Traffic 2008; 9: 665–677 Blackwell Munksgaard
# 2008 The Authors Journal compilation # 2008 Blackwell Publishing Ltd
doi: 10.1111/j.1600-0854.2008.00736.x
Identification of Trafficking Determinants for Polytopic Rhomboid Proteases in Toxoplasma gondii Lilach Sheiner1, Timothy J. Dowse1,2 and Dominique Soldati-Favre1,* 1
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland 2 Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, UK *Corresponding author: Dominique Soldati-Favre,
[email protected] Rhomboids (ROMs) constitute a family of polytopic serine proteases conserved throughout evolution. The obligate intracellular parasite Toxoplasma gondii possesses six genes coding for ROM-like proteases that are targeted to distinct subcellular compartments: TgROM1 localizes to regulated secretory organelles, micronemes, TgROM2 is present in the Golgi, while TgROM4 and TgROM5 are found in the pellicle of the parasite. The targeting mechanism/s of ROM proteins is an aspect that has not yet been assessed. The existence of TgROM family members localized to different subcellular compartments provides a convenient system to study their sorting mechanisms in a genetically tractable organism that possesses an elaborate secretory pathway and conserved trafficking machineries. In this study, we experimentally established the topology of TgROM1 and TgROM4 at the plasma membrane and applied domain-exchange and site-directed mutagenesis approaches to identify critical sorting determinants on the N-terminal cytosolic domains of TgROM2 and TgROM1 that confer their Golgi and post-Golgi localizations, respectively. Key words: adaptor protein, Golgi, microneme, polytopic, protease, rhomboid, Toxoplasma gondii, trafficking Received 17 September 2007, revised and accepted for publication 9 March 2008, uncorrected manuscript published online 11 March 2008, published online 27 March 2008
Compartmentalization in eukaryotic cells is an elegant solution used in nature to control the organization of a vast number of proteins and their associated biological functions. Numerous studies have established the importance of short amino acid sequences within the targeted protein and their recognition by components of the vesicular trafficking machinery in mediating selective sorting. Wellcharacterized motifs dictate the localization of proteins along the secretory pathway. Endoplasmic reticulum (ER) localization is mediated by retention as well as by retrieval signals (1,2). Retrieval motifs are recognized by sorting re-
ceptors of the coat protein (COP)I-coated vesicles that mediate retrograde transport. ER export, however, is mediated by COPII-coated vesicles. Di-acidic or di-hydrophobic motifs are commonly involved in the interaction of cargo proteins with COPII (3). After traveling from the ER to the Golgi, and through the Golgi itself, the trans Golgi network (TGN) has a central role as a platform of protein sorting. Post-Golgi sorting is mediated by clathrin-coated vesicles where signals on the cargo protein are recognized by adaptor proteins (APs), such as the Golgi-localizing gammaadaptins (GGAs) and the AP complexes (4). Among the peptide-sorting motifs recognized by components of the AP complexes are the tyrosine-based sorting signals such as NPXY and YXXF as well as the dileucine-based motif (LL/LI) often preceded by a patch of acidic residues. Dileucine motifs (DXXLL) are also recognized by the GGA adaptors (5). Studies on the sorting of polytopic proteins often identified more than one determinant involved in their trafficking to secretory organelles. Examples include the yeast plasma membrane (PM) protein Yor1p, whose anterograde trafficking is directed by two signals, at least one of which (DXE) is a peptide-based sorting motif (6). Additionally, the trafficking of the neuronal polytopic ceroid lipofuscinosis (CLN)-related protein, CLN3, has been thoroughly investigated. Two motifs target CLN3 to the lysosomes of mammalian cells, the widespread LI as well as an unconventional M(X)9G motif (7). Surface targeting of gammaaminobutyric acid type B receptor, GABABR1, is mediated by two sorting determinants: RSR(R) and LL (8). In plants, the anterograde transport of an 80-kD binding protein (BP80) from the Golgi to the prevacuolar compartment involves a YXXF motif (9). Finally, two regions of CLN6 contribute to its retention within the ER, and this involves at least one peptide-sorting motif, KK (10). The protozoan parasite Toxoplasma gondii belongs to the large phylum Apicomplexa, which includes the malaria parasite Plasmodium falciparum. Toxoplasma and other apicomplexans have preserved the compartmental organization found in free-living eukaryotes and have additionally developed specialized compartments (reviewed in 11). The secretory pathway of Toxoplasma comprises a single polarized interconnected ER network and a single Golgi stack (12). Like other members of the phylum, including the Plasmodium species, this parasite also contains a set of specialized apical secretory organelles named rhoptries and micronemes as well as dense granules distributed throughout the cell. The pellicle of the parasite is composed of the PM and an inner membrane complex (IMC), formed by a patchwork of flattened vesicles positioned underneath the www.traffic.dk
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PM. These organelles display clearly distinctive morphologies easily identified by immunofluorescence microscopy (13). As in other eukaryotes, protein traffic through the ER and the Golgi depends on both COPI- and COPII-coated vesicles and is regulated by anterograde targeting signals as well as ER retrieval motifs (13). Tyrosine and dileucine motifs have been identified on single transmembrane (TM) proteins as targeting signals to the rhoptries (14) and to the micronemes (15–17). Targeting to the rhoptries was shown to be APs dependent (14). Disruption of these targeting signals generally resulted in mistargeting to the surface (16,18). It is therefore suggested that the parasite surface constitutes a default destination, and transport to this location may simply occur in the absence of any specific targeting signals (19). Nothing is known about the trafficking and sorting of polytopic proteins in T. gondii. Rhomboid (ROM)-like proteins have been described in organisms from Escherichia coli to humans and have been assigned various biological roles, for which the common denominator is intramembrane proteolysis (20,21). Human ROMs are found in the endosomes, the Golgi and at the PM (22), and Drosophila ROM-1 localizes in late endosomes (23). Despite extensive research on ROM proteases, the mechanisms implicated in their sorting are currently elusive. The T. gondii genome codes for six ROM-like proteases, TgROM1 to TgROM6, and at least one of them is predicted
to be involved in the essential process of host cell invasion and to act at the parasite PM (24,25). Two smaller ROMs (TgROM1 and TgROM2) and two larger ROMs (TgROM4 and TgROM5) contain seven membrane-spanning domains and localize to discrete compartments of the secretory pathway (summarized in Figure 1). In contrast, TgROM6 is predicted to possess only six TM domains and is localized to the mitochondrion (unpublished data). TgROMs along with the other apicomplexan ROM-like proteins cluster into distinct phylogenetic groups (26). The localization of TgROMs was studied by expression of epitope-tagged proteins in transgenic parasites, and experimental evidence suggests that those proteases belonging to the same phylogenetic group also localize to the same subcellular compartment in different apicomplexans. TgROM1 and the phylogenetically related P. falciparum ROM, PfROM1, have been localized to the micronemes (24,25,27,28). A recent study indicates, however, that PfROM1 may localize to a distinct novel secretory organelle (29). TgROM4, TgROM5 and PfROM4 belong to the same phylogenetic group and are all found in the pellicle of the parasite (24,25,28). TgROM2, the only described apicomplexan ROM localized to the Golgi, does not cluster with high bootstrap support to any of the main phylogenetic groups (26) [except with the orthologous gene NcROM2 of the most closely related apicomplexan, Neospora caninum (unpublished data)]. This raises the possibility that comparative sequence analyses may help to identify common targeting motifs.
Figure 1: TgROMs in the secretory pathway of Toxoplasma gondii. Schematic representation of T. gondii secretory organelles. Sorting signals previously described in T. gondii are indicated. Dashed arrows represent data achieved with exogenous cargo. Golgi in blue, Rhoptries (Rhop) and Rhoptry precursor compartment (PC) in green, micronemes (MIC) in red and dense granules (DG) in black. IFAs were performed with parasites expressing Ty-tagged TgROM1 or TgROM4 and Myc-tagged TgROM2 using aTy or aMyc (green, left panel in each pair, indicated by arrows) and aMLC (red in the merge panel) antibodies. Merge also contains 40 ,6-diamidino-2-phenylindole (DAPI) staining. Small illustration is a schematic representation of the four parasites in their parasitophorous vacuole as seen in the IFAs. Scale bar, 1 mm. GPI, glycosyl-phosphatidylinositol.
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Given the importance of TgROMs compartmentalization in controlling accessibility to their substrates (24,25) and in light of the general poor description of ROM targeting in eukaryotic cells, we herein undertook domain exchange followed by mutagenesis approaches in order to assess the basis of TgROMs localization and to identify sorting determinants. The N-terminal (Nt) extensions of the TgROMs show the highest level of sequence variability, and according to topology predictions [(http:// www.ch.embnet.org/software/TMPRED_form.html) and (24)], they are expected to be cytoplasmic. We first experimentally confirmed the topology of TgROM1 and TgROM4 and then performed exchanges of the cytosolic Nt domains between differently localized TgROMs. Our results led to the identification of signals in the Nt of TgROM1 and TgROM2 that are involved in their Golgi and post-Golgi localizations.
Results TgROM4 is a PM protein with a cytosolic Nt Epitope-tagged TgROM4 has been previously localized to the pellicle of Toxoplasma, but its precise topology and whether this protein was embedded in the PM or in the IMC remained unclear (24,25). A serum was raised against the Nt region of TgROM4 to confirm the localization of the endogenous protein, thus validating the epitope-tagging approach and clarifying its topology. This serum recognized a single band at the predicted size in a Western blot of the insoluble fraction of T. gondii total lysates (Figure 2A) and confirmed the subcellular distribution of endogenous TgROM4 at the cell periphery by indirect immunofluorescence assays (IFAs) (Figure 2B). The scheme in Figure 2C shows the predicted topology of TgROM4 at the PM. To challenge this model experimentally, a parasite line was
Figure 2: TgROM4 topology and localization. Confirmation of the antiTgROM4 antibody specificity. A) Lysate from RH parasites was separated into soluble and insoluble phases in PBS and analyzed by Western blot using antiTgROM4 serum (aR4). Host cells (Host) lysate was loaded as a control for aR4 specificity. Antibodies recognizing the soluble protein catalase were used as a control for the soluble fraction. Size markers in kilo Dalton are indicated on the left. B) IFA performed with aR4 (red) on RH parasites. Merge panel also shows 40 ,6-diamidino-2-phenylindole (DAPI) staining. Bar, 0.5 mm. C) Schematic representation of TgROM4 topology in the parasite PM. The antibodies used in the experiment described in panel D are indicated. D–E) TgROM4Ty staining during invasion. D) Staining with anti-TgSAG1 (green) and aR4 (red) prior to permeabilization. E) Both rows show staining with aTy prior to permeabilization in green (large arrowhead) and aR4 after permeabilization in red (small arrowhead). Scale bar, 1 mm. Schematic illustration of the merge panel is on the right. F) TgROM1Ty staining during invasion. Staining with aTy prior to permeabilization in red (large arrowhead) and aMLC (to visualize the parasite) after permeabilization in green (small arrowhead). Scale bar, 1 mm.
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generated expressing TgROM4 with a Ty-1 epitope tag fused to the C-terminus (Ct). IFAs were performed before or after permeabilization on parasites in the process of invasion. The antibodies recognizing the Nt of TgROM4 readily stained permeabilized parasites (Figure 2B), whereas extracellular, non-permeabilized parasites were not stained (Figure 2D). In contrast, anti-Ty antibodies selectively labeled the extracellular part of non-permeabilized parasites, while the protected portion of the parasite surface already contained within the forming parasitophorous vacuole could not be detected (Figure 2E). These observations confirmed that TgROM4 localizes to the PM and validated the topology prediction. Microneme proteins are exocytosed onto the parasite surface during invasion. This has been shown specifically for PfROM1 (29) and should also hold true for TgROM1. An identical topology to TgROM4 was therefore confirmed for TgROM1 during parasite invasion using a similar approach (Figure 2F). Although the topology of TgROM2 cannot be examined by the same method because of its Golgi localization,
we postulate, based on the overall similarities between the TgROMs, that the Nt domain of TgROM2 is also cytosolic. The Nt of TgROM2 contains a double phenylalanine motif acting as an ER export signal Based on the topology prediction and the above data, TgROM1, 2, 4 and 5 can be divided into discrete domains: the cytosolic Nt domain, seven TM domains, six loops and the lumenal/extracellular Ct tail. A first approach to resolve the question of TgROMs targeting was therefore to narrow down the sequences containing sorting information. We hypothesized that the cytosolic Nts (Figure 3A) contain this information and in consequence designed a strategy that consisted of exchanging the corresponding domains between the microneme TgROM1, the Golgi TgROM2 and the PM TgROM4 and TgROM5. The nomenclature and schematic description of all chimeras produced in this study are listed in Figure 7. TgROM4 lacking its Nt domain (R4DNt) was fused to the TgROM2 Nt domain (R2Nt), and the epitope tag Ty was
Figure 3: TgROMs Nt domain swap. Alignments of the Nt domains (A) and the Ct domain (B) of the apicomplexan homologues of the short ROMs. Charged residues in red, aromatic residues in blue and residues with bulky and hydrophobic side chain in green. Tmpredpredicted first TM domains (A) and sixth and/or seventh TM domains (B) are highlighted in gray. Amino acids that have been substituted but that do not seem to be implicated in targeting are in broken black box. TgROM2-proposed Golgi-targeting motif in blue box and TgROM1proposed post-Golgi-sorting motif in red box. EtROM3- and NcROM1,2,3-predicted annotation was achieved using the http:// www.sanger.ac.uk/Projects/E_tenella/ and http://www.sanger.ac.uk/sequencing/Neospora/caninum/genome databases. Accession numbers for the other sequences are found in Dowse and Soldati (26).
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inserted at the Ct to create the chimera R2NtR4DNtTy. Similarly, R2Nt was fused to TgROM1 lacking its Nt domain (R1DNt) to create the chimera R2NtR1DNtTy (Figure 7). These constructs were introduced stably into the parasite genome, and correct expression was verified by Western blot (Figure 4A). Polyclonal antibodies raised specifically against the Nt domain of TgROM2 (Figure S1), and anti-Ty antibodies detected bands corresponding to the predicted size for these chimeras (Figure 4A). IFA analysis performed on the transgenic parasites resulted in staining at the apical side of the nucleus, typical for the Golgi apparatus in T. gondii. This was confirmed by colocalization with the Golgi marker Golgi reassembly stacking protein (GRASP)–yellow fluorescent protein (YFP) (30) (Figure 4B). The localization of R2NtR4DNtTy is in close proximity to but do not overlap perfectly with GRASP–YFP, suggesting that the chimera accumulates in the Trans Golgi Network (TGN) recently defined with TgGalNac marker (51). These results are in agreement with the previously reported localization for TgROM2 (24) and provide the first evidence that R2Nt is sufficient to confer Golgi localization to related ROM proteins that are normally targeted to the micronemes or to the PM. In order to identify a sorting motif within R2Nt independently of the rest of the protein, the R2NtR21TMGFPTy fusion was constructed (Figure 7). For this chimera, green fluorescent protein (GFP) was fused downstream of the Ct of a truncated form of TgROM2 consisting of the Nt cytosolic domain and the first TM domain (R2NtR21TM). The first TM domain was included in order to preserve initial targeting to the ER and insertion into the membrane. The accurate expression of R2NtR21TMGFPTy was verified by Western blot (Figure 4A). As is frequently observed for GFP fusions targeted to the secretory pathway of T. gondii, R2NtR21TMGFPTy was not fluorescent, thus allowing the use of YFP–GRASP for colocalization. The presence of R2NtR21TMGFPTy in the Golgi (Figure 4B) reinforced the conclusion that R2Nt possesses a determinant sufficient for targeting to this compartment. By comparing the Nt tails of ROMs from different apicomplexans, we identified two adjacent phenylalanine residues in TgROM2, F36 and F37 (Figure 3A, blue box) that are not found in the other TgROMs and that could correspond to the double phenylalanine (FF) motif described previously in proteins of the cis Golgi network (31). These consecutive amino acids are also present in the closely related protein NcROM2. The contribution of this motif to targeting was examined by site-directed mutagenesis. Substitution of FF to two alanine residues (mutation M1) was performed in R2NtR21TMGFPTy to generate R2NtM1R21TMGFPTy. As shown by coexpression with GRASP–YFP (Figure 4C), this mutation abolished the Golgi localization and led to redistribution of the chimera to a granular compartment along with additional accumulation around the nucleus. The appearance of R2NtM1R21TMGFPTy also in the parasitophorous vacuole supports the possibility that it localizes to the dense granules and is constitutively secreted (Figure Traffic 2008; 9: 665–677
4C, upper panel). These observations suggest that the FF sequence is a determinant for the Golgi localization within R2Nt. To support this conclusion, substitution of FF to AA in R2Nt within the chimera R2NtR1DNtTy (see below) also abolished Golgi targeting and resulted in accumulation of the chimera in the ER (Figure 5D). More than one signal controls the targeting of TgROM2 To determine if the FF is necessary for the Golgi localization of TgROM2, transgenic parasites expressing fulllength TgROM2 bearing the FF to AA substitution (MycR2M1) were generated. Unexpectedly, MycR2M1 was still targeted efficiently to the Golgi compartment (Figure 5A), indicating that at least one additional determinant for localization to the Golgi is present in TgROM2. To identify the domain containing the second signal, TgROM2 was truncated downstream of the fifth loop. The truncated protein MycR2DCt extends from Met1 to Asp226 and is predicted to comprise an Nt cytosolic domain, five membrane-spanning domains and a lumenal tail (http://www.ch.embnet.org/software/TMPRED_form.html). MycR2DCt is targeted to the Golgi (Figure 5B), suggesting that it still adopts a fold and membrane insertion compatible with proper targeting. Substitution of FF with AA impaired the targeting of MycR2M1DCt, resulting in ER accumulation (Figure 5C). Taken together, these data establish that TgROM2 relies on two targeting signals to ensure Golgi localization and that the second determinant is in the Ct portion of the protein, which includes the sixth cytosolic loop and the luminal Ct domain. Interestingly, we noticed that upon expression of the chimera R1NtR2DNtTy, this second sorting determinant overrides the post-Golgi sorting signal of R1Nt (documented below) and the resulting chimera still localized to the Golgi (data not shown). The second Golgi determinant is unique to TgROM2 because the introduction of the FF to AA substitution in R2NtM1R1DNtTy abrogated the Golgi localization of that chimera (Figure 5D). As a sorting signal was previously reported to be included within a loop of a polytopic protein (7), we searched for sequence features within the sixth loop of TgROM2 that are not shared by TgROM1. Two amino acids, D256R257 (Figure 3B, dashed box), restricted to TgROM2 were substituted with two alanine residues in MycR2M2. As expected, MycR2M2 resulted in Golgi localization (Figure 5E), but simultaneous substitutions of FF and DR in MycR2M1M2 still did not affect Golgi localization (Figure 5F). This result suggests that D256R257 are not critical for Golgi targeting or retention of TgROM2, leaving the identity of the second determinant elusive. The Nt domain of TgROM1 is sufficient for post-Golgi sorting Involvement of the TgROM1 Nt in sorting was examined by fusing it to TgROM5 lacking its Nt domain (R5DNt). The 669
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Figure 4: Legend on next page.
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product of this transgene, R1NtR5DNtTy (Figure 7), was first analyzed by Western blot to verify the expression of the protein at the predicted size (Figure 6A, left). In transiently transfected parasites, R1NtR5DNtTy localized to the ER, Golgi and also to the apical organelle, rhoptries (Figure S2A), residents of which were shown to posses sorting signals directing anterograde trafficking from the Golgi. In stable parasite lines, R1NtR5DNtTy only showed accumulation to an apical compartment exhibiting partial colocalization with a typical microneme marker (TgMIC4) but mainly concentrated in a post-Golgi compartment (Figure 6B). This staining pattern is very similar to the previously reported localization of MycTgROM1 (24) and for hemagglutinin–TgROM1 (27), and colocalization of the R1NtR5DNtTy and MycTgROM1 is shown in Figure S2A. A second chimera was constructed where R1Nt was fused to TgROM4 lacking its Nt domain (R4DNt), resulting in the construction of R1NtR4DNtTy (Figure 7). We failed to generate stable parasite lines with this construct, possibly suggesting a deleterious effect of targeting TgROM4 to the late secretory pathway, perhaps because of premature cleavage of microneme adhesins involved in invasion (24,25,28). In transient transfection, R1NtR4DNtTy accumulated in an apical compartment (Figure 6A) and as previously observed in transient expression of microneme proteins (32) also localized to the ER (data not shown). Altogether, these data suggest that the Nt domain of TgROM1 is sufficient to bring ROMs that are normally targeted to the PM to an apical post-Golgi compartment. The sequence FPHF within the Nt of TgROM1 is essential for its localization The Nt sequences of the apicomplexan ROM1 proteins were scanned in search for sequence similarities with previously described peptide-sorting motifs. As a truncated form of TgROM1, starting at amino acid 45, also localizes to the micronemes (25), we focused on sequences found downstream of Met45. Two amino acid combinations were recognized: a sequence of two consecutive leucine residues, L52 and L53, and the FLEL sequence (amino acids 49–52) consisting of an aromatic residue, two random residues and then a residue with a bulky hydrophobic side chain. These two sequences are reminiscent of the wellcharacterized post-Golgi-sorting motifs, the dileucine LL and tyrosine-based YXXF motifs (5,33). The five amino acids FLELL containing these two potential, overlapping
signals (Figure 3A) were mutated to AASAA (M1) in a construct of TgROM1 containing four Ct Ty tags, TgROM1M1Tys (Figure 7). When stably expressed in parasites, this construct trafficked normally and accumulated in the micronemes (Figure 6C). In search of an as-yet undescribed sorting motif, we looked for conserved sequences in the Nt domains of all apicomplexan ROM1 orthologues available to date. A stretch of residues conserved among the ROM1s but absent from other ROMs was identified (Figure 3A, red box). This sequence, FPHF (amino acids 54–57), is positioned just upstream of the first predicted membrane-spanning domain. A second mutant, TgROM1M2Tys, was constructed to assess the importance of FPHF by changing this sequence to ASAA. In contrast to TgROM1M1Tys, TgROM1M2Tys failed to traffic to the micronemes and instead accumulated in the ER (Figure 6C). Western blot analysis confirmed that TgROM1wtTys, TgROM1M1Tys and TgROM1M2Tys are expressed with the predicted size (Figure 6A, right). Taken together, these observations indicate that the conserved FPHF motif is necessary for targeting TgROM1 to the micronemes.
Discussion Peptide-sorting motif-dependent trafficking is a widespread strategy to accurately direct proteins to their destination, and several lines of evidence suggest that the trafficking signals and machineries found in mammalian cells and in yeast are also largely conserved in T. gondii (11,14). Indeed, the complete sequences of T. gondii and P. falciparum genomes are available and offer the opportunity to establish the repertoire of putative genes that compose the trafficking machineries in apicomplexans (http://toxodb.org/toxo/, http://plasmodb.org/plasmo/). A few components were previously characterized and include COPI coat members, b-COP and ADP ribosylation factor 1 that were shown to be present in the Golgi stack of T. gondii (12,34) and also the small guanosine triphosphatase, rab1, which regulates ER–Golgi recycling (35). The fact that T. gondii contains all the genes coding for subunits of the AP-1, AP-2, AP-3 and AP-4 complexes (Table S1) as well as the rabs (36), with the exception of a-adaptin and Rab9, indicates that the trafficking machineries are well conserved across the evolution of eukaryotes. This notion is further supported by the finding of
Figure 4: The Nt domain of TgROM2 is sufficient for Golgi targeting. An FF motif is necessary. A) Lysates from stable cell lines expressing the different TgROM2 Nt domain-containing chimeras were separated and analyzed by Western blot using aTy and aR2 antibodies. Size markers in kilo Dalton are indicated on the left. The names of each chimera as well as endogenous TgROM2 are indicated. B) IFAs were performed with cell lines stably expressing chimeras containing R2Nt and a Ct Ty-tag using anti-Ty antibody (red, arrowhead). These cells were transiently transfected with YFP–GRASP Golgi marker (green). The name and scheme of each chimera are mentioned above the corresponding three IFA panels. An arrow connects an IFA and its corresponding Western blot. Merge also contains 40 ,6diamidino-2-phenylindole (DAPI) staining. Scale bar, 2 mm. C) IFAs were performed with a cell line stably expressing R2Nt1TMGFP_Ty chimera where FF has been substituted with AA using aTy antibody (red, arrowhead). These cells were transiently transfected with YFP– GRASP Golgi marker (green). The name and scheme are mentioned on the left. Merge also contains 40 ,6-diamidino-2-phenylindole (DAPI) staining. Scale bar, 1 mm.
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Figure 5: TgROM2 possesses a second Golgi-targeting signal not found in TgROM1. IFAs were performed with cell lines stably expressing Myc-tagged TgROM2, which bears FF to AA substitution (A) or is truncated of its Ct domain (B) or both (C). Panels (D–F) represent IFAs performed with pools of stably transfected parasites with R2NtM1R1DNtTy (D) or Myc-tagged TgROM2 bearing DR to AA substitution (E) or both FF to AA and DR to AA substitutions (F). In all panels, aTy is in red (arrowheads). In panels (A–D), aTgMLC1 is in green, and in panels (E and F), parasites where cotransfected with GRASP–YFP (green). The name and scheme of each recombinant protein are mentioned on the left of the corresponding three IFA panels. Scale bar, 1 mm.
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Figure 6: The Nt domain of TgROM1 is sufficient for post-Golgi sorting. The FPHF motif is necessary. A) Left: lysate from the cell line stably expressing R1NtR5DNtTy chimera was analyzed by Western blot using aTy antibody. Size markers in kilo Dalton are indicated on the left. Right: lysates from cell line expressing the different R1_Tys mutants as well as wild-type R1_Tys were analyzed by Western blot using aTy. Size markers in kilo Dalton are indicated on the left. B) IFAs were performed with cell lines stably expressing R1NtR5DNtTy (upper three panels) and transiently transfected parasites expressing R1NtR4DNtTy chimeras (lower three panels). The name and scheme of each chimera are mentioned above the corresponding three IFA panels. Antibodies used are aTy (upper panel – red and lower panel – green, arrowheads) and aTgMIC4 (upper panel – green and lower panel – red). A blowup of the apical region of parasites expressing R1NtR5DNtTy is on the right. Scale bar, 1 mm. C) IFAs were performed with cell lines stably expressing R1M1_Tys and R1M2_Tys, and the name and scheme of each mutant are mentioned above the corresponding three IFA panels. Antibodies used are aTy (red, arrowhead) and aMIC4/aMLC (green, upper and lower panels, respectively). Merge also contains 40 ,6diamidino-2-phenylindole (DAPI) staining. Scale bar, 1 mm.
components of the trafficking machineries in the genomes of other protozoans (37,38). The presence of members of the ROM family in T. gondii and their discrete localization to distinct secretory compartments constituted an attractive system to study the Traffic 2008; 9: 665–677
topology and trafficking of these polytopic proteins, which has so far not been assessed in any other organism. We established here for the first time the membrane topology of eukaryotic ROM-like proteins, confirming experimentally the predicted topology for TgROM4 and TgROM1. By swapping the Nts between various TgROMs, we showed 673
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Figure 7: Summary of chimerical constructs and their intracellular localization.
that these domains play a critical role in the targeting of TgROM1 and TgROM2 to the micronemes and Golgi, respectively. Site-directed mutagenesis revealed that the FF motif present in the Nt domain of TgROM2 is necessary for Golgi targeting of chimeras bearing this domain but is dispensable for TgROM2 Golgi localization. An FF motif was previously described as essential for targeting of proteins from the gp25L/emp24/p24 family to the cis Golgi network by binding to Sec23 of COPII in rat liver and HeLa cells (31). Substitution of the FF present in the cytosolic domain of a few of these family members resulted in inaccurate targeting. The localization of ER–Golgi intermediate compartment-53 to the ER–Golgi intermediate compartment also involves an FF motif (39,40). This raises the possibility that the FF motif is functionally conserved in 674
T. gondii. An as-yet uncharacterized second determinant in the Ct portion of TgROM2 is also involved in determining its localization. Our experimental attempt to identity more precisely this motif failed. It is plausible that the second signal could lie within TM domains 6 or 7. Indeed, a membrane-spanning domain has been reported to contribute to the Golgi localization of the polytopic copper-transporting adenosine triphosphatase, ATP7A (41). Alternatively, the targeting or retention signal might be present in the lumenal Ct tail of TgROM2 and involve interactions with other Golgi-resident proteins. The second trafficking determinant identified in this study is essential for TgROM1 targeting to the micronemes. Fusion of R1Nt to the core of the surface proteins TgROM4 and TgROM5 resulted in their localization to an apical compartment, suggesting that R1Nt is sufficient for trafTraffic 2008; 9: 665–677
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ficking beyond the Golgi. Stable expression of R1NtR5DNtTy allowed a close examination of its localization and demonstrated a partial colocalization with TgROM1 itself in the late secretory pathway but failed to show targeting to the extreme apical end of the parasite where TgMIC4 is found. This discrepancy could be because of a structural feature of R5DNt that may prevent further targeting. For example, TgROM5 contains a much larger Ct tail and loops compared with TgROM1. Moreover, loop-1 and the Ct tail of TgROM5 contain many cysteine residues that may promote dissimilar folding to that of TgROM1. Another explanation may be that, as in the case of TgROM2, a second sorting signal is present in the core of TgROM1. In that case, the arrest of the chimera at a late apical compartment, but prior to the micronemes, could reflect the absence of this signal in R5DNt. The resulting ER accumulation because of the substitution of the FPHF sequence present in R1Nt further supports the conclusion that the Nt is required for proper microneme localization. Although the mutation is positioned in the cytosolic domain of TgROM1 and despite no evidence for protein degradation, we cannot exclude that the ER accumulation observed is a result of interference with the proper protein folding. Nevertheless, as this sequence is exclusively present in apicomplexan ROM1 homologues including PfROM1, we suggest that it may represent a sorting signal for apical secretory organelles, either the micronemes (28) or the mononemes (29). This latter novel organelle has not been described so far in T. gondii, and to date, attempts to raise specific antibodies capable of localizing endogenous TgROM1 by IFA have been unsuccessful (unpublished data). Targeting signals based on residues consisting of aromatic side chains such as the tyrosine-based motif have been extensively characterized. Notably, it was shown that the aromatic side chain of tyrosine is involved in hydrophobic interactions with residues in the m2-binding pocket, a component of the AP-2 complex known to be involved in postGolgi sorting in eukaryotic cells (42). AP complexes have also been found to act in protein trafficking in T. gondii (18). It is thus plausible that one of the aromatic side chains of the sequence FPHF promotes a similar interaction between it and the corresponding sorting receptor and thus facilitates trafficking. Identification of interaction of this sequence with adapter molecules will shed light on its precise role in sorting.
Materials and Methods Reagents and parasite culture Restriction enzymes were from New England Biolabs. Secondary antibodies for Western blots and IFA were from Molecular Probes. T. gondii tachyzoites (RH strain wild type and RH hxgprt) were grown in human foreskin fibroblasts (HFF) or Vero cells in DMEM (GIBCO) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and 25 mg/mL gentamicin.
Cloning of DNA constructs Plasmids pT8TgROM4Ty and pT8TgROM1Ty were constructed by cloning the corresponding complementary DNA (amplified using primers 1483/4
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and 843/4, respectively; Table S2) between the EcoRI and the NsiI sites on the plasmid pTUB8MycGFPPf.myotailTy-1-HX (43). The plasmid pT8TgROM4Ty was used as a template for reverse polymerase chain reaction (PCR) using primers 1530/1. These primers were designed to amplify the whole plasmid excluding TgROM4 Nt domain and allow religation through EcoRI to create the plasmid pT8TgROM4DNtTy. This plasmid is the backbone for most of the constructs described in this study. The primers and cloning strategies are all summarized in Table S2. Primers 1645 and 1533 were used to amplify a PCR product corresponding to amino acid 1–86 of TgROM2. This was cloned using EcoRI and NsiI sites in the plasmid pT8IRPNtGFPTy (44) to create pT8TgR2Nt1TMGFPTy. Plasmid pTetO7SAG1mycTgROM2 was constructed by cloning the PCR product of primers 897 and 1785, corresponding to amino acid 1–233 of TgROM2, between the NsiI sites on pTetO7mycTgROM2 [based on pTetO7mycGFP (45)].
Expression and purification of recombinant fusion proteins in E. coli Primers 1053/4 and 827/8 were used to amplify the sequence corresponding to TgROM2 and TgROM1 Nt domains, respectively. These sequences were cloned into the bacterial expression plasmid pGEX-4T1-His between the SalI and BamHI sites to drive expression of the Nt domain–glutathione S-transferase (GST) fusion. Similarly, primers 1081 and 1082 were used to amplify the sequence corresponding to TgROM4 Nt domain, which was then cloned between the SalI and the BglII sites into pGEX-4T1-His also for expression as a GST fusion. Expression of recombinant proteins was induced with 1 mM isopropyl-beta-D-thiogalactopyranoside. The GST fusion proteins were purified by affinity chromatography on glutathione agarose according to the manufacturer’s protocol (Stratagene).
Generation of a serum specific for the Nt domain of TgROM1, 2 and 4 Antibodies against TgROM1, TgROM2 and TgROM4 were raised in rabbits by Eurogentec S.A. and according to their standard protocol. The antibody reactivity was tested by immunoblotting and IFA. Antibodies were raised against the purified Nt soluble part of the three ROMs (mentioned above).
Mutated constructs To mutate the FF or DR on the plasmids pT7SAG1mycTgROM2, pT7SAG1mycTgROM2DCt, pT8TgR2Nt1TMGFPTy and pT8TgR2NtR4DNtTy, primers 1746/7 were used in a site-directed mutagenesis reaction using the commercial QuikChange II Site-Directed Mutagenesis Kit (Stratagene) and according to manufacturer’s instructions. To introduce the mutations in the Nt domain of TgROM1, primers 843 and 1787/9 as well as primers 844 and 1786/8 were used to amplify the sequences corresponding to amino acids 1–52 and 53–298, each containing the mutant amino acids. The fragment 843-1787 was digested by EcoRI and XhoI, the fragment 1786-844 by XhoI and NsiI, and the two were ligated at once into the EcoRI/NsiI sites on pTUB8TgROM1_4Ty. Similarly, the fragment 8431789 was digested by EcoRI and EagI, the fragment 1788-844 by EagI and NsiI, and the two were ligated into pTUB8TgROM1_4Ty. All mutated constructs were sequenced along the entire open-reading frame (ORF) to confirm the correct sequence.
Parasite transfection and selection of clonal stable lines Parasite transfection was performed by electroporation as previously described (46). The hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene was used as a positive selectable marker in the presence of mycophenolic acid and xanthine as described before (47). Briefly, freshly released parasites (5 107) of RH hxgprt or TATi-1 (45) strains were resuspended in cytomix buffer in presence of 50–80 mg of plasmid carrying the selectable marker gene and the expression cassette containing the TgROMs chimera sequences. Parasites were electroporated at 2 kV, 25 mF and 48 V using a BTX electroporator (Harvard biosciences) before being added to a monolayer of HFF cells in the presence of mycophenolic acid (25 mg/mL) and xanthine (50 mg/mL) and cloned by
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Sheiner et al. limiting dilution on 96-well microtiter plates. For transient transfection, the procedure was the similar excluding DNA linearization and parasite cloning.
Western blots About 2 107 freshly lysed parasites were harvested after complete lysis of the host cells. Protein extracts were prepared by five consecutive freeze/thaw cycles with intermediate vortexing and then two consecutive sonications. SDS–PAGE was performed using standard methods. The suspension was mixed or resuspended with SDS–PAGE-loading buffer containing 100 mM DTT, and proteins were separated by electrophoresis in a 10% polyacrylamide gel. Separated proteins were transferred to a nitrocellulose membrane using a semi-dry electroblotter. Western blots were performed using primary antibodies antibody raised against the Nt domains of TgROM4 or TgROM2 or anti-catalase (48) or anti-Myc (hybridoma GE10) and anti-Ty1 (49) antibodies in PBS/5% nonfat milk powder. Secondary antibody was a peroxidase-conjugated goat anti-mouse or anti-rabbit antibody (Molecular Probes). Bound antibodies were visualized using the enhanced chemiluminescence system (Amersham Corp.). All antibodies where used diluted 1:1000.
IFA and confocal microscopy All manipulations were carried out at room temperature. Intracellular parasites grown in HFF seeded on glass coverslips were fixed with 4% paraformaldehyde for 20 min. Following fixation, slides were quenched in PBS–0.1 M glycine. Cells were then permeabilized in PBS–0.02% Triton-X100 (PBS/Triton) for 20 min and blocked in the same buffer supplemented with 2%1% BSA (PBS/Triton/BSA). Slides were incubated for 60 min with primary antibodies [anti-MIC6 (32), anti-MIC4 (50), anti-myosin light chain (MLC) (43), anti-Myc, anti-Ty diluted in PBS/Triton/FCS], washed and incubated for 40 min with Alexa488- or Alexa594-conjugated goat antimouse or goat anti-rabbit immunoglobulin Gs diluted in PBS/Triton/FCS. After 40 ,6-diamidino-2-phenylindole (DAPI) staining, slides were mounted in Fluoromount G (Southern Biotech) and stored at 48C in the dark. Micrographs were obtained the Zeiss Axioskop 2 equipped with an Axiocam color chargecoupled device camera. Images were recorded and treated on computer through the AXIOVISIONä software. ADOBE PHOTOSHOP (Adobe Systems) was used for processing of images. Confocal images were collected with a Leica laser scanning confocal microscope (TCS-NT DM/IRB) using a 63 Plan-Apo objective with numerical aperture (NA) 1.40. Optical sections were recorded at 250 nm per vertical step and four times averaging.
Invasion assay A high concentration of extracellular parasites stably expressing TgROM4Ty (5 106 parasites/mL) was used to infect host cells grown on glass coverslips. Cells were fixed with Paraformaldehyde (PFA) at 2-, 5or 10-min incubation in 378C after addition of parasites. Following quenching for 3 min in 0.1 M glycine in PBS, cells were washed with PBS before incubating in 2% BSA in PBS for 20 min. Cells were then incubated with one primary anti-Ty or anti-SAG1 antibodies diluted in 2% BSA in PBS for 1 h. Following wash in PBS, cells were incubated with secondary Alexa488 antibodies in 2% BSA in PBS for 45 min. Up to this point, no permeabilizing agent has been used. After this initial staining, IFA was performed as described above, with anti-ROM4 primary antibodies and Alexa-594 secondary antibodies.
Acknowledgments This work is part of the activities of the BioMalPar European Network of Excellence supported by a European grant (LSHP-CT-2004-503578) from the Priority 1 ‘Life Sciences, Genomics and Biotechnology for Health’ in the 6th Framework Program, from supports to D. S.-F. by the Swiss National Foundation and the Howard Hughes Medical Institutes and from support by a Wellcome Trust Studentship to T. J. D. Thanks to Natacha Klages and Rebecca Shepers for their technical support. We are thankful to Thierry
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Soldati for helpful discussions and for helping with the documentation and to Mike Shea for critical reading of the manuscript.
Supplementary Materials Figure S1: Anti-TgROM2 antibody (aR2) specifically recognizes recombinant and endogenous TgROM2 by Western blot analysis. Lysates from a cell line expressing Myc-tagged TgROM2 under the control of a tetracycline-regulated promoter were collected after 48 h incubation with (þ) or without () ATc and were analyzed by Western blot using aMyc and aR2 antibodies. Size markers in kilo Dalton are indicated on the left. Bands corresponding to Myc-tagged and endogenous TgROM2 are indicated. Figure S2: The Nt domain of TgROM1 is sufficient for targeting to apical organelles. A) IFAs were performed with a pool of parasites transiently expressing R1NtR5DNtTy chimera. The name and scheme of the chimera are mentioned above the corresponding IFA panels. The organelle showing staining is mentioned above the corresponding IFAs. Antibodies used are aTy (red, arrowheads) and aTgMIC4 (green). Bar, 1 mm. B) Parasites stably transfected with R1NtR5DNtTy chimera and transiently transfected with MycTgROM1 are presented. The name and scheme of the two chimeras are mentioned above the corresponding IFA panels [in red (aTy) and in green (aMyc)]. Bar, 1 mm. Table S1: Toxoplasma gondii and Plasmodium falciparum AP homologues Table S2: List of primers Supplemental materials are available as part of the online article at http:// www.blackwell-synergy.com
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