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3969 Journal of Cell Science 113, 3969-3977 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS1679 Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system Amy DeRocher1,2, Christopher B. Hagen2, John E. Froehlich3, Jean E. Feagin1,2 and Marilyn Parsons1,2,* 1Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, WA 98195, USA 2Seattle Biomedical Research Institute, 4 Nickerson St., Seattle, WA 98109, USA 3MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA *Author for correspondence at address 2 (e-mail: [email protected]) Accepted 14 September; published on WWW 31 October 2000 SUMMARY Apicomplexan parasites possess a plastid-like organelle called the apicoplast. Most proteins in the Toxoplasma gondii apicoplast are encoded in the nucleus and imported post-translationally. T. gondii apicoplast proteins often have a long N-terminal extension that directs the protein to the apicoplast. It can be modeled as a bipartite targeting sequence that contains a signal sequence and a plastid transit peptide. We identified two nuclearly encoded predicted plastid proteins and made fusions with green fluorescent protein to study protein domains required for apicoplast targeting. The N-terminal 42 amino acids of the apicoplast ribosomal protein S9 directs secretion of green fluorescent protein, indicating that targeting to the apicoplast proceeds through the secretory system. Large sections of the S9 predicted transit sequence can be deleted with no apparent impact on the ability to direct green fluorescent protein to the apicoplast. The predicted transit peptide domain of the S9 targeting sequence directs protein to the mitochondrion in vivo. The transit peptide can also direct import of green fluorescent protein into chloroplasts in vitro. These data substantiate the model that protein targeting to the apicoplast involves two distinct mechanisms: the first involving the secretory system and the second sharing features with typical chloroplast protein import. INTRODUCTION indicates that apicoplasts are the site of non-mevolonate isoprene synthesis, a pathway not found in metazoans (Jomaa et al., 1999). Fosmidomycin is a specific inhibitor of this pathway and is an effective therapy for Plasmodium vinckei infection in rats (Jomaa et al., 1999). Likewise, enzymes of type II fatty acid biosynthesis reside in the plastid, and thiolactomycin, which inhibits this pathway in other organisms, is toxic to malaria parasites but has little effect on the mammalian host cell (Waller et al., 1998). Only a small fraction of the proteins in plastids are encoded by the corresponding plastid genome; most plastid proteins are encoded in the nucleus, and imported post-translationally from the cytoplasm into the plastid (Keegstra and Cline, 1999). Proteins destined to reside in plastids that have two membranes, such as the chloroplasts of green plants, typically have an N-terminal transit peptide, which is cleaved upon import. This transit peptide, typically rich in hydroxylated and basic residues, is both necessary and sufficient for translocation. In contrast to plant chloroplasts, the plastids of several other organisms are surrounded by either three or four membranes. These plastids are thought to have been acquired through a secondary endosymbiosis (McFadden and Gilson, 1995; Palmer and Delwiche, 1996). Protein transport to these plastids Toxoplasma gondii is an obligate intracellular parasite that is capable of infecting all mammalian cell types and establishing a latent infection in the host organism. T. gondii is a unicellular parasite and during the tachyzoite phase of its life cycle it resides in a parasitophorous vacuole within the host cell where it divides synchronously every 8-10 hours. The host cell then lyses, releasing up to 28 tachyzoites, which infect new host cells. Tachyzoites can also differentiate into bradyzoites, which are a quiescent cyst-forming stage that can persist for the lifetime of the host (Lüder and Gross, 1998). The infection is suppressed by the immune system in healthy individuals, but T. gondii causes significant morbidity and mortality in immunocompromised individuals, including AIDS and chemotherapy patients. T. gondii is also a leading cause of birth defects. It is used as a model system for other apicomplexan parasites including Plasmodium sp., the causative agent of malaria. T. gondii, like most other apicomplexan parasites, contains an organelle that is related to plastids such as chloroplasts. This organelle, called the apicoplast, is thought to be an essential organelle (Fichera and Roos, 1997) and thus is an attractive candidate as a therapeutic target. For example, recent evidence Key words: Toxoplasma gondii, Apicoplast, Protein targeting, Mitochondrion, Chloroplast 3970 A. DeRocher and others is mediated by an N-terminal bipartite targeting sequence composed of an ER signal sequence followed by a chloroplast transit peptide-like domain (Schwartzbach et al., 1998). Like the chloroplasts of diatoms and euglenoids, the T. gondii apicoplast appears to have arisen by secondary endosymbiosis (Köhler et al., 1997). The inner two membranes are presumably derived from the chloroplast inner and outer membranes, the third from the endosymbiont plasma membrane, and the outer membrane from the phagocytic vacuole of the ancestral apicomplexan. Sequence analysis suggests that the apicomplexan endosymbiont was a green alga (Köhler et al., 1997). The 35 kb apicoplast genome is the smallest plastid genome described to date, with only a handful of protein coding genes. Thus, it is likely that most apicoplast proteins are encoded in the nucleus. A few nuclearly encoded plastid proteins of T. gondii have been identified and these contain an N-terminal extension with characteristics of a bipartite targeting peptide (Waller et al., 1998). The N-terminal segment has characteristics of an ER signal sequence, and the region between the putative signal sequence cleavage site and the predicted mature protein is rich in hydroxylated and basic residues, characteristics of chloroplast transit peptides. The Nterminal extension of acyl carrier protein (ACP) has been shown previously to direct a reporter protein to the apicoplast (Waller et al., 1998). We demonstrate that, as predicted, the targeting sequence of ribosomal protein S9 (S9) is functionally bipartite, with the N-terminal portion targeting a reporter protein for secretion. The transit portion is required for localization to the apicoplast; however, much of the transit peptide is dispensable for targeting to the organelle. The transit peptide alone can target a reporter protein to the T. gondii mitochondrion in vivo, and it is capable of directing import into higher plant chloroplasts in vitro. MATERIALS AND METHODS Cloning and vector construction A partial ribosomal protein S9 sequence (rps9 gene) was identified from the T. gondii EST database, and the corresponding cDNA, zy33g01, was obtained from Dr L. D. Sibley, rescued and sequenced. The 5′ end of the rps9 cDNA was obtained by PCR amplification from a cDNA library, TgME49 (gift of Drs J. Boothroyd and I. Manger), with primers SC1 and SC2 (Table 1), cloned into pGEM-T and sequenced (GenBank accession no. AF087139). The T. gondii green fluorescent protein (GFP) expression vector ptubP30-GFP/sag-CAT (Striepen et al., 1998), a gift from Drs D. Roos and B. Streipen, was modified for better GFP fluorescence by replacing the GFP coding sequence with GFP(S65T) provided by Dr Kami Kim. An AvrII site (CCTAGG) was added to the 5′ end of the GFP coding sequence to assist cloning. The initial S9 constructs were prepared by polymerase chain reaction (PCR) amplification from TgME49 DNA using primers that inserted a BglII site at the 5′ end and an AvrII site at the 3′ end of the fragment. Fragments encoding the indicated residues of S9 were amplified using the following primers: S9(1-42), SA1s and SA5a; S9(1-159), SA1s and SA2a; and S9(33-272), SA3s and SA4a. Primer sequences are listed in Table 1. The fragments were cloned into pCR2.1TOPO (Invitrogen, Carlsbad, CA, USA) and the sequences verified. The inserts were removed by digestion with AvrII and BglII and cloned into the modified ptubP30GFP/sag-CAT vector that had been digested with the same enzymes to excise P30. The resulting plasmids pS9(1-42)-GFP, pS9(1-159)GFP and pS9(33-272)-GFP, encode GFP fusion proteins with the T. gondii S9 sequence at the N terminus. The S9 and junction regions were verified by DNA sequencing. Sequences encoding residues 1-143 of the predicted apicoplast ribosomal protein L28 were amplified from cDNA library TgME49 using primers LA1s and LA2a. The acpP coding sequence was PCR amplified using primers AA1s and AA2a (Table 1) as described (Waller et al., 1998). The resulting fragments were cloned into pCR2.1TOPO, verified by sequencing, and then cloned into the T. gondii GFP expression plasmid as described above. Deletion constructs were generated from pS9(1-159)-GFP using the Quick Change Site Directed Mutagenesis kit (Stratagene, LaJolla, CA, USA) according to the manufacturer’s protocol. The construct pS9(1-132)-GFP was generated using primers SM1s and SM2a, pS9(1-95)-GFP was generated using SM3s and SM4a, and pS9(146,79-159)-GFP was generated using SM5s and SM6a. T. gondii transfection The RH strain of T. gondii was grown in human foreskin fibroblasts as described (Roos et al., 1994). Extracellular parasites were transfected with 50 µg of circular plasmid (Roos et al., 1994) via electroporation in a 2 mm cuvette at 2 kV, 25 µF using a Biorad Gene Pulser II. A portion of the cells were used to inoculate chamber slides for viewing 18-24 hours post-transfection, and the rest were subjected to selection with 20 µM chloramphenicol to isolate stable transfectants. Microscopy T. gondii were grown overnight on chamber slides and either viewed directly, or processed for immunofluorescence assays. Slides were fixed in 4% paraformaldehyde in PBS for 5 minutes, then processed as described (Das et al., 1998). Primary antibodies were rabbit antiGFP (a gift from Dr Jim Cregg) (1:1000) and mouse anti-T. gondii mitochondrial HSP60 (a gift from Dr Stanislas Tomavo) (1:100). Secondary antibodies were Texas Red-coupled anti-rabbit IgG and fluorescein isothiocyanate (FITC)-coupled anti-mouse IgG (Southern Biotechnology, Birmingham, AL, USA). Where indicated, slides were incubated with 10 mM 4,6-diamidino-2-phenylindole (DAPI) for 10 minutes. Slides were mounted with antifade (Molecular Probes, Eugene, OR, USA) and viewed on a Nikon Microphot FX microscope equipped with a Photometrics SenSys camera and MetaMorph software. The DAPI filter had excitation and emission peaks of 360 and 460 nm, the Texas Red filter had excitation and emission peaks of 560 and 630 nm, and the FITC filter had excitation and emission peaks of 484 and 510, nm respectively. Extracellular parasites were stained with 1 µg/ml MitoTracker Orange CM-H2-TMRos (Molecular Probes, Eugene OR, USA) for 30 minutes, rinsed, then incubated with Table 1. PCR primers Name Oligonucleotide sequence* SC1 SC2 SA1s SA2a SA3s SA4a SA5a SM1s SM2a SM3s SM4a SM5s SM6a LA1s LA2a AA1s AA2a CGGCTGCGTCTCTGTTGTTG GGAACAGCTATGACCATG AGATCTAAAATGGCCCTCGAACGTTG CCTAGGCCTTGCATAAGACCTTTTCCG AGATCTAAAATGACTGCCTTCATAGTCCCTCAAC CCTAGGCCGTTTGCTGTACTGTTCCTTC CCTAGGCAGGTTGCGTTGAGGGACT GATGCGACACTCTCAATGCATAAAGGAGAAC GTTCTCCTTTATGCATTGAGAGTGTCGCATC CGGCCTGGTCTGCGATGCATAAAGGAGAAC GTTCTCCTTTATGCATCGCAGACCAGGCCG CTGCATAGCTTTACCTCCCCTCGGTCACTC GAGTGACCGAGGGGAGGTAAAGCTATGCAG AGATCTAAAATGCAACGACCAAGGAGCGGG CCTAGGGCACTCCATAGCCTGAAGAGG GGAAGATCTAAAATGGAGATGCATCCCCGCAACGC TGGACCTAGGCCGATCATCAGAACTCGCCTCGT *Restriction endonuclease recognition sites are underlined. Targeting to the T. gondii plastid 3971 fibroblasts for 2 hours prior to viewing. This procedure avoided the staining of host cell mitochondria, which cluster around the parasite (Sinai et al., 1997). Where indicated, cells were incubated with 10 µM cycloheximide for 1 hour prior to viewing. This concentration of cycloheximide has been shown to be sufficient to inhibit >95% protein synthesis in parasites (Beckers et al., 1995). Longer cycloheximide treatment resulted in an increasing proportion of cells (of both cell lines) showing atypical morphology and aberrant fluorescence patterns. In vitro transcription and translation For in vitro translation reactions, the GFP fusion inserts were PCRamplified from the T. gondii GFP expression plasmids using primers SA3s and SA4a. The PCR products were cloned into pGEM easyT (Promega, Madison, WI, USA) and sequenced. In vitro transcription/translation reactions were performed using either a T7 TnT-coupled wheat germ extract system (Promega, Madison WI, USA) (for the experiments shown in Fig. 5), or sequential T7 transcription and translation using a wheat germ extract (for the experiments shown in Fig. 6). For import assays, synthesized proteins were labeled with [35S]methionine (NEN, Boston, MA, USA; 43 TBq/mmol) according to the manufacturer’s protocol. In vitro translation of chloroplast ribulose-1,5-bis phosphate carboxylase/ oxygenase small subunit (RSS) from Pisum sativum was performed using the rabbit reticulocyte system (Promega, Madison WI, USA). Immunoblot analysis Lysates were prepared from 107 T. gondii stably transfected with the indicated constructs by suspending washed parasites in SDS-sample buffer and boiling for 2 minutes. Samples were analyzed by SDSPAGE, and immunoblotted using anti-GFP (1:10,000 dilution). Bound antibody was detected with 125I-protein A (NEN, Boston MA, USA). Protein import into chloroplasts Proteins were translated in vitro and then imported into isolated pea chloroplasts as previously described (Bruce et al., 1994). Intact chloroplasts were recovered by sedimentation through a 40% (v/v) Percoll cushion and treated with thermolysin as described (Cline et al., 1984) in the presence or absence of 1% v/v Triton X-100 (final concentration). The proteolytic digestion was stopped by the addition of EDTA to 10 mM. Chloroplasts were sedimented through 40% Percoll containing 5 mM EDTA and the resulting pellets were resuspended in lysis buffer (25 mM Hepes-KOH, pH 8.0, 4 mM MgCl2), and incubated on ice for 15 minutes. After ultracentrifugation at 100,000 g, crude membrane and soluble fractions were obtained and solubilized in 2× SDS-PAGE sample buffer. All fractions were analyzed by SDS-PAGE and fluorography. RESULTS The N-terminal extension of apicoplast proteins confers targeting We searched for the T. gondii database for sequences related to known plant chloroplast proteins and identified several possible apicoplast proteins, including ribosomal proteins S9 and L28, and ACP. BLAST analysis (http:// www.ncbi.nln.nih.gov/BLAST/) (Altschul et al., 1997) showed that the T. gondii S9 and L28 sequences were most similar to chloroplast and bacterial homologues. T. gondii ACP was most similar to bacterial ACPs, and had lower but significant similarity to ACP from algae and plants. Recently, Waller et al. (Waller et al., 1998) used specific antisera to show that ACP and S9 localize to the plastid and suggested that L28 was also a plastid protein. All three predicted proteins include over 100 amino acids (aa) N-terminal to the region of interspecies conservation, consistent with the presence of N-terminal targeting domains (Fig. 1A). SignalP analysis (http:// www.cbs.dtu.dk/services/SignalP/) (Nielsen et al., 1997) predicts that S9, L28, and ACP are all targeted to the ER using a cleavable signal sequence. ChloroP (http:// www.cbs.dtu.dk/services/ChloroP/) (Emanuelsson et al., 1999) predicts that the region between the signal sequence and conserved core of S9, L28 and ACP each could function as chloroplast transit peptides. Unmodified GFP is cytoplasmic when expressed in T. gondii (Striepen et al., 1998). In order to analyze plastid targeting in T. gondii, GFP fusion constructs were prepared in a T. gondii expression vector (Striepen et al., 1998) (Fig. 1B). The encoded proteins contain full-length ACP, aa 1-159 of S9 or aa 1-143 of L28 fused to the N terminus of GFP. The S9 fusion protein included the predicted signal and transit sequences plus approximately ten downstream residues to maintain context. As expected from the studies of Waller et al. (Waller et al., 1998), ACP-GFP localized to a single, dot-like region of the cell (Fig. 2, pseudocolored green). Fig. 2 also shows DAPI staining (pseudocolored red), which detects the nuclear and apicoplast DNA but not the uncharacterized mitochondrial genome (Feagin, 2000). The merged red and green signals yield a yellow dot-like structure, demonstrating that this compartment is indeed the apicoplast. The first 159 aa of S9 and the first 143 aa of L28 also efficiently targeted GFP to the apicoplast (Fig. 2). This observation confirms that the Nterminal extension of several apicoplast proteins is sufficient to direct a reporter protein to the apicoplast. Deletion analysis of the S9 transit sequence A series of deletion constructs was prepared to assess the importance of different parts of the S9 transit peptide domain in targeting GFP to the apicoplast. This region of S9 is over 100 aa long. We examined this region for consensus cleavage sites for processing of proteins imported into chloroplasts. A green algal consensus cleavage site (V X A ↓ X) (Franzén et al., 1990) occurs at aa 92, and a dinoflagellate consensus cleavage site (A X A ↓ X) (Sharples et al., 1996) occurs at aa 129 (Fig. 1A). While it is uncertain if T. gondii recognizes these sites, we prepared deletion constructs specifying fusion proteins spanning each site: pS9(1-95)-GFP and pS9(1-132)GFP. The encoded proteins include three residues downstream of the possible cleavage site to maintain the potential cleavage context. An additional deletion construct, pS9(1-46, 79-159)GFP, eliminated the region coding for 32 aa that had the least similarity to canonical transit peptides. S9(1-95)-GFP (Fig. 2), S9(1-46, 79-159)-GFP (Fig. 2) and S9(1-132)-GFP (data not shown) all localized to the apicoplast. However in the parasites expressing S9(1-46, 79-159)-GFP, some green fluorescence was observed outside the plastid as well, suggesting that this fusion protein was not targeted as efficiently as the others. This evidence argues that a large segment of the T. gondii S9 transit peptide is dispensable for targeting to the organelle. We tested a further deletion of the transit sequence by constructing pS9(1-42)-GFP, which encodes GFP fused to the predicted signal sequence plus 10 aa of the transit peptide. If the predicted signal sequence is functional, this protein would be expected to be targeted for secretion. As a control, we used P30-GFP (Fig. 3A,B), which is known to be secreted into the 3972 A. DeRocher and others A Ribosomal protein S9 MALERWCRGV NLHSFTRNFR RSLPGSVGQV EPTALGADAT RLPPGSGKLT NEFDIIAEAH RRAGFLTVDA RCSLPSWAVM VLPLVGKRWF TAWSAVTEGC LSSIPSELPE INNRDAADYL GGGLGGQSGA RKVERKKFGL CLAYFLCGPA AGDAAVQHCK PSFEDAVHET PGTPRNSFSW QDNPWWIHNC IMLAVAREIV RKARKKEQYS VGTAFIVPQR GWFQQTGLSP SLPSDWVERS GTRKRSYARV IAPLMELQLE RQRPELRPPL KR 272 40 80 120 160 200 240 KTLLALALFM GVCATPSTAS SALFAADEAS FIKDLDADSL ALSYIEKAKS ATSIASSYGF LGTLGQPAGT SDDRPLLERV DSVELVMAFE ATA 183 VSPGLIRFNY VARRPGPFRS KDVVADQLGV EKFGVSIPDE 40 80 120 160 FRLSRWSASA PSGCTNEFFW STVRRHYGIK GKMDNTKARN RLRLSVKGIR RMVSRLSLAG KM 272 40 80 120 160 200 240 ACP MEMHPRNAGR RYGTCPNMSS VSANVIGSPS DRARINPESN EASKIATVQD Ribosomal protein L28 MQRPRSGRVC LCPAQSPSCV RRHGSQTALG GRGGGKLRLG RSHSRVATHK TIKKYGLQRA GTAGHINADE VYLVTNVLLA GTAHRLSTPL ISSRRHLQVI KHTGVPLQAM VQRLNLHWKR ADKFGLNLKK IAGPLSGQRE LSIWLSRASA IAAGCELGSF RRKKYRTKGT ECPARRCMLL LWWPEAGYYV KKYYAGYSHR TSTIARVDAA B Loc. S9(1-159)-GFP P S9(1-132)-GFP P S9(1-95)-GFP P S9(1-46,79-159)-GFP P S9(1-42)-GFP S S9(33-159)-GFP M S9(33-272)-GFP M ACP(1-183)-GFP P L28(1-43)-GFP P P30-GFP S 50 aa Fig. 1. T. gondii apicoplast proteins and GFP fusions. (A) The predicted aa sequences of S9, ACP and L28 are shown. The font indicates the approximate region of the protein as predicted by sequence analysis: signal sequence in bold; transit in italics; mature protein in regular font. The latter was deduced by homology to the corresponding mature proteins as follows: Arabidopsis thaliana S9, Cryptomonas ACP and A. thaliana L28. The transit peptide cleavage site predicted by ChloroP, which applies to land plants, is bold underlined. The residues in each sequence in the –1 position of putative green algal and dinoflagellate transit peptide cleavage consensus sites are single and double underlined, respectively. (B) GFP fusion proteins expressed in T. gondii. Regions corresponding to the putative signal sequence of each protein are diagonally striped, the putative transit peptide is stippled, and the mature domain is solid. GFP is fused at the C terminus of the depicted segments. Observed targeting of the proteins in T. gondii is shown at the right: plastid (P), secretory system/parasitophorous vacuole (S) and mitochondrion (M). parasitophorous vacuole by dense granules (the GFP fusion protein lacks the P30 glycophosphatidyl inositol anchor site; Striepen et al., 1998). In cells expressing pS9(1-42)-GFP, fluorescence was clearly observed in the parasitophorous vacuole, indicating that some of the S9(1-42)-GFP had been secreted. Additionally, fluorescence was seen within the parasite itself (Fig. 3E,F). When these cells were treated with 10 µM cycloheximide for 1 hour, less intracellular fluorescence was seen (Fig. 3G,H). This observation suggests that the intracellular fluorescence seen in the pS9(1-42)-GFP cell line may represent proteins en route to the parasitophorous vacuole. Cycloheximide treatment had little effect on the fluorescence pattern of cells expressing P30-GFP, since at steady state the vast majority of that protein has already reached its destination (Fig. 3C,D). Similarly, cycloheximide had no effect on the fluorescence pattern of cells expressing apicoplast-targed GFP fusions (not shown). Secretion of the S9(1-42)-GFP fusion protein strongly argues that protein targeting to the apicoplast utilizes the secretory system. The data also indicate that, in addition to the signal sequence, information in the transit peptide is required for S9 to reach its proper destination. The S9 transit peptide targets to the mitochondrion To further investigate the importance of the signal peptide in apicoplast targeting, we prepared two constructs encoding S9GFP fusion proteins lacking the signal peptide: S9(33-272)GFP and S9(33-159)-GFP. Surprisingly, parasites expressing these fusion proteins (Fig. 4C,D,I,J) did not produce the cytoplasmic fluorescence seen with GFP alone (Fig. 4A,B). Instead, fluorescence was confined to a thread-like structure, which typically was located near the plasma membrane of the parasite. This compartment is reminiscent of the T. gondii mitochondrion (Seeber et al., 1998). In eukaryotes, the HSP60 proteins are restricted to plastids or mitochondria and are not present in the secretory system or cytosol. Recently the classical organelle marker HSP60 has been identified in T. gondii (Yahiaoui et al., 1999) and has been shown to be mitochondrial by microscopic analysis and subcellular fractionation (Toursel et al., 2000). The pSORT plant matrix (http://psort.nibb.ac.jp/) (Nakai and Kanehisa, 1992), which predicts the compartments in a cell to which a given protein are most likely to be targeted, predicts T. gondii HSP60 to target to the mitochondrial matrix (0.50). We performed colocalization studies of S9(33-272)-GFP and HSP60. GFP was revealed with rabbit anti-GFP followed by Texas Red-goat anti-rabbit IgG while HSP60 was revealed with mouse anti-T. gondii HSP60 followed by FITC-goat anti-mouse IgG. S9(33272)-GFP (Fig. 4D) and HSP60 (Fig. 4E) colocalize within the cell (Fig. 4F). In these experiments, the cells were fixed and permeabilized prior to antibody treatment, which eliminated intrinsic GFP fluorescence (Fig. 4G) while allowing staining with anti-GFP (Fig. 4H). Recently, the mitochondrion of T. gondii was shown to stain with Mitotracker (Melo et al., 2000). In live cells intrinsic fluorescence of S9(33-159)-GFP (Fig. 4K) colocalizes with Mitotracker staining (Fig. 4L, merged in Fig. 4M), confirming the identity of the compartment as the mitochondrion. Similar colocalization was seen for S9(33272)-GFP and Mitotracker and for S9(33-159)-GFP and HSP60 (not shown). Thus, the transit peptide of S9, when not preceded by a signal sequence, efficiently targets GFP to the mitochondrion. Targeting to the T. gondii plastid 3973 Fig. 2. Subcellular localization of GFP fusion proteins in T. gondii. The location of the indicated GFP fusion protein in T. gondii was examined by immunofluorescence analysis using anti-GFP. Cells were counterstained with DAPI. The images were pseudocolored green for GFP and red for DAPI; overlapping signals result in yellow in the overlay. (Red pseudocoloring was used for DAPI since it results in a more obvious overlay than blue pseudocoloring). The experiments used stable transfectants expressing ACP-GFP, S9(1-159)-GFP and S9(1-95)-GFP, and transient transfectants expressing S9(1-46,79-159)-GFP and L28(1-143)-GFP. All images are at the same magnification; bar, 10 µm. A positively charged, amphipathic alpha helix is characteristic of mitochondrial targeting sequences (Rosie et al., 1988). Helical wheel projection of the transit peptide domain of S9 (Protean module, DNAStar software) reveals such a helix between aa 40 and 52 (data not shown). The transit peptide domains of L28 and ACP can also be modeled as amphipathic alpha helices, albeit shorter helices than that of S9. Protein sequences C-terminal to the predicted signal sequence cleavage sites of these proteins were analyzed using the PSORT plant matrix. These regions of T. gondii S9 and L28 were both predicted to have a strong propensity to target to the mitochondrion (0.64 and 0.73, respectively), while T. gondii and P. falciparum ACP had a lower (0.1) likelihood. GFP fused to the transit peptide of the latter was recently shown to be cytoplasmic in P. falciparum (Waller et al., 2000). Similar analysis of the sequences of proteins targeted to other compartments in the secretory system (Gra1, Gra2, Gra3, Gra4, Gra5, Gra6, Gra7, Gra8, Bip, apyrase, NTPase II, Mic1, and Rop2) showed that they had a low (≤0.1) likelihood of targeting to the mitochondrion. In vivo processing To assess whether the fusion proteins were proteolytically processed, S9-GFP fusion proteins from transfected parasites were analyzed by SDS-PAGE and immunoblotting with antiGFP. The migration of the in vivo expressed fusion proteins (Fig. 5A) were then compared to the migration of in vitro translated proteins (Fig. 5B). The in vivo expressed proteins with an encoded signal sequence migrated similarly to in vitro translation products lacking the signal sequence. For example, S9(1-42)-GFP expressed in vivo migrated similarly to GFP translated in vitro, and S9(1-95)-GFP and S9(1-159)-GFP migrated similarly to in vitro translation products of S9(33-95)GFP and S9(33-159)-GFP, respectively. These data indicate that the S9 signal sequence is removed in vivo. Since the majority of S9(1-95)-GFP and S9(1-159)-GFP are not processed to the size of GFP in vivo, it appears that much of the transit peptide domain remained attached to GFP even when the protein was localized to the apicoplast. Four immunoreactive proteins were detected in parasites expressing the mitochondrially targeted S9(33-159)-GFP (Fig. 5A), the largest of which is slightly smaller than the in vitro translated molecule, while the smallest comigrates with GFP. From the gel profiles, it is clear that the fusion proteins, particularly those with large segments of the S9 leader, migrated anomalously slowly. The sequence does not show any obvious clues as to why this should be. Cloning artifacts were ruled out by DNA sequencing. Chloroplast targeting The bipartite targeting sequence model predicts that the transit peptide domain of the targeting signal is derived from an ancestral chloroplast transit peptide. We therefore tested the ability of the transit peptide fusions S9(33-132)-GFP and 3974 A. DeRocher and others Fig. 3. The S9 signal sequence directs secretion of GFP. T. gondii were stably transformed with constructs encoding P30-GFP (A-D) and S9(1-42)-GFP (E-H). Cells shown in (C,D,G,H) were treated with cycloheximide for 1 hour prior to viewing. Green fluorescence and transmitted light pictures of live cells are shown. Bar, 10 µm. S9(33-159)-GFP to be imported into pea chloroplasts in vitro (Fig. 6). The nuclearly encoded chloroplast protein RSS and GFP were used as positive and negative controls, respectively. Proteins were synthesized by in vitro translation in the presence of [35S]methionine and then incubated with purified chloroplasts under import conditions. In each panel, the first lane (T) shows the total in vitro translated protein profile. In addition to the full-length product seen in the in vitro translations, a few smaller species were also seen for the S9GFP fusions. Since the transcripts do not encode any methionines between the start codon engineered for the transit sequence and the in-frame methionine at the beginning of GFP, we suggest that these species may represent premature termination products. The next pair of lanes shows the radiolabelled proteins that associated with chloroplasts (P lanes contain the membrane proteins and S lanes contain the soluble chloroplast proteins). The subsequent lanes show the accessibility of the proteins to protease in the presence or absence of detergent, used to test for translocation into a membrane-bound compartment. Like RSS, S9(33-132)-GFP and S9(33-159)-GFP associated with chloroplasts and were Fig. 4. The S9 transit peptide directs proteins to the mitochondrion. Parasites expressing GFP were viewed with transmitted light (A) and for green fluorescence (B). T. gondii stably transfected with pS9(33-272)-GFP were fixed and viewed under visible light (C), or stained with anti-GFP and Texas Red-anti-rabbit IgG (D); or with anti-HSP60 and FITC anti-mouse IgG (E). Signals from D and E are merged in (F). (G,H) Fixed cells stained with anti-GFP and Texas Red secondary antibody, viewed in the green and red channels, respectively. Live parasites transfected with S9(33-159)-GFP were viewed under visible light (I) and green fluorescence (J), or stained with Mitotracker then viewed under red (K) and green (L) fluorescence and overlayed (M). Bars, 10 µm. found in the soluble fraction. S9(33-132)-GFP and S9(33-159)GFP proteins were also protected from digestion with thermolysin, but were sensitive to protease upon addition of detergent. These data demonstrate that S9(33-132)-GFP and S9(33-159)-GFP were imported and were not merely associated with the chloroplasts. In contrast to the above proteins, GFP was not imported into chloroplasts (Fig. 6) nor Targeting to the T. gondii plastid 3975 Fig. 5. Processing of GFP fusion proteins in T. gondii. (A) GFP fusion proteins in T. gondii. Lysates were prepared from parasites stably transformed with the indicated clones and proteins were separated by SDS-PAGE on a 10% acrylamide gel. The gel was processed by immunoblotting with anti-GFP and bound antibodies were revealed with 125I protein A and autoradiography. Numbers at the left mark the migration of molecular mass markers (kDa). The subcellular location of each GFP fusion protein is indicated at the bottom: cytoplasm (C), secretory/parasitophorous vacuole (S), plastid (P) and mitochondrion (M). (B) In vitro translation products. The inserts of pGEM plasmids encoding GFP, S9(33-95)-GFP, and S9(33-159)-GFP were transcribed, translated in vitro and separated by SDS-PAGE. The gels were processed by immunoblotting with anti-GFP antiserum as above. was S9(33-46, 79-159)-GFP (not shown). Thus, the ability of the transit peptide deletion constructs to direct GFP to chloroplasts in vitro generally, but not perfectly, reflects the ability of the same peptides to direct GFP to the T. gondii apicoplast in vivo. For the imported GFP fusion proteins, the major species (marked by arrowheads) is approximately 10 kDa smaller than the full-length in vitro translation product. A smaller imported species (approximately the size of GFP) is also seen, as well as some intermediately sized molecules. The ChloroP program predicted that the transit peptide domain of S9 could direct import into the chloroplast with a proposed processing protease cleavage site at aa 89 using the land plant consensus. Our data are consistent with the hypothesis that the S9-GFP fusions are imported into pea chloroplasts with an initial cleavage site near that predicted by ChloroP, followed by additional proteolysis within the chloroplast. DISCUSSION Multiple factors make the study of the T. gondii plastid particularly interesting. Protein targeting to the apicoplast is probably essential for parasite viability, and as such may be a useful target for novel pharmaceuticals. Additionally, understanding protein targeting to plastids in evolutionarily divergent organisms will help highlight the essential shared features of this process in chloroplasts and apicomplexan parasites. Since T. gondii tachyzoites are haploid and are amenable to targeted gene replacement, T. gondii is a potential Fig. 6. Chloroplast targeting. The indicated proteins were translated in vitro in the presence of [35S]methionine and incubated with chloroplasts. After import, chloroplasts were sedimented through a Percoll cushion, and incubated with or without thermolysin and Triton X-100 as indicated. The chloroplasts were hypotonically lysed, and then separated into pellet (P) and supernatant (S) fractions. The pellet fractions made in the absence of Triton X-100 contain the membranes. Protein samples were analyzed by SDSPAGE on a 7.5%-15% acrylamide gradient gel and viewed by fluorography. Arrowheads indicate migration of the largest imported protein. Total in vitro translation products are shown in lane T. model system for understanding protein import into complex plastids. Two groups have demonstrated that chimeric proteins expressed in T. gondii can target to the apicoplast (Waller et al., 1998; Jomaa et al., 1999). Waller et al. demonstrated that the N-terminal 104 aa of ACP is sufficient to target a reporter protein to the apicoplast. They also proposed that, as seen in other plastids with more than two membranes, a bipartite targeting sequence directs targeting to the apicoplast. We have extended this work by demonstrating the ability of the N terminus of additional proteins, S9 and L28, to target GFP to the apicoplast in vivo. More importantly, we have shown that the targeting sequence can be functionally separated into two domains that direct a reporter protein to different compartments in vitro and in vivo. The predicted signal sequence of S9, when fused to GFP, directed secretion of the reporter protein. The signal sequence was removed from GFP in parasites transfected with S9(1-42)GFP, consistent with import into the ER. Since the signal sequence is both sufficient for ER targeting and necessary for apicoplast targeting, transport to the apicoplast must proceed through the endomembrane system. Shortly before submission of this work, Waller et al. reported that the predicted signal sequence of P. falciparum ACP targets GFP for secretion in 3976 A. DeRocher and others that organism (Waller et al., 2000). Taken together, the data indicate that trafficking through secretory system is likely to be a general feature of apicoplast targeting. Our data also show the necessity of a transit sequence to direct the reporter to the plastid once it has entered the secretory system. Like chloroplast transit peptides, the T. gondii plastid transit peptides have no primary sequence similarity, yet are rich in basic and, to a somewhat lesser extent, hydroxylated amino acids. We found that substantial portions of the T. gondii S9 transit peptide could be deleted with no apparent effect on targeting to the apicoplast, raising the possibility that some information may be redundant or extraneous. It is also possible that these partial transit sequences target GFP to one of the apicoplast membranes or intermembrane spaces, as this would probably be indistinguishable from lumenal targeting in our immunofluorescence assays. The most abundant products in parasites expressing S9(1-95)-GFP or S9(1-159)-GFP comigrate with the corresponding in vitro translation products lacking the signal sequence. Thus, these fusion proteins are apparently not processed further upon localization to the apicoplast. Whether this finding results from inefficient processing, improper presentation of the cleavage site, or intermediate localization as mentioned above remains unclear. Interestingly, immunoblots of the endogenous protein using anti-S9 show relatively high levels of precursor (Waller et al., 1998), suggesting that plastid processing of S9 may be inefficient. The need for both signal sequence and transit peptide in apicoplast targeting can be traced in an evolutionary flow diagram incorporating a secondary endosymbiosis (Bhaya and Grossman, 1991). The secondary endosymbiont theory posits that the progenitor apicomplexan internalized a photosynthetic alga already containing a plastid (Köhler et al., 1997). Plastid proteins encoded in the algal nucleus already had a transit peptide to direct them to the chloroplast. Captured within a phagocytic vacuole of the parasite’s endomembrane system, the endosymbiont transferred genes for plastid proteins to the apicomplexan nucleus. At this point, they acquired sequences specifying an N-terminal signal peptide to direct them into the endomembrane system. From there, the proteins would be targeted to the apicoplast using their transit peptide. Our data show that the transit peptide of apicoplast S9 can direct import of a reporter protein into isolated pea chloroplasts, emphasizing the functional similarity of the transit peptides for the two organelles. Consistent with the model above, bipartite signal sequences have also been described for other organisms whose plastids are thought to have been acquired through secondary endosymbioses (reviewed by Schwartzbach et al., 1998). Examples include diatoms, which possess chloroplasts with four membranes, and euglenoids, whose chloroplasts have three membranes. In diatom bipartite targeting sequences, the ER signal sequence function has been demonstrated by its ability to mediate import into microsomes (Bhaya and Grossman, 1991; Lang and Apt, 1998). The transit peptide portion of at least one protein, the ATPase γ subunit, can direct translocation into pea chloroplasts in vitro. In euglenoids, chloroplast proteins that are encoded in the nucleus, such as light harvesting complex protein II, have a bipartite targeting sequence with an N-terminal ER signal sequence and a region resembling a typical chloroplast transit peptide (Kishore et al., 1993). Transport of this protein through the Golgi has been detected by immunoelectron microscopy (Osafune et al., 1991) and by density gradient sedimentation (Sulli and Schwartzbach, 1995). These data suggest that proteins destined for euglenoid plastids are transported through the Golgi to the plastid via vesicles that fuse with the outer of the three chloroplast membranes, and then the proteins are imported in a conventional manner. In T. gondii and diatoms, however, a fourth membrane must be passed; how this occurs is still unknown. The transit peptide segment of the S9 targeting sequence directed GFP to the T. gondii mitochondrion. This crosstargeting emphasizes the similarity of chloroplast and mitochondrial targeting sequences: both are at the N terminus, rich in serine and threonine, and poor in acidic residues (von Heijne et al., 1989). However, the secondary structure of the two types of targeting sequences differ, with mitochondrial targeting sequences generally forming an amphipathic alpha helix. The vast majority of targeting peptides direct proteins exclusively to a single organelle, but there are a few examples of transit peptides that direct proteins to both chloroplasts and mitochondria in vivo. Arabidopsis thaliana has a single methionyl-tRNA synthase, which is imported into both chloroplasts and mitochondria both in vivo and in vitro (Menand et al., 1998). Other examples of dual targeting include the leader sequences of a yeast mitochondrial cytochrome oxidase subunit Va and pea chloroplast glutathione reductase, each of which can target to both chloroplasts and mitochondria in transgenic tobacco in vivo (Huang et al., 1990; Creissen et al., 1995). The rarity of dual targeting corroborates the intuitively obvious importance of maintaining distinct targeting mechanisms to the two organelles in higher plants. T. gondii has achieved this in part by sequestering the plastid within the secretory system. The ability of the S9 transit peptide to direct a reporter protein to the mitochondrion in vivo, the chloroplast in vitro, and the apicoplast when preceded by a signal peptide is a striking example of the similarity of certain organellar import signals. Our data suggest that the characteristics recognized by the apicoplast import apparatus and those recognized by the T. gondii mitochondrial import apparatus are not mutually exclusive. 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