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A Plasmodium falciparum homologue of the Ran binding protein 1, a protein involved in nucleocytoplasmic transport
Molecular & Biochemical Parasitology 123 (2002) 67 /71 www.parasitology-online.com
Short communication
A Plasmodium falciparum homologue of the Ran binding protein 1, a protein involved in nucleocytoplasmic transport Vandana Ramachandran a,b,1, Dominique Dorin b,1, Chan Woon Khiong a, Ursula A. Kara a, Christian Doerig b,* b
a Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 119764, Singapore INSERM U511, Wellcome Centre for Molecular Parasitology, The Anderson College, 56 Dunbarton Road, Glasgow G11 6NN, Scotland, UK
Received 13 December 2001; received in revised form 21 January 2002; accepted 11 April 2002 Keywords: Plasmodium falciparum ; RanBP1; Ran/TC4; Nucleocytoplamsic transport; GTPase
Nucleocytoplasmic transport of key molecules such as cyclins and cyclin-dependent (and other) kinases is crucial to the eukaryotic cell division process. Plasmodium schizogony raises interesting questions regarding this particular aspect of cellular regulation (reviewed in Refs. [1 /3]). First, unlike that of higher eukaryotes, the nuclear membrane is maintained during nuclear divisions, except at the immediate vicinity of spindle poles (at least in erythrocytic schizogony) [4]; does this imply specific mechanisms for nuclear shuttling of M-phase regulators? Second, it appears that nuclei in a given schizont divide asynchronously [5]. This is in contrast to the synchrony of nuclear division observed in other instances of schizogony (for example in Drosophila early embryogenesis, see Ref. [2]), and strongly suggests that progression of the Plasmodium nuclear division cycle is not dictated solely by the temporally regulated expression, and hence cytoplasmic availability, of regulators such as cyclins. Rather, this phenomenon of asynchronicity points to a specific regulatory role for the machinery ensuring import/export of cell cycle molecules into/from the nucleus. Nucleocytoplasmic transport is controlled by sets of proteins functioning as transporters (e.g. importins), indicator proteins, and recycling factors. The small GTP-binding protein Ran (ras-related nuclear protein) is the principal indicator protein, whose function is to
The sequence data reported herein has been submitted to GenBankTM and assigned the accession number AJ420904. * Corresponding author. Tel.: /44-141-339-8855; fax: /44-141330-5422 E-mail address: [email protected] (C. Doerig). 1 Contributed equally to this work.
distinguish cytoplasmic versus nuclear environments (reviewed in Ref. [6]). The other core components of the Ran pathway are (i) GTPase activating protein (RanGAP), which is cytoplasmic and stimulate hydrolysis of GTP to GDP by Ran, (ii) guanine-nucleotide exchange factor (RanGEF), also called regulator of chromosome condensation (RCC1), which is nuclear and ensures GTP binding to Ran, and (iii) Ran binding protein 1 (RanBP1), which interacts specifically with the GTP-bound form of Ran and acts as a co-activator of RanGAP. The GTP-bound form of Ran is present in the nucleus and the GDP-bound form is found predominantly in the cytoplasm. This asymmetric repartition is a consequence of the presence of RanGAP in the cytoplasm and RCC1 in the nucleus. Thus the concentration of RanGTP is a marker of a nuclear or cytoplasmic environment, and drives the uptake and release of cargo proteins by transporters such as importins. After export from the nucleus, the transporters are bound to RanGTP and need to be released from it. The dissociation of transporter-RanGTP complexes is accomplished by RanBP1 [7,8], and GTP hydrolysis is triggered by RanGAP on the transient complex RanGTP /RanBP1. At this point another round of transport can be initiated. Our knowledge of nucleocytoplasmic transport in Plasmodium is rather limited. P. falciparum genes encoding homologues of the Ran [9,10] and RCC1 [11] proteins were identified and shown to be expressed during schizogony; furthermore, PfRCC1 was demonstrated to be localised predominantly in the nucleus [11]. Considering the importance of nuclear shuttling in cellular processes, we have now started to search genomic databases for other components of the Ran
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transport pathway, with the purpose of generating biochemical tools that will allow us to study this process in detail in P. falciparum . BLAST analysis of the PlasmoDB database [12] using RanBP1 sequences from a variety of organisms allowed us to identify a sequence located on a contig from chromosome 4, which potentially encoded a protein with maximal homology to RanBP1 family members. Alignment (Fig. 1a) of the P. falciparum sequence (PfRanBP1) with that of RanBP1 family members suggested that the coding region was interrupted by an intron, in agreement with gene prediction data on PlasmoDB. This was confirmed by sequencing the PCR products resulting from amplification of the region located between the predicted Start and Stop codons using 3D7 genomic DNA and a cDNA library: this demonstrated the presence of a 294-bp intron between codons 23 and 24, as predicted on PlasmoDB (Fig. 1b). The 843-bp coding region of the cDNA encodes a predicted 280 amino acid protein with a molecular weight of 33.2 kDa. The polypeptide displays 45% identity with human RanBP1 in its N-terminal region (aminoacids 1/150). The C-terminal region (residues 150 /280) is very rich in charged aminoacids, and the SAPS programme (Statistical Analysis of Protein Sequences) [13] detects in this region degenerate copies of motifs such as KEKVEEKKD or EKDKDDKTKDD (such extensions rich in charged residues and repeated motifs are commonly found in P. falciparum proteins). RT-PCR using total RNA extracted from asynchronous blood stage asexual parasites yielded the 843-bp (intronless) product which was absent if the reverse transcriptase had been omitted from the reaction (data not shown). This excludes the possibility that the signal arose from contaminating genomic DNA, and demonstrates that PfRanBP1 mRNA accumulates in blood stage parasites, like the PfRan [9,10] and PfRCC1 [11] mRNAs. We next wanted to determine whether PfRanBP1 was able to bind to PfRan, as expected from analogy with the behaviour of these proteins in other organisms [14 / 16]. To this end, we expressed an N-terminal His-tagged PfRanBP1 in Escherischia coli . Coomassie blue staining and Western blot analysis of the recombinant protein purified on nickel-agarose beads using an anti-His antibody (Fig. 2panels a and b) showed a band of approximately 43 kDa apparent molecular mass (instead of the expected 34 kDa), and a ladder-like pattern of bands ranging from 20 to 32 kDa. All these bands were absent from non-induced E. coli cells (not shown) and from induced cells expressing Pfmap-2, another Histagged protein (lane 2), indicating that they originate from the His-PfRanBP1 expression plasmid. Our interpretation of these data is that (i) full-length HisPfRanBP1 has an anomalous migration, which is presumably due to the content in charged aminoacids
of its C-terminus, and (ii) the ladder-like pattern results from partial proteolysis of the degenerate repeats at the C-terminus. We also expressed the entire PfRan sequence fused to the maltose-binding protein (MBP) in E. coli (lane 3). Interaction between these two proteins was investigated using the overlay approach [17], where one of the polypeptides is run on a polyacrylamide gel, transferred to nitrocellulose, renatured, and probed with the other protein. We exploited the previously demonstrated [9] GTP-binding property of PfRan to generate a radiolabelled PfRan-[g-32P-GTP] complex, which was used to probe a membrane carrying renatured PfRanBP1. The autoradiogramme (Fig. 2 panel d) shows that a signal is associated with the bands corresponding to recombinant His-PfRanBPl (lane 5), but not with His-Pfmap-2 [18], an unrelated His-tagged recombinant protein used here as a negative control (lane 4) (other recombinant proteins used as negative control also gave no signal (data not shown)). Coomassie staining of an identically loaded gel run in parallel (panel C) shows that similar amounts of each protein were used. Data in panel D are consistent with a specific interaction between GTP-bound PfRan and PfRanBP1; however, we wanted to verify that the signal was not due to direct non-specific binding of free radiolabelled GTP, which might have leaked from the PfRan-GTP probe, to PfRanBP1. To exclude this possibility, we probed a similar membrane with the same amount of free radiolabelled GTP as had been used to generate the GTPPfRan complex, and used an exposure time 32-fold longer than that used in panel D. In these conditions, no signal corresponding to PfRanBP1 was observed (panel E), although GTP binding by PfRan (used as a positive control) was readily detected ([9] and panel F). This demonstrates that the radiolabel associated with PfRanBP1 in panel D is dependent on the presence of PfRan. Hence, PfRanBP1 possesses the property of binding to Ran-GTP, as expected from its primary structure. Moreover, the PfRan-PfRanBP1 interaction was independently confirmed by screening a P. falciparum yeast two-hybrid cDNA library with PfRan: two library clones containing different sized fragments of PfRanBP1 were isolated multiple times (/5 times each) in the screen (D. LaCount, M. Vignali, and S. Fields, personal communication). The Ran binding domain (RanBD) of PfRanBP1 possesses most of the motifs that are highly conserved in RDBs from other Ran-binding proteins (black bars on Fig. 1a) [19]. Furthermore, most residues shown by crystallographic analysis of a human RanBD-Ran complex to directly interact with Ran [20] are conserved in PfRanBP1 (Fig. 1a). The conservation is not absolute, however, which may indicate that some of the relevant residues are not required for the interaction in P. falciparum , or that the corresponding region in PfRan is itself divergent. In the GTP-bound conformation of
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Fig. 1. (a) Amino acid sequence alignment of RanBP1 homologues from Schizosaccharomyces pombe Sp (D86381), human RanBPl (X83617), Xenopus laevis RanBP1 (U09128) and P. falciparum RanBPl (AJ420904). The solid bars indicate motifs that are conserved in the Ran binding domain (RBD) of various proteins interacting with Ran (including RanBPl and RanBP2) [19,20]. Residues that have been shown by crystallographic analysis of the human Ran-RanBP2 complex to be in contact with the Ran protein [20] are indicated by a dot (+ ) below the sequence. Residues whose mutation impairs binding to Ran in other systems are indicated by a triangle (n). An arrow indicates the position where an intron interrupts the coding sequence (between K23 and V24). (b) PCR amplification of the PfRanBP1 coding region using primers spanning the Start and the Stop codons. The templates were: lane 2, P. falciparum clone 3D7 genomic DNA; lane 3, a plasmid cDNA library prepared from erythrocytic stages; lane 4, negative control (water). Lane 1 is the molecular weight marker (SmartLadder, Eurogentec).
Ran, the acidic C-terminal region hinders access of RanGAP to the active site. Binding of RanBP1 to Ran occurs on this acidic C-terminal domain and causes its displacement, allowing an increased affinity between
RanGAP and Ran-GTP and hence hydrolysis of GTP [19 /21]. The good level of conservation of the RanBD motifs in PfRanBP1 and the presence of a C-terminal acidic domain on PfRan [9,10] suggest that a similar
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Fig. 2. (A, B) Analysis of recombinant proteins. The entire PfRanBP1 coding region was amplified from cDNA and cloned into the pQE30 vector (Quiagen). Expression in E. coli and purification of His-PfRanBP1 (lane 1) and His-Pfmap-2 [18] (lane 2) on nickel-agarose beads were performed as previously described [22]. PfRan fused to the MBP (lane 3) was expressed in E. coli from the pMALp-c2x vector (New England Biolabs) and purified on amylose beads. (A) Coomassie blue staining of a gel showing the three recombinant proteins. (B) Western blot analysis of a gel loaded identically and run in parallel. The membrane was probed with anti-His antibody (Quiagen) and a peroxidase-conjugated secondary antibody, and revealed by chemiluminescence (ECL, Amersham) following the suppliers’ recommendations. The full-length PfRanBP1 signal is somewhat weaker than expected from the Coomassie staining (in comparison with the lower bands). This may be due to the fact that the full-length protein transferred not as efficiently, or that the charged C-terminal tail somehow interferes with Ab binding. (C /F). Ran-overlay assay. Two micrograms of recombinant HisPfmap-2 (lane 4) and His-PfRanBP1 [18] (lane 5) were run on a polyacrylamide gel, transferred onto a nitrocellulose membrane and renatured [17]. The membrane was then incubated with PfRan bound to radiolabelled GTP (generated by loading 3 mg of recombinant PfRan with 10 mCi [g-32P]GTP (3000 Ci mmol 1, Amersham) for 20 min on ice in the presence of 10 mM MOPS, pH 7.1, 1 mM EDTA and 1 mg ml 1 bovine serum albumin in a 15 ml reaction). After washing, the membrane was exposed for autoradiography. Panel C is a Coomassie staining of an identically loaded gel run in parallel, and panel D shows the autoradiogramme (exposure time 30 min). Panel E is an autoradiogramme (exposure time 16 h) of the control experiment in which 10 mCi unbound [g-32P]-GTP were used as a probe [9]. Panel F is an autoradiogramme showing that in the same conditions free GTP is able to bind to MBP-PfRan (lane 1) but not to His-Pfmap-2 (lane 2) or another preparation of PfRanBP1 (lane 3).
mechanism may operate in P. falciparum . Furthermore, mutations affecting highly conserved residues and which impair binding to Ran have been characterised in various RDBs ([20] and references therein); these residues are either identical (E30, L48, F141) or similar (D136 instead of E) in PfRanBP1 (Fig. 1a).
This series of experiments demonstrates that PfRanBP1 interacts with GTP-bound PfRan, confirming a biochemical property predicted by analogy from the primary structure of this protein. Our present data do not allow us to determine whether PfRanBP1 binds specifically to the GTP-bound (versus the GDP-bound)
V. Ramachandran et al. / Molecular & Biochemical Parasitology 123 (2002) 67 /71
form of PfRan. Although this still requires experimental evidence, it is likely that PfRanBP1, PfRan and PfRCC1 are involved in nucleocytoplasmic transport. We are now attempting to (i) isolate additional putative players in this phenomenon, such as importins and RanGAP homologues, and (ii) generate batteries of antibodies against these molecules. Such tools should allow us to address questions concerning the nuclear transport of specific gene products such as protein kinases and cyclins, whose subcellular location and nucleocytoplasmic transport regulation presumably play an important role in the control of the Plasmodium cell cycle.
Acknowledgements We wish to thank the scientists and funding agencies comprising the international Malaria Genome Project for making sequence data from the genome of P. falciparum (3D7) public prior to publication of the completed sequence. The Sanger Centre (UK) provided sequence for chromosomes 1, 3/9 and 13, with financial support from the Wellcome Trust. A consortium composed of The Institute for Genome Research, along with the Naval Medical Research Center (USA), sequenced chromosomes 2, 10, 11 and 14, with support from NIAID/NIH, the Burroughs Wellcome Fund, and the Department of Defence. The Stanford Genome Technology Center (USA) sequenced chromosome 12, with support from the Burroughs Wellcome Fund. The Plasmodium Genome Database is a collaborative effort of investigators at the University of Pennsylvania (USA) and Monash University (Melbourne, Australia), supported by the Burroughs Wellcome Fund. We thank Alistair Craig and Pietro Alano for P. falciparum cDNA libraries, and David Arnot for an anti-PfRan antiserum to monitor PfRan protein expression in E. coli . This work was supported by INSERM, The French Ministries of Research (PAL/ and PRFMMIP programmes) and Defence (De´le´gation Ge´ne´rale pour 1’Armement), and the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR). V.R. is supported by a National University of Singapore postgraduate research scholarship.
References [1] Doerig C, Chakrabarti D, Kappes B, Matthews K. The cell cycle in protozoan parasites. Prog Cell Cycle Res 2000;4:163 /83. [2] Leete TH, Rubin H. Malaria and the cell cycle. Parasitol Today 1997;12:442 /4. [3] Arnot DE, Gull K. The Plasmodium cell-cycle: facts and questions. Ann Trop Med Parasitol 1998;92:361 /5.
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[4] Aikawa M. The fine structure of the erythrocytic stages of three avian malarial parasites, Plasmodium fallax , P. lophurae and P. cathemerium . Am J Trop Med Hyg 1966;15:449 /70. [5] Read M, Sherwin T, Holloway SP, Gull K, Hyde JE. Microtubular organisation visualised by immunofluorescence microscopy during erythrocytic schizogony in Plasmodium falciparum and investigation of post-translational modifications of parasite tubulin. Parasitology 1993;106:223 /32. [6] Sazer S, Dasso M. The Ran decathlon: multiple roles of Ran. J Cell Sci 2000;113:1111 /8. [7] Coutavas E, Ren M, Oppenhcim JD, D’Eustachio, Rush MG. Characterisation of proteins that interact with the cell-cycle regulatory protein Ran/TC4. Nature 1993;366:585 /7. [8] Plafker K, Macara IG. Facilitated nucleocytoplasmic shuttling of the ran binding protein RanBPl. Mol Cell Biol 2000;20:3510 /20. [9] Dontfraid F, Chakrabarti D. Cloning and expression of a cDNA encoding the homologue of Ran/TC4 GTP-binding protein from Plasmodium falciparum . Biochem Biophys Res Comm 1994;20:423 /9. [10] Sultan AA, Richardson WA, Alano P, Arnot DE, Doerig C. Cloning and characterisation of a Plasmodium falciparum homologue of the Ran/TC4 signal transducing GTPase involved in cell cycle control. Mol Biochem Parasitol 1994;65:331 /8. [11] Ji DD, Sultan AA, Chakrabarti D, Horrocks P, Doerig C, Arnot DE. An RCC1-type guanidine exchange factor for the Ran G protein is found in the Plasmodium falciparum nucleus. Mol Biochem Parasitol 1998;95:165 /70. [12] The Plasmodium Genome Database Collaborative. PlasmoDB: An integrative database of the P. falciparum genome. Tools for accessing and analyzing finished and unfinished sequence data. Nucleic Acids Res 2001;29:66 /9. [13] Brendel V, Bucher P, Nourbakhsh I, Blaisdell BE, Karlin S. Methods and algorithms for statistical analysis of protein sequence’s. Proc Natl Acad Sci USA 1992;89:2002 /6. [14] Kunzler M, Trueheart J, Sctte C, Hurt E, Thoner J. Mutations in the YRB1 gene encoding yeast ran binding protein 1 that impair Nucleocytoplasmic transport and suppress yeast mating defects. Genetics 2001;157:1089 /105. [15] Petersen C, Orem N, Trueheart J, Thorner JW, Macara IG. Random mutagenesis and functional analysis of the Ran-binding protein, RanBP1. J Biol Chem 2000;275:4081 /91. [16] Lounsbury KM, Macara IG. Ran-binding protein 1 (RanBP1) forms a ternary complex with Ran and karyopherin beta and reduces Ran GTPase-activating protein (RanGAP) inhibition by karyopherin beta. J Biol Chem 1997;272:551 /5. [17] Lounsbury KM, Beddow AL, Macara IG. A family of proteins that stabilize the Ran/TC4 GTPase in its GTP-bound conformation. J Biol Chem 1994;289:11285 /90. [18] Dorin D, Alano P, Boccaccio I, et al. An atypical mitogen activated protein kinase (MAPK) homologue expressed in gametocytes of the human malaria parasite Plasmodium falciparum . J Biol Chem 1999;274:29912 /20. [19] Beddow AL, Richards SA, Orem NR, Macara IG. The Ran/TC4 GTPase-binding domain: identification by expression cloning and characterisation of a conserved sequence motif. Proc Natl Acad Sci USA 1995;92:3328 /32. [20] Vetter IR, Nowak C, Nishimoto T, Kuhlmann J, Wittinghoffer A. Structure of Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport. Nature 1999;398:39 /46. [21] Bischoff FR, Gorlich D. RanBP1 is crucial for the release of RanGTP from importin b-related nuclear transport factors. FEBS Lett 1997;419:254 /94. [22] Le Roch K, Scstier C, Dorin D, et al. Activation of a Plasmodium. falciparum cdc related kinase by heterologous p25 and cyclin H. Functional characterisation of a P. falciparum cyclin homologue. J Biol Chem 2000;275:8952 /8.
Short communication
A Plasmodium falciparum homologue of the Ran binding protein 1, a protein involved in nucleocytoplasmic transport Vandana Ramachandran a,b,1, Dominique Dorin b,1, Chan Woon Khiong a, Ursula A. Kara a, Christian Doerig b,* b
a Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 119764, Singapore INSERM U511, Wellcome Centre for Molecular Parasitology, The Anderson College, 56 Dunbarton Road, Glasgow G11 6NN, Scotland, UK
Received 13 December 2001; received in revised form 21 January 2002; accepted 11 April 2002 Keywords: Plasmodium falciparum ; RanBP1; Ran/TC4; Nucleocytoplamsic transport; GTPase
Nucleocytoplasmic transport of key molecules such as cyclins and cyclin-dependent (and other) kinases is crucial to the eukaryotic cell division process. Plasmodium schizogony raises interesting questions regarding this particular aspect of cellular regulation (reviewed in Refs. [1 /3]). First, unlike that of higher eukaryotes, the nuclear membrane is maintained during nuclear divisions, except at the immediate vicinity of spindle poles (at least in erythrocytic schizogony) [4]; does this imply specific mechanisms for nuclear shuttling of M-phase regulators? Second, it appears that nuclei in a given schizont divide asynchronously [5]. This is in contrast to the synchrony of nuclear division observed in other instances of schizogony (for example in Drosophila early embryogenesis, see Ref. [2]), and strongly suggests that progression of the Plasmodium nuclear division cycle is not dictated solely by the temporally regulated expression, and hence cytoplasmic availability, of regulators such as cyclins. Rather, this phenomenon of asynchronicity points to a specific regulatory role for the machinery ensuring import/export of cell cycle molecules into/from the nucleus. Nucleocytoplasmic transport is controlled by sets of proteins functioning as transporters (e.g. importins), indicator proteins, and recycling factors. The small GTP-binding protein Ran (ras-related nuclear protein) is the principal indicator protein, whose function is to
The sequence data reported herein has been submitted to GenBankTM and assigned the accession number AJ420904. * Corresponding author. Tel.: /44-141-339-8855; fax: /44-141330-5422 E-mail address: [email protected] (C. Doerig). 1 Contributed equally to this work.
distinguish cytoplasmic versus nuclear environments (reviewed in Ref. [6]). The other core components of the Ran pathway are (i) GTPase activating protein (RanGAP), which is cytoplasmic and stimulate hydrolysis of GTP to GDP by Ran, (ii) guanine-nucleotide exchange factor (RanGEF), also called regulator of chromosome condensation (RCC1), which is nuclear and ensures GTP binding to Ran, and (iii) Ran binding protein 1 (RanBP1), which interacts specifically with the GTP-bound form of Ran and acts as a co-activator of RanGAP. The GTP-bound form of Ran is present in the nucleus and the GDP-bound form is found predominantly in the cytoplasm. This asymmetric repartition is a consequence of the presence of RanGAP in the cytoplasm and RCC1 in the nucleus. Thus the concentration of RanGTP is a marker of a nuclear or cytoplasmic environment, and drives the uptake and release of cargo proteins by transporters such as importins. After export from the nucleus, the transporters are bound to RanGTP and need to be released from it. The dissociation of transporter-RanGTP complexes is accomplished by RanBP1 [7,8], and GTP hydrolysis is triggered by RanGAP on the transient complex RanGTP /RanBP1. At this point another round of transport can be initiated. Our knowledge of nucleocytoplasmic transport in Plasmodium is rather limited. P. falciparum genes encoding homologues of the Ran [9,10] and RCC1 [11] proteins were identified and shown to be expressed during schizogony; furthermore, PfRCC1 was demonstrated to be localised predominantly in the nucleus [11]. Considering the importance of nuclear shuttling in cellular processes, we have now started to search genomic databases for other components of the Ran
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transport pathway, with the purpose of generating biochemical tools that will allow us to study this process in detail in P. falciparum . BLAST analysis of the PlasmoDB database [12] using RanBP1 sequences from a variety of organisms allowed us to identify a sequence located on a contig from chromosome 4, which potentially encoded a protein with maximal homology to RanBP1 family members. Alignment (Fig. 1a) of the P. falciparum sequence (PfRanBP1) with that of RanBP1 family members suggested that the coding region was interrupted by an intron, in agreement with gene prediction data on PlasmoDB. This was confirmed by sequencing the PCR products resulting from amplification of the region located between the predicted Start and Stop codons using 3D7 genomic DNA and a cDNA library: this demonstrated the presence of a 294-bp intron between codons 23 and 24, as predicted on PlasmoDB (Fig. 1b). The 843-bp coding region of the cDNA encodes a predicted 280 amino acid protein with a molecular weight of 33.2 kDa. The polypeptide displays 45% identity with human RanBP1 in its N-terminal region (aminoacids 1/150). The C-terminal region (residues 150 /280) is very rich in charged aminoacids, and the SAPS programme (Statistical Analysis of Protein Sequences) [13] detects in this region degenerate copies of motifs such as KEKVEEKKD or EKDKDDKTKDD (such extensions rich in charged residues and repeated motifs are commonly found in P. falciparum proteins). RT-PCR using total RNA extracted from asynchronous blood stage asexual parasites yielded the 843-bp (intronless) product which was absent if the reverse transcriptase had been omitted from the reaction (data not shown). This excludes the possibility that the signal arose from contaminating genomic DNA, and demonstrates that PfRanBP1 mRNA accumulates in blood stage parasites, like the PfRan [9,10] and PfRCC1 [11] mRNAs. We next wanted to determine whether PfRanBP1 was able to bind to PfRan, as expected from analogy with the behaviour of these proteins in other organisms [14 / 16]. To this end, we expressed an N-terminal His-tagged PfRanBP1 in Escherischia coli . Coomassie blue staining and Western blot analysis of the recombinant protein purified on nickel-agarose beads using an anti-His antibody (Fig. 2panels a and b) showed a band of approximately 43 kDa apparent molecular mass (instead of the expected 34 kDa), and a ladder-like pattern of bands ranging from 20 to 32 kDa. All these bands were absent from non-induced E. coli cells (not shown) and from induced cells expressing Pfmap-2, another Histagged protein (lane 2), indicating that they originate from the His-PfRanBP1 expression plasmid. Our interpretation of these data is that (i) full-length HisPfRanBP1 has an anomalous migration, which is presumably due to the content in charged aminoacids
of its C-terminus, and (ii) the ladder-like pattern results from partial proteolysis of the degenerate repeats at the C-terminus. We also expressed the entire PfRan sequence fused to the maltose-binding protein (MBP) in E. coli (lane 3). Interaction between these two proteins was investigated using the overlay approach [17], where one of the polypeptides is run on a polyacrylamide gel, transferred to nitrocellulose, renatured, and probed with the other protein. We exploited the previously demonstrated [9] GTP-binding property of PfRan to generate a radiolabelled PfRan-[g-32P-GTP] complex, which was used to probe a membrane carrying renatured PfRanBP1. The autoradiogramme (Fig. 2 panel d) shows that a signal is associated with the bands corresponding to recombinant His-PfRanBPl (lane 5), but not with His-Pfmap-2 [18], an unrelated His-tagged recombinant protein used here as a negative control (lane 4) (other recombinant proteins used as negative control also gave no signal (data not shown)). Coomassie staining of an identically loaded gel run in parallel (panel C) shows that similar amounts of each protein were used. Data in panel D are consistent with a specific interaction between GTP-bound PfRan and PfRanBP1; however, we wanted to verify that the signal was not due to direct non-specific binding of free radiolabelled GTP, which might have leaked from the PfRan-GTP probe, to PfRanBP1. To exclude this possibility, we probed a similar membrane with the same amount of free radiolabelled GTP as had been used to generate the GTPPfRan complex, and used an exposure time 32-fold longer than that used in panel D. In these conditions, no signal corresponding to PfRanBP1 was observed (panel E), although GTP binding by PfRan (used as a positive control) was readily detected ([9] and panel F). This demonstrates that the radiolabel associated with PfRanBP1 in panel D is dependent on the presence of PfRan. Hence, PfRanBP1 possesses the property of binding to Ran-GTP, as expected from its primary structure. Moreover, the PfRan-PfRanBP1 interaction was independently confirmed by screening a P. falciparum yeast two-hybrid cDNA library with PfRan: two library clones containing different sized fragments of PfRanBP1 were isolated multiple times (/5 times each) in the screen (D. LaCount, M. Vignali, and S. Fields, personal communication). The Ran binding domain (RanBD) of PfRanBP1 possesses most of the motifs that are highly conserved in RDBs from other Ran-binding proteins (black bars on Fig. 1a) [19]. Furthermore, most residues shown by crystallographic analysis of a human RanBD-Ran complex to directly interact with Ran [20] are conserved in PfRanBP1 (Fig. 1a). The conservation is not absolute, however, which may indicate that some of the relevant residues are not required for the interaction in P. falciparum , or that the corresponding region in PfRan is itself divergent. In the GTP-bound conformation of
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Fig. 1. (a) Amino acid sequence alignment of RanBP1 homologues from Schizosaccharomyces pombe Sp (D86381), human RanBPl (X83617), Xenopus laevis RanBP1 (U09128) and P. falciparum RanBPl (AJ420904). The solid bars indicate motifs that are conserved in the Ran binding domain (RBD) of various proteins interacting with Ran (including RanBPl and RanBP2) [19,20]. Residues that have been shown by crystallographic analysis of the human Ran-RanBP2 complex to be in contact with the Ran protein [20] are indicated by a dot (+ ) below the sequence. Residues whose mutation impairs binding to Ran in other systems are indicated by a triangle (n). An arrow indicates the position where an intron interrupts the coding sequence (between K23 and V24). (b) PCR amplification of the PfRanBP1 coding region using primers spanning the Start and the Stop codons. The templates were: lane 2, P. falciparum clone 3D7 genomic DNA; lane 3, a plasmid cDNA library prepared from erythrocytic stages; lane 4, negative control (water). Lane 1 is the molecular weight marker (SmartLadder, Eurogentec).
Ran, the acidic C-terminal region hinders access of RanGAP to the active site. Binding of RanBP1 to Ran occurs on this acidic C-terminal domain and causes its displacement, allowing an increased affinity between
RanGAP and Ran-GTP and hence hydrolysis of GTP [19 /21]. The good level of conservation of the RanBD motifs in PfRanBP1 and the presence of a C-terminal acidic domain on PfRan [9,10] suggest that a similar
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Fig. 2. (A, B) Analysis of recombinant proteins. The entire PfRanBP1 coding region was amplified from cDNA and cloned into the pQE30 vector (Quiagen). Expression in E. coli and purification of His-PfRanBP1 (lane 1) and His-Pfmap-2 [18] (lane 2) on nickel-agarose beads were performed as previously described [22]. PfRan fused to the MBP (lane 3) was expressed in E. coli from the pMALp-c2x vector (New England Biolabs) and purified on amylose beads. (A) Coomassie blue staining of a gel showing the three recombinant proteins. (B) Western blot analysis of a gel loaded identically and run in parallel. The membrane was probed with anti-His antibody (Quiagen) and a peroxidase-conjugated secondary antibody, and revealed by chemiluminescence (ECL, Amersham) following the suppliers’ recommendations. The full-length PfRanBP1 signal is somewhat weaker than expected from the Coomassie staining (in comparison with the lower bands). This may be due to the fact that the full-length protein transferred not as efficiently, or that the charged C-terminal tail somehow interferes with Ab binding. (C /F). Ran-overlay assay. Two micrograms of recombinant HisPfmap-2 (lane 4) and His-PfRanBP1 [18] (lane 5) were run on a polyacrylamide gel, transferred onto a nitrocellulose membrane and renatured [17]. The membrane was then incubated with PfRan bound to radiolabelled GTP (generated by loading 3 mg of recombinant PfRan with 10 mCi [g-32P]GTP (3000 Ci mmol 1, Amersham) for 20 min on ice in the presence of 10 mM MOPS, pH 7.1, 1 mM EDTA and 1 mg ml 1 bovine serum albumin in a 15 ml reaction). After washing, the membrane was exposed for autoradiography. Panel C is a Coomassie staining of an identically loaded gel run in parallel, and panel D shows the autoradiogramme (exposure time 30 min). Panel E is an autoradiogramme (exposure time 16 h) of the control experiment in which 10 mCi unbound [g-32P]-GTP were used as a probe [9]. Panel F is an autoradiogramme showing that in the same conditions free GTP is able to bind to MBP-PfRan (lane 1) but not to His-Pfmap-2 (lane 2) or another preparation of PfRanBP1 (lane 3).
mechanism may operate in P. falciparum . Furthermore, mutations affecting highly conserved residues and which impair binding to Ran have been characterised in various RDBs ([20] and references therein); these residues are either identical (E30, L48, F141) or similar (D136 instead of E) in PfRanBP1 (Fig. 1a).
This series of experiments demonstrates that PfRanBP1 interacts with GTP-bound PfRan, confirming a biochemical property predicted by analogy from the primary structure of this protein. Our present data do not allow us to determine whether PfRanBP1 binds specifically to the GTP-bound (versus the GDP-bound)
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form of PfRan. Although this still requires experimental evidence, it is likely that PfRanBP1, PfRan and PfRCC1 are involved in nucleocytoplasmic transport. We are now attempting to (i) isolate additional putative players in this phenomenon, such as importins and RanGAP homologues, and (ii) generate batteries of antibodies against these molecules. Such tools should allow us to address questions concerning the nuclear transport of specific gene products such as protein kinases and cyclins, whose subcellular location and nucleocytoplasmic transport regulation presumably play an important role in the control of the Plasmodium cell cycle.
Acknowledgements We wish to thank the scientists and funding agencies comprising the international Malaria Genome Project for making sequence data from the genome of P. falciparum (3D7) public prior to publication of the completed sequence. The Sanger Centre (UK) provided sequence for chromosomes 1, 3/9 and 13, with financial support from the Wellcome Trust. A consortium composed of The Institute for Genome Research, along with the Naval Medical Research Center (USA), sequenced chromosomes 2, 10, 11 and 14, with support from NIAID/NIH, the Burroughs Wellcome Fund, and the Department of Defence. The Stanford Genome Technology Center (USA) sequenced chromosome 12, with support from the Burroughs Wellcome Fund. The Plasmodium Genome Database is a collaborative effort of investigators at the University of Pennsylvania (USA) and Monash University (Melbourne, Australia), supported by the Burroughs Wellcome Fund. We thank Alistair Craig and Pietro Alano for P. falciparum cDNA libraries, and David Arnot for an anti-PfRan antiserum to monitor PfRan protein expression in E. coli . This work was supported by INSERM, The French Ministries of Research (PAL/ and PRFMMIP programmes) and Defence (De´le´gation Ge´ne´rale pour 1’Armement), and the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR). V.R. is supported by a National University of Singapore postgraduate research scholarship.
References [1] Doerig C, Chakrabarti D, Kappes B, Matthews K. The cell cycle in protozoan parasites. Prog Cell Cycle Res 2000;4:163 /83. [2] Leete TH, Rubin H. Malaria and the cell cycle. Parasitol Today 1997;12:442 /4. [3] Arnot DE, Gull K. The Plasmodium cell-cycle: facts and questions. Ann Trop Med Parasitol 1998;92:361 /5.
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[4] Aikawa M. The fine structure of the erythrocytic stages of three avian malarial parasites, Plasmodium fallax , P. lophurae and P. cathemerium . Am J Trop Med Hyg 1966;15:449 /70. [5] Read M, Sherwin T, Holloway SP, Gull K, Hyde JE. Microtubular organisation visualised by immunofluorescence microscopy during erythrocytic schizogony in Plasmodium falciparum and investigation of post-translational modifications of parasite tubulin. Parasitology 1993;106:223 /32. [6] Sazer S, Dasso M. The Ran decathlon: multiple roles of Ran. J Cell Sci 2000;113:1111 /8. [7] Coutavas E, Ren M, Oppenhcim JD, D’Eustachio, Rush MG. Characterisation of proteins that interact with the cell-cycle regulatory protein Ran/TC4. Nature 1993;366:585 /7. [8] Plafker K, Macara IG. Facilitated nucleocytoplasmic shuttling of the ran binding protein RanBPl. Mol Cell Biol 2000;20:3510 /20. [9] Dontfraid F, Chakrabarti D. Cloning and expression of a cDNA encoding the homologue of Ran/TC4 GTP-binding protein from Plasmodium falciparum . Biochem Biophys Res Comm 1994;20:423 /9. [10] Sultan AA, Richardson WA, Alano P, Arnot DE, Doerig C. Cloning and characterisation of a Plasmodium falciparum homologue of the Ran/TC4 signal transducing GTPase involved in cell cycle control. Mol Biochem Parasitol 1994;65:331 /8. [11] Ji DD, Sultan AA, Chakrabarti D, Horrocks P, Doerig C, Arnot DE. An RCC1-type guanidine exchange factor for the Ran G protein is found in the Plasmodium falciparum nucleus. Mol Biochem Parasitol 1998;95:165 /70. [12] The Plasmodium Genome Database Collaborative. PlasmoDB: An integrative database of the P. falciparum genome. Tools for accessing and analyzing finished and unfinished sequence data. Nucleic Acids Res 2001;29:66 /9. [13] Brendel V, Bucher P, Nourbakhsh I, Blaisdell BE, Karlin S. Methods and algorithms for statistical analysis of protein sequence’s. Proc Natl Acad Sci USA 1992;89:2002 /6. [14] Kunzler M, Trueheart J, Sctte C, Hurt E, Thoner J. Mutations in the YRB1 gene encoding yeast ran binding protein 1 that impair Nucleocytoplasmic transport and suppress yeast mating defects. Genetics 2001;157:1089 /105. [15] Petersen C, Orem N, Trueheart J, Thorner JW, Macara IG. Random mutagenesis and functional analysis of the Ran-binding protein, RanBP1. J Biol Chem 2000;275:4081 /91. [16] Lounsbury KM, Macara IG. Ran-binding protein 1 (RanBP1) forms a ternary complex with Ran and karyopherin beta and reduces Ran GTPase-activating protein (RanGAP) inhibition by karyopherin beta. J Biol Chem 1997;272:551 /5. [17] Lounsbury KM, Beddow AL, Macara IG. A family of proteins that stabilize the Ran/TC4 GTPase in its GTP-bound conformation. J Biol Chem 1994;289:11285 /90. [18] Dorin D, Alano P, Boccaccio I, et al. An atypical mitogen activated protein kinase (MAPK) homologue expressed in gametocytes of the human malaria parasite Plasmodium falciparum . J Biol Chem 1999;274:29912 /20. [19] Beddow AL, Richards SA, Orem NR, Macara IG. The Ran/TC4 GTPase-binding domain: identification by expression cloning and characterisation of a conserved sequence motif. Proc Natl Acad Sci USA 1995;92:3328 /32. [20] Vetter IR, Nowak C, Nishimoto T, Kuhlmann J, Wittinghoffer A. Structure of Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport. Nature 1999;398:39 /46. [21] Bischoff FR, Gorlich D. RanBP1 is crucial for the release of RanGTP from importin b-related nuclear transport factors. FEBS Lett 1997;419:254 /94. [22] Le Roch K, Scstier C, Dorin D, et al. Activation of a Plasmodium. falciparum cdc related kinase by heterologous p25 and cyclin H. Functional characterisation of a P. falciparum cyclin homologue. J Biol Chem 2000;275:8952 /8.