Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii

Peroxisomes are central to eukaryotic metabolism, including the oxidation of fatty acids—which subsequently provide an important source of metabolic energy—and in the biosynthesis of cholesterol and plasmalogens. However, the presence and nature of peroxisomes in the parasitic apicomplexan protozoa remains controversial. A survey of the available genomes revealed that genes encoding peroxisome biogenesis factors, so-called peroxins (Pex), are only present in a subset of these parasites, the coccidia. The basic principle of peroxisomal protein import is evolutionarily conserved, proteins harbouring a peroxisomal-targeting signal 1 (PTS1) interact in the cytosol with the shuttling receptor Pex5 and are then imported into the peroxisome via the membrane-bound protein complex formed by Pex13 and Pex14. Surprisingly, whilst Pex5 is clearly identifiable, Pex13 and, perhaps, Pex14 are apparently absent from the coccidian genomes. To investigate the functionality of the PTS1 import mechanism in these parasites, expression of Pex5 from the model coccidian Toxoplasma gondii was shown to rescue the import defect of Pex5-deleted Saccharomyces cerevisiae. In support of these data, green fluorescent protein (GFP) bearing the enhanced (e)PTS1 known to efficiently localise to peroxisomes in yeast, localised to peroxisome-like bodies when expressed in Toxoplasma. Furthermore, the PTS1-binding domain of Pex5 and a PTS1 ligand from the putatively peroxisome-localised Toxoplasma sterol carrier protein (SCP2) were shown to interact in vitro. Taken together, these data demonstrate that the Pex5–PTS1 interaction is functional in the coccidia and indicate that a nonconventional peroxisomal import mechanism may operate in the absence of Pex13 and Pex14.


Introduction
Peroxisomes were first biochemically characterised and named by Christian de Duve and colleagues following purification from rat liver tissue, and later the protozoa [1,2]. Within the isolated, membrane-bound microbodies, the y identified several oxidases that oxidize substrates whilst reducing oxygen to hydrogen peroxide (H 2 O 2 ), as well as two enzyme classes able to reduce this damaging H 2 O 2 : peroxidases and catalases [2]. The following decades saw peroxisomes identified as apparently ubiquitous, functionally diverse, eukaryotic organelles involved in many catabolic functions including lipid, amino acid and purine metabolism [3,4]. This biogenesis and functionality of peroxisomes is dependent on a group of proteins known as peroxins (Pex). Furthermore, the topogenic import signals for the import of matrix proteins is conserved across evolution [5]. For most imported proteins this motif comprises a short tripeptide sequence at the extreme C-terminus. Prototypically, this motif, serine-lysine-leucine (-SKL) or neutral variants, is known as peroxisomal-targeting signal 1 (PTS1) [6]. The PTS1 receptor is the peroxin Pex5, a shuttling receptor, which translocates the PTS1-bearing ligand from the cytosol to the peroxisome matrix via interaction with a membrane docking complex of Pex13 and Pex14 (and Pex17 in yeast) [7,8].
Illustrative of the essential function of peroxisomes in many eukaryotes is the association of mutations in the Pex5-PTS1 import system with human diseases such as Zellweger syndrome [9]. However, despite the crucial roles these organelles can play, eukaryotic microbes such as the intestinal parasite Entamoeba histolytica have been reported to lack peroxisomes, presumably due to their existence in low-oxygen environments and the consequent lack of H 2 O 2 [10]. Other intracellular parasites such as the fungal Microsporidia and the protozoan Apicomplexa (including the malaria parasite Plasmodium spp.), as well as intestinal parasitic helminths, have also been reported to lack peroxisomes and encoded Pex proteins [4,11]. However, as the genome databases have developed it has become clear that some apicomplexan parasites, the coccidia which include Eimeria spp. (coccidiosis in poultry) and Toxoplasma gondii (toxoplasmosis), encode many of the Pex proteins synonymous with the presence of these organelles [5].
Obligate intracellular organisms rely on the host cell for many of their nutritional requirements. For example, Toxoplasma is auxotrophic for cholesterol and is reliant on that scavenged from the host via endocytosis of low-density lipoprotein (LDL) [12,13], a process that has been proposed as a possible drug target [14]. This salvage of LDL requires molecular mechanisms for coordinated delivery to the desired organelles, and a sterol carrier protein (SCP2) has been characterised as playing a key role in this process [15]. In the Eukaryota, SCP2 is specifically targeted to peroxisomes via a PTS1, a process important for the β-oxidation of fatty acids [16]. Similarly, the Toxoplasma SCP2, which harbours a putative PTS1 (SRL), is localised to peroxisomes in mammalian cells, and to the lumen of well-defined vesicles in the parasite [15]. However, the identity of these vesicles as peroxisomes is controversial, with the predicted PTS1 signal of Toxoplasma catalase failing to direct green fluorescent protein (GFP) to membrane-bound microbodies in one study [17]. However, a parallel study showed the parasite catalase localising to vesicles in Toxoplasma and to mammalian peroxisomes, with the putative PTS1 signal responsible [18].
Clearly, the nature and/or presence of peroxisomes in the Apicomplexa requires further analyses. However, bioinformatic studies have very recently provided evidence, by virtue of the presence of encoded Pex and PTS-bearing proteins, that Toxoplasma and related coccidia harbour peroxisomes [5,19,20]. Here, we have taken a functional biology approach to define the Toxoplasma Pex5 as a functional orthologue of the yeast, but not human, protein. In addition, we have shown that the enhanced (e)PTS1 identified from Saccharomyces cerevisiae [21] efficiently localises GFP to peroxisome-like bodies when expressed in Toxoplasma. Finally, we have biochemically demonstrated that the PTS1 ligand domain of the parasite SCP2 interacts with the PTS1-binding domain from Toxoplasma Pex5 in vitro. Taken together with previous studies, the se data strongly suggest the presence of peroxisomes in this group of parasites.
After incubation, human cells were washed with Dulbecco Phosphate-Buffered Saline (DPBS; Sigma Aldrich) and fixed in 4% v/v paraformaldehyde (Roth, Karsruhe, Germany) in DPBS for 20 min at room temperature. Cells fixed on cover-slips were then mounted on glass slides under the anti-fade reagent Mowiol (Sigma Aldrich) containing 4 ,6-ciamidino-2-phenylindole (DAPI; ThermoFisher Scientific). Yeast cells were directly visualized under microscopy. For imaging, wide-field fluorescence microscopy was performed using an Axioplan 2 microscope with an AxioCam MR digital camera and Axiovision software version 4.6.3 (Zeiss, Oberkochen, Germany). Green fluorescent protein signal was visualized using 450-490 nm band pass excitation filter, a 510 nm dichromatic mirror and a 515-565 nm band pass emission filter.

Growth Analyses of Toxoplasma Pex5 Complementation of Yeast Mutant Line
Serial 10-fold dilutions of exponentially growing UTL-7A Pex5 deleted yeast, transformed with TgPex5, ScPex5 or empty vector, were spotted onto −Ura glucose or oleate plates and incubated at 30 • C for 3 to 4 days before image capture as previously described [31,32].
Images were obtained using a DeltaVision Core microscope (GE Heathcare, Chicago, IL, USA) and processed using Softworx (GE Heathcare) and FIJI software [35]. Parasites with heavily distorted cell shape and fragmented mitochondria were excluded from the analysis.

Analyses of the Interaction of Toxoplasma Pex5 and SCP2
Regions encoding domains for the predicted PTS1 of TgSCP2 (TgSCP2 PTS1 : TgSCP2 499-625) and the PTS1-binding region of receptor TgPex5 (TgPex5C: TgPex5 581-899) were synthesized with an Escherichia coli codon bias and cloned into the bacterial expression vector pETM-30 by GenScript. The resulting pETM-30-TgSCP2 PTS1 and pETM-30-TgPex5C were transformed into E. coli BL21 (DE3) (ThermoFisher Scientific), and grown in pH 7.5 buffered Luria-Bertani (LB) medium, supplemented with 1% (w/v) glucose and 50 mg/mL kanamycin (Sigma-Aldrich) at 37 • C. When exponential growth was reached, the temperature was reduced to 20 • C for 1 h, before induction with 0.5 mM IsoPropyl β-D-1-ThioGalactopyranoside (IPTG; Sigma Aldrich) and overnight incubation at 30 • C. Cell pellets were flash frozen before resuspension in Buffer A: 100 mM KH 2 PO 4 pH 7.4, 5 mM 2-mercaptoethanol, EDTA-free complete protease inhibitor cocktail, 1 mg/mL lysozyme, 1 mg/mL DNase I and 1 mg/mL RNAse A (Sigma-Aldrich). The bacteria were then sonicated (Electronic UW2200; Bandelin, Berlin, Germany) and, following centrifugation, the supernatant collected through a 0.45 µm filter (Sigma Aldrich). This supernatant was then applied to a 1 mL Glutathione Sepharose 4B resin column (GSTrap) (GE Healthcare), which was pre-equilibrated with Buffer A. Following a Buffer A wash, the protein was eluted with Buffer A containing 20 mM reduced glutathione. The TgPex5C HIS-GST-tag was cleaved using TEV protease (Sigma Aldrich) in accordance with manufacturer's instructions, before dialysis into Buffer A and application to a 1 mL Ni-NTA (ThermoFisher Scientific) column equilibrated in Buffer A and eluted with Buffer A supplemented with 250 mM imidazole, pH 7.4. To analyse the interaction, TgSCP2 PTS1 containing His 6 (HIS)-GST-tag was bound to a GSTrap column and isolated TgPex5C applied in Buffer A. Following a Buffer A wash, the proteins were eluted with Buffer A containing 20 mM reduced glutathione before analyses by SDS-PAGE.

Functional Complementation Analyses of TgPex5 in Yeast and Human Cell Lines
Bioinformatic analyses of Toxoplasma, yeast and human Pex5 protein sequences indicated that parasite Pex5 contains structural features characteristic of Pex5 proteins. The se include (i) C-terminal TetratricoPeptide Repeat (TPR) motifs forming the PTS1-binding site; (ii) within the probably unstructured N-terminal domain, three so-called WxxxF motifs required for receptor docking; and (iii) a single cysteine at a conserved position, which becomes ubiquitinated to initiate recycling of the receptor into the cytosol ( Figure 1A). Toxoplasma Pex5 was heterologously expressed in human or yeast cells for functional complementation analysis. In contrast to the positive control, human HsPex5L [24], expression of Toxoplasma TgPex5 in Pex5-deficient human fibroblasts was unable to restore the PTS1-mediated trafficking of eGFP to peroxisomal compartments ( Figure 2). However, parallel analyses in S. cerevisiae demonstrated that TgPex5 could clearly restore GFP-PTS1 targeting to peroxisomes in a cellular context (Figure 3). The results of this localisation assay were augmented by the observation that TgPex5, like ScPex5 [23], was able to restore peroxisomal function and β-oxidation as characterised by increased growth and the utilisation of fatty acid oleate, which results in halo formation around the colonies on oleate plates ( Figure 4) [31,32]. Growth complementation indicates that the heterologous TgPex5 targets not only the artificial cargo GFP-PTS1, but also the endogenous yeast enzymes, into peroxisomes. It is remarkable that TgPex5 restored translocation into the organellar lumen of not only the β-oxidation enzymes with typical PTS1, but also of the non-PTS acyl-CoA oxidase Fox1, the initial enzyme required for the fatty acid catabolic pathway. The Fox1 binding regions have been assigned to the N-terminal half of ScPex5 [36]. Sequence alignments between yeast and Toxoplasma Pex5 suggest that the key residues required for Fox1 interaction are largely conserved ( Figure 1B). Taken together, the se data indicate that Toxoplasma TgPex5 is a functional orthologue of the yeast Pex5, and heterologous expression of this protein can functionally replace its yeast but not its human counterpart. between yeast and Toxoplasma Pex5 suggest that the key residues required for Fox1 interaction are largely conserved ( Figure 1B). Taken together, these data indicate that Toxoplasma TgPex5 is a functional orthologue of the yeast Pex5, and heterologous expression of this protein can functionally replace its yeast but not its human counterpart.

Localization of GFP Tagged with Enhanced PTS1 (GFP-ePTS1) in Toxoplasma
Enhanced (e)PTS1 was identified by screening a randomised library for linker sequences (directly upstream of PTS1) for the ability to more efficiently localise fluorescent protein to peroxisomes in S. cerevisiae [21]. When GFP-ePTS1 was expressed in Toxoplasma, it localised to punctate structures with diameters ranging from 129-311 nm ( Figure 5), this concurs with the diameter of peroxisomes (100-200 nm) and previous observations in this parasite [15,18]. This observation supports (i) the interaction of PTS1 (from yeast, as well as Toxoplasma) with Pex5 in the parasite; (ii) the existence of peroxisomes with the canonical translocation machinery in the coccidia.

Localization of GFP Tagged with Enhanced PTS1 (GFP-ePTS1) in Toxoplasma
Enhanced (e)PTS1 was identified by screening a randomised library for linker sequences (directly upstream of PTS1) for the ability to more efficiently localise fluorescent protein to peroxisomes in S. cerevisiae [21]. When GFP-ePTS1 was expressed in Toxoplasma, it localised to punctate structures with diameters ranging from 129-311 nm ( Figure 5), this concurs with the diameter of peroxisomes (100-200 nm) and previous observations in this parasite [15,18]. This observation supports (i) the interaction of PTS1 (from yeast, as well as Toxoplasma) with Pex5 in the parasite; (ii) the existence of peroxisomes with the canonical translocation machinery in the coccidia.

In Vitro Analyses of the Interaction of TgPex5 and TgSCP2
Regions encoding the following domains were expressed in E. coli: (i) predicted TgSCP2 PTS1 (TgSCP2PTS1: TgSCP2 499-625) containing the PTS1-targeting signal; and (ii) TgPex5 PTS1 receptor (TgPex5C: TgPex5 581-899), harbouring the predicted PTS1-binding domain. The recombinant proteins were isolated and subjected to in vitro binding studies. Using HIS-GST-tagged TgSCP2 PTS1 (43.4 kDa) bound to a GSTrap column as bait, the binding of TgPex5C (34.5 kDa) was analysed. Following stringent washes, the elution of bound material demonstrated interaction with TgSCP2PTS1, showing that the Toxoplasma Pex5 can bind to the parasite PTS1 ( Figure 6).

Discussion
With recent bioinformatic analyses demonstrating the presence of a suite of Pex proteins in the coccidian apicomplexans, but not in other subclasses [5,19,20], functional analyses are demanded.

Localization of GFP Tagged with Enhanced PTS1 (GFP-ePTS1) in Toxoplasma
Enhanced (e)PTS1 was identified by screening a randomised library for linker sequences (directly upstream of PTS1) for the ability to more efficiently localise fluorescent protein to peroxisomes in S. cerevisiae [21]. When GFP-ePTS1 was expressed in Toxoplasma, it localised to punctate structures with diameters ranging from 129-311 nm ( Figure 5), this concurs with the diameter of peroxisomes (100-200 nm) and previous observations in this parasite [15,18]. This observation supports (i) the interaction of PTS1 (from yeast, as well as Toxoplasma) with Pex5 in the parasite; (ii) the existence of peroxisomes with the canonical translocation machinery in the coccidia.

In Vitro Analyses of the Interaction of TgPex5 and TgSCP2
Regions encoding the following domains were expressed in E. coli: (i) predicted TgSCP2 PTS1 (TgSCP2PTS1: TgSCP2 499-625) containing the PTS1-targeting signal; and (ii) TgPex5 PTS1 receptor (TgPex5C: TgPex5 581-899), harbouring the predicted PTS1-binding domain. The recombinant proteins were isolated and subjected to in vitro binding studies. Using HIS-GST-tagged TgSCP2 PTS1 (43.4 kDa) bound to a GSTrap column as bait, the binding of TgPex5C (34.5 kDa) was analysed. Following stringent washes, the elution of bound material demonstrated interaction with TgSCP2PTS1, showing that the Toxoplasma Pex5 can bind to the parasite PTS1 ( Figure 6).

Discussion
With recent bioinformatic analyses demonstrating the presence of a suite of Pex proteins in the coccidian apicomplexans, but not in other subclasses [5,19,20], functional analyses are demanded.

Discussion
With recent bioinformatic analyses demonstrating the presence of a suite of Pex proteins in the coccidian apicomplexans, but not in other subclasses [5,19,20], functional analyses are demanded. However, previous studies in Toxoplasma have proven to be controvertible. The Toxoplasma catalase was localised to peroxisome-like vesicles in one study [18], however, in the same year it was reported that the putative catalase PTS1 failed to direct GFP to these membrane-bound microbodies [17]. The sterol carrier protein SCP2 has also been shown to localise to peroxisome-like bodies in Toxoplasma [15].
To improve the molecular understanding of the putative peroxisomal bodies in Toxoplasma and the related coccidians, here we report a series of cellular and biochemical studies of the parasite Pex5 orthologue. Like human, plant and nematode [37], the Toxoplasma protein clearly rescues the targeting of GFP bearing a canonical PTS1 sequence (SKL) in yeast, indicating that TgPex5 is a functional peroxin involved in the trafficking of PTS1-bearing proteins to the peroxisomes. In addition, yeast-derived ePTS1 was sufficient to localise GFP to peroxisome-like bodies in Toxoplasma [15,18], and TgSCP PTS1 (-SRL) clearly binds to the parasite TgPex5 PTS1-binding domain in vitro. Furthermore, heterologous expression of TgPex5 restored the utilisation of oleate in Pex5-deficient yeast, clearly demonstrating that a complete set of endogenous fatty acid-degrading enzymes with, and without, typical PTS1 sequences are recognized and targeted to peroxisomes by the parasite peroxin. This common cargo selectivity of the yeast and Toxoplasma receptor will include Fox1, which is recognized by a PTS1-independent binding domain N-terminal of the yeast Pex5 PTS1-binding region. Importantly, unlike the mammalian orthologue, TgPex5 maintains a Fox1-binding region. In contrast to yeast, TgPex5 was unable to direct eGFP-SKL to human fibroblast peroxisomes and thus rescue the import defect of an equivalent mammalian Pex5-deficient cell line. Taken together, the se data indicate that TgPex5 is a functional orthologue of the yeast but not the human peroxin. A difference that may be exploited by consideration of the TgPex5-PTS1 interaction as a drug target, as has been described in the protozoan pathogen Trypansoma brucei [38].
Therefore, in summary we have demonstrated that the Toxoplasma Pex5 is functional in the context of a ∆pex5 yeast strain and interacts with the protozoan PTS1 in vitro. Coupled with the localization of ePTS1-tagged GFP to peroxisome-like bodies in Toxoplasma, this strongly suggests that the coccidia harbour peroxisomes. The relative lack of structural evidence may be due to lifecycle-dependent expression, with the peroxins characterised by upregulation during the extracellular stages of the lifecycle, oocysts and sporozoites. Future studies should be directed here, rather than at the more tractable, intracellular, tachyzoite forms [20]. In addition, in all eukaryotic systems studied to date, a membrane-bound docking complex of Pex13 and Pex14 is required for peroxisomal protein import [7,8]. However, an obvious Pex13 is missing in the genome of Toxoplasma and related coccidian parasites [19,20]. Furthermore, the identity and presence of Pex14 in Toxoplasma is disputed [5,19,20]. The refore, the exact mode of Pex5-mediated protein import into peroxisomes in the coccidia is unclear and further bioinformatic and functional analyses are warranted.