching effects in different subcellular fractions. In fact, considerable quenching of luciferase activity by hemoglobin has been reported recently (24Colin M. Moritz S. Schneider H. Capeau J. Coutelle C. Brahimi-Horn M.C. Gene Ther. 2000; 7: 1333-1336Crossref PubMed Scopus (62) Google Scholar). An experimental approach that allows a selective fractionation of iRBCs is based on the different lytic properties of SLO and saponin (4Ansorge I. Benting J. Bhakdi S. Lingelbach K. Biochem. J. 1996; 315: 307-314Crossref PubMed Scopus (162) Google Scholar). SLO forms pores of 30 nm in diameter within the erythrocyte plasma membrane, but it does not insert into the PVM. After the treatment of iRBCs with SLO and subsequent centrifugation, the supernatant contains soluble proteins of the erythrocyte cytosol only. Saponin disintegrates both the erythrocyte plasma membrane and PVM. Soluble proteins contained in both compartments are released into the supernatant fraction. Routinely, complete disintegration of the erythrocyte plasma membrane is determined by the complete release of hemoglobin. The integrity of the parasite plasma membrane and the PVM is determined by the segregation of marker proteins contained within the parasite cytosol and vacuolar space, respectively. Infected erythrocytes were transfected with either pHLH1, which encodes luciferase, or pHx1luc, which codes for a chimeric luciferase that contains the N-terminal secretory signal sequence of the parasite protein EXP1. Routinely, transfections with a particular plasmid were carried out in triplicate and processed individually in parallel. The cells were harvested 72 h after transfection and fractionated by treatment with either saponin or SLO. In each sample, complete disintegration of the erythrocyte plasma membrane was monitored by quantification of hemoglobin in the supernatant and the respective pellet fractions. Consistently more than 97% of total hemoglobin was released upon treatment. To assess the integrity of the vacuolar membrane and the parasite plasma membrane in each sample, the segregation of two parasite marker proteins was determined in each experiment. Parasite aldolase is a cytosolic protein within the parasite, and SERP is localized within the vacuolar space. Fig.5 shows exemplary results of these controls from two separate experiments conducted with iRBCs transfected with pHx1luc. After permeabilization with SLO, SERP was found predominantly in the pellet fraction (Fig. 5 A, lanes 1 and 2) with only minor traces in the supernatant (Fig. 5 A, lanes 3 and 4). Treatment with saponin resulted in an opposite distribution of the vacuolar
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