149
Molecular and Biochemical Parasitology, 46 (1991) 149-158 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100048Z MOLBIO 01519
An exported protein of Plasmodiumfalciparumis synthesized as an integral membrane protein Kathrin G0nther 1, M e i k e TiJmmler ~, Hans-Henning Arnold 1, Robert Ridley 2, Michael G o m a n 2, John G. Scaife 2 and Klaus Lingelbach 1 ~Fraunhofer Institute of Toxicology, Hamburg, F.R.G.; and 2Department of Molecular Biology, University of Edinburgh, King' s Buildings, Edinburgh, U.K. (Received 13 August 1990; accepted 27 November 1990)
Exp- 1 is an antigen of Plasmodiumfalciparum which is transported from the parasite cell to the membrane of the parasitophorous vacuole and to membranouscompartments in the erythrocyte. To investigate how this protein is transported, we studied the synthesis and membrane translocation of exp-I in a cell-free system. The protein was translocated into canine pancreatic microsomes. Its Nterminal half was thus protected from proteinase K digestion, suggesting that exp-1 is an integral membrane protein with its N-terminus facing the lumen of the microsomes. This conclusion has been confirmed in vivo. In parasitized erythrocytes, exp-1 is membrane-associated and resistant to extraction with alkali, as would be expected for an integral membrane protein. Moreover, using segment-specific monoclonal antibodies, we have shown that here again the N-terminus of exp-1 faces the inside of vesicles, inaccessible to proteases, whereas the C-terminus is degraded. We conclude that exp- 1 is an integral membrane protein and infer that it is transported by vesicles from the parasite to a compartment in the host cell cytoplasm. Key words: Plasmodiumfalciparum; Membrane protein; Intracellular transport; Secretion; Segment-specific'antibodies
Introduction
Plasmodiumfalciparumis an intracellular parasite of the human red blood cell (RBC). The parasite resides within the parasitophorous vacuole which is formed during merozoite invasion. The membrane of the parasitophorous vacuole (PVM) separates the parasite from the cytoplasm of the host cell. Many proteins are exported from the parasite cell and some of them are subsequently found associated with membranous structures in the cytoplasm of the RBC and with the erythrocyte membrane Correspondence address: Klaus Lingelbach, Fraunhofer Institute of Toxicology; Grindelalle e 117, D-2000 Hamburg 13, F.R.G.
Abbreviations: ER, endoplasmic reticulum; HPRT, hypoxanthine phosphoribosyl transferase; PPM, parasite plasma membrane; PV, parasitophorous vacuole; PVM, PV membrane; RBC, red blood cell; RBCM, RBC membrane; SRP, signal recognitionparticle.
(RBCM) [1,2]. Proteins destined for locations within the RBC or the RBCM have to be released from the parasite, and they have to cross the PVM. The molecular mechanisms and protein signals required for translocation of parasite proteins across multiple membranes and their intra-erythrocytic transport are not well understood. To unravel this aspect of host-parasite interaction and to follow the different steps of protein transport within the host, we have begun to study marker proteins of known sequences found in defined locations within the host cell. Exp- 1, also called QF 116, was first identified as a 23-kDa blood stage antigen by immunoreaction with a monoclonal antibody [3]. Isolation and sequencing of cDNA clones encoding exp- 1 revealed a protein which contained a putative N-terminal signal sequence for secretion and a segment of hydrophobic amino acid residues which has been proposed to act as stop-transfer sequence [4,5]. The 5.1 epitope which is recognized by the monoclonal
150
antibody mAb5.1 is located C-terminally to the hydrophobic segment [6]. In the infected RBC, extensive immunogold labeling studies have shown that exp-1 is found predominantly in the PVM and in vesicle-like structures in the RBC cytoplasm [6-8]. Myristilation of exp-1 has recently been reported [9]. Exp- 1 is thus one of the best studied proteins exported by the parasite. The work presented here uses a heterologous cell-free system and studies on infected RBCs to show that exp-1 is an integral membrane protein, with its N-terminus oriented towards the lumen of membrane vesicles isolated from parasitized erythrocytes. Knowledge of the orientation of this protein in the membrane will contribute to our understanding of its function in the parasite-infected RBC. In addition, exp-1 provides a model system for further studies on the mechanism of parasite protein export.
tracted sequentially with high salt and alkali by a modification of the procedure of Fuijiki et al. [ 14]. Briefly, 150 ~tl of high salt buffer (50 mM Hepes, pH 7.5/0.5 M KC1/5 mM DTT/50 mM lysine/3 mM MgC12) were added to a 50 ~tl translation/ translocation reaction and centrifuged in an Eppendoff centrifuge at 4°C for 15 min. The microsomal pellet was extracted once more as described above, then resuspended in 50 ~tl of 0.1 M NazCO3, pH 11, and left on ice for 15 min. Subsequently, it was centrifuged through 0.25 M sucrose/0.1 M Na2CO3, pH 11 for 5 min in a Beckman Airfuge at 24 psi. Proteins from the combined supernatants of the salt extractions and from the supernatant of the alkali extraction were precipitated in 10% trichloroacetic acid (TCA) and solubilized in SDS sample buffer for SDS-polyacrylamide gel electrophoresis (SDSPAGE). The pellet of extracted microsomes was solubilized in 25 ~tl sample buffer.
Materials and Methods Enzymes for DNA modification and reagents for in vitro transcription were from Boehringer Mannhelm. [L- S]Methlonme was obtained from Amersham, and horseradish peroxidase-conjugated second antibodies from Dako. Canine pancreatic microsomes and signal recognition particle (SRP) were isolated according to standard procedures [10,11]. Schizont-infected RBC from P. falciparum isolate FCBR (Columbia) were obtained as described [ 12] and stored frozen. •
35
•
•
Cell-free biosynthesis of parasite proteins and membrane translocation. The experimental procedures for in vitro transcription of cloned DNA sequences, the translation of transcripts in wheatgerm lysate, and the co-translational translocation of polypeptides across pancreatic microsomes have recently been described in detail [ 13 ].
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Proteins were separated on 7.5% and 15% Laemmli-type SDS-polyacrylamide ~slS, respectively. Proteins radiolabeled with [L]methionine were visualized by fluorography using Na-salicylate.
Carbonate extraction.
Following the translation/translocation reaction, microsomes were ex-
Membrane preparation of parasitized red blood cells• Schizont-infected RBC were collected by centrifugation and frozen. The pellet was resuspended in ice-cold phosphate-buffered saline containing a mixture of protease inhibitors (1 ~tg/ml each ofpepstatin A, leupeptin, antipain, elastatinal, trasylol, Na-EDTA, and soybean trypsin inhibitor). During this process cells were lysed. An equivalent of 1.5 x 10 erythrocytlc stage parasites was solubllized in SDS sample buffer. For extraction with carbonate, approximately 6 × 108 parasites were extracted with high salt buffer and Na2CO3 as described above. For protease digestion, proteinase K was added to the lysate at a concentration of 1.8 mg ml -~, either in the absence or in the presence of 0.3% Triton X-100 and left on ice for 1 h. The reaction was stopped by adding phenylmethylsulfonylfluoride to a final concentration of 2 mg ml , proteins were precipitated in 10% TCA, and solubilized in SDS sample buffer. 8
•
•
•
Western blotting analysis.
•
.
•
-1
Proteins separated by SDS-PAGE were transferred to nitrocellulose filters [15]. Subsequently the filters were processed, incubated with antibodies, and immunostained using horseradish peroxidase-conjugated second antibodies, following standard procedures [ 16].
151 Results Construction ofpGem3/exp-1. Recent studies on the serine-rich protein, SERP, of P. falciparum indicate the value of a cell-free system for studying membrane translocation of parasite proteins [ 13]. We have applied this approach to the exp-1 protein. To generate transcripts of the gene for translocation in vitro, a 600-bp cDNA [4] was inserted into the EcoRI site of the polylinker region of pGem3 (Fig. 1). For in vitro transcription, the plasmid was linearized at the unique SmaI site and transcribed from the T7 promoter. The precursor form of exp-1 is processed in the presence of SRP and microsomes. In eukaryotic cells, secreted proteins and proteins of the plasma membrane require the cytosolic signal recognition particle (SRP) for their translocation across the membrane of the endoplasmic reticulum (ER). This particle first interacts with the signal sequence of the nascent polypeptide chain and subsequently with its receptor in the membrane of the ER. In gen-
era1, translocation occurs cotranslationally and is accompanied by cleavage of an N-terminal signal sequence from the precursor form (for reviews, see refs. 17 and 18). The main elements of this pathway can be studied in a reconstituted cell-free system, which also provides an approach to investigate membrane targetting of parasite proteins. When exp-1 was expressed in vitro, a major translation product of an apparent Mr of 24000 was synthesized (Fig. 2, lane 1). If translation was carried out in the presence of microsomal membranes only, the same primary translation product was obtained (Fig. 2, lane 2), and it was susceptible to digestion with proteinase K (Fig. 2, lane 3,4). Synthesis of exp-1 in the presence of microsomes and SRP resulted in an overall increased translational efficiency, including the synthesis of proteins
Molecular and Biochemical Parasitology, 46 (1991) 149-158 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100048Z MOLBIO 01519
An exported protein of Plasmodiumfalciparumis synthesized as an integral membrane protein Kathrin G0nther 1, M e i k e TiJmmler ~, Hans-Henning Arnold 1, Robert Ridley 2, Michael G o m a n 2, John G. Scaife 2 and Klaus Lingelbach 1 ~Fraunhofer Institute of Toxicology, Hamburg, F.R.G.; and 2Department of Molecular Biology, University of Edinburgh, King' s Buildings, Edinburgh, U.K. (Received 13 August 1990; accepted 27 November 1990)
Exp- 1 is an antigen of Plasmodiumfalciparum which is transported from the parasite cell to the membrane of the parasitophorous vacuole and to membranouscompartments in the erythrocyte. To investigate how this protein is transported, we studied the synthesis and membrane translocation of exp-I in a cell-free system. The protein was translocated into canine pancreatic microsomes. Its Nterminal half was thus protected from proteinase K digestion, suggesting that exp-1 is an integral membrane protein with its N-terminus facing the lumen of the microsomes. This conclusion has been confirmed in vivo. In parasitized erythrocytes, exp-1 is membrane-associated and resistant to extraction with alkali, as would be expected for an integral membrane protein. Moreover, using segment-specific monoclonal antibodies, we have shown that here again the N-terminus of exp-1 faces the inside of vesicles, inaccessible to proteases, whereas the C-terminus is degraded. We conclude that exp- 1 is an integral membrane protein and infer that it is transported by vesicles from the parasite to a compartment in the host cell cytoplasm. Key words: Plasmodiumfalciparum; Membrane protein; Intracellular transport; Secretion; Segment-specific'antibodies
Introduction
Plasmodiumfalciparumis an intracellular parasite of the human red blood cell (RBC). The parasite resides within the parasitophorous vacuole which is formed during merozoite invasion. The membrane of the parasitophorous vacuole (PVM) separates the parasite from the cytoplasm of the host cell. Many proteins are exported from the parasite cell and some of them are subsequently found associated with membranous structures in the cytoplasm of the RBC and with the erythrocyte membrane Correspondence address: Klaus Lingelbach, Fraunhofer Institute of Toxicology; Grindelalle e 117, D-2000 Hamburg 13, F.R.G.
Abbreviations: ER, endoplasmic reticulum; HPRT, hypoxanthine phosphoribosyl transferase; PPM, parasite plasma membrane; PV, parasitophorous vacuole; PVM, PV membrane; RBC, red blood cell; RBCM, RBC membrane; SRP, signal recognitionparticle.
(RBCM) [1,2]. Proteins destined for locations within the RBC or the RBCM have to be released from the parasite, and they have to cross the PVM. The molecular mechanisms and protein signals required for translocation of parasite proteins across multiple membranes and their intra-erythrocytic transport are not well understood. To unravel this aspect of host-parasite interaction and to follow the different steps of protein transport within the host, we have begun to study marker proteins of known sequences found in defined locations within the host cell. Exp- 1, also called QF 116, was first identified as a 23-kDa blood stage antigen by immunoreaction with a monoclonal antibody [3]. Isolation and sequencing of cDNA clones encoding exp- 1 revealed a protein which contained a putative N-terminal signal sequence for secretion and a segment of hydrophobic amino acid residues which has been proposed to act as stop-transfer sequence [4,5]. The 5.1 epitope which is recognized by the monoclonal
150
antibody mAb5.1 is located C-terminally to the hydrophobic segment [6]. In the infected RBC, extensive immunogold labeling studies have shown that exp-1 is found predominantly in the PVM and in vesicle-like structures in the RBC cytoplasm [6-8]. Myristilation of exp-1 has recently been reported [9]. Exp- 1 is thus one of the best studied proteins exported by the parasite. The work presented here uses a heterologous cell-free system and studies on infected RBCs to show that exp-1 is an integral membrane protein, with its N-terminus oriented towards the lumen of membrane vesicles isolated from parasitized erythrocytes. Knowledge of the orientation of this protein in the membrane will contribute to our understanding of its function in the parasite-infected RBC. In addition, exp-1 provides a model system for further studies on the mechanism of parasite protein export.
tracted sequentially with high salt and alkali by a modification of the procedure of Fuijiki et al. [ 14]. Briefly, 150 ~tl of high salt buffer (50 mM Hepes, pH 7.5/0.5 M KC1/5 mM DTT/50 mM lysine/3 mM MgC12) were added to a 50 ~tl translation/ translocation reaction and centrifuged in an Eppendoff centrifuge at 4°C for 15 min. The microsomal pellet was extracted once more as described above, then resuspended in 50 ~tl of 0.1 M NazCO3, pH 11, and left on ice for 15 min. Subsequently, it was centrifuged through 0.25 M sucrose/0.1 M Na2CO3, pH 11 for 5 min in a Beckman Airfuge at 24 psi. Proteins from the combined supernatants of the salt extractions and from the supernatant of the alkali extraction were precipitated in 10% trichloroacetic acid (TCA) and solubilized in SDS sample buffer for SDS-polyacrylamide gel electrophoresis (SDSPAGE). The pellet of extracted microsomes was solubilized in 25 ~tl sample buffer.
Materials and Methods Enzymes for DNA modification and reagents for in vitro transcription were from Boehringer Mannhelm. [L- S]Methlonme was obtained from Amersham, and horseradish peroxidase-conjugated second antibodies from Dako. Canine pancreatic microsomes and signal recognition particle (SRP) were isolated according to standard procedures [10,11]. Schizont-infected RBC from P. falciparum isolate FCBR (Columbia) were obtained as described [ 12] and stored frozen. •
35
•
•
Cell-free biosynthesis of parasite proteins and membrane translocation. The experimental procedures for in vitro transcription of cloned DNA sequences, the translation of transcripts in wheatgerm lysate, and the co-translational translocation of polypeptides across pancreatic microsomes have recently been described in detail [ 13 ].
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Proteins were separated on 7.5% and 15% Laemmli-type SDS-polyacrylamide ~slS, respectively. Proteins radiolabeled with [L]methionine were visualized by fluorography using Na-salicylate.
Carbonate extraction.
Following the translation/translocation reaction, microsomes were ex-
Membrane preparation of parasitized red blood cells• Schizont-infected RBC were collected by centrifugation and frozen. The pellet was resuspended in ice-cold phosphate-buffered saline containing a mixture of protease inhibitors (1 ~tg/ml each ofpepstatin A, leupeptin, antipain, elastatinal, trasylol, Na-EDTA, and soybean trypsin inhibitor). During this process cells were lysed. An equivalent of 1.5 x 10 erythrocytlc stage parasites was solubllized in SDS sample buffer. For extraction with carbonate, approximately 6 × 108 parasites were extracted with high salt buffer and Na2CO3 as described above. For protease digestion, proteinase K was added to the lysate at a concentration of 1.8 mg ml -~, either in the absence or in the presence of 0.3% Triton X-100 and left on ice for 1 h. The reaction was stopped by adding phenylmethylsulfonylfluoride to a final concentration of 2 mg ml , proteins were precipitated in 10% TCA, and solubilized in SDS sample buffer. 8
•
•
•
Western blotting analysis.
•
.
•
-1
Proteins separated by SDS-PAGE were transferred to nitrocellulose filters [15]. Subsequently the filters were processed, incubated with antibodies, and immunostained using horseradish peroxidase-conjugated second antibodies, following standard procedures [ 16].
151 Results Construction ofpGem3/exp-1. Recent studies on the serine-rich protein, SERP, of P. falciparum indicate the value of a cell-free system for studying membrane translocation of parasite proteins [ 13]. We have applied this approach to the exp-1 protein. To generate transcripts of the gene for translocation in vitro, a 600-bp cDNA [4] was inserted into the EcoRI site of the polylinker region of pGem3 (Fig. 1). For in vitro transcription, the plasmid was linearized at the unique SmaI site and transcribed from the T7 promoter. The precursor form of exp-1 is processed in the presence of SRP and microsomes. In eukaryotic cells, secreted proteins and proteins of the plasma membrane require the cytosolic signal recognition particle (SRP) for their translocation across the membrane of the endoplasmic reticulum (ER). This particle first interacts with the signal sequence of the nascent polypeptide chain and subsequently with its receptor in the membrane of the ER. In gen-
era1, translocation occurs cotranslationally and is accompanied by cleavage of an N-terminal signal sequence from the precursor form (for reviews, see refs. 17 and 18). The main elements of this pathway can be studied in a reconstituted cell-free system, which also provides an approach to investigate membrane targetting of parasite proteins. When exp-1 was expressed in vitro, a major translation product of an apparent Mr of 24000 was synthesized (Fig. 2, lane 1). If translation was carried out in the presence of microsomal membranes only, the same primary translation product was obtained (Fig. 2, lane 2), and it was susceptible to digestion with proteinase K (Fig. 2, lane 3,4). Synthesis of exp-1 in the presence of microsomes and SRP resulted in an overall increased translational efficiency, including the synthesis of proteins
