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

Abstract

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.

The acyl-CoA synthetase Tg ACS1 allows neutral lipid metabolism and extracellular motility in Toxoplasma gondii through relocation via its peroxisomal targeting sequence (PTS) under low nutrient conditions

“…However, the existence of peroxisome per se in T. gondii has long been questioned and remains a debated topic as (i) the typical organelle’s presence remains elusive or not fully defined, and (ii) there is no clear experimental evidence for biochemical activities attributed to such predicted organelle compartment(s). Interestingly the PTS found in Tg ACS1 is similar to the one identified in other Toxoplasma proteins and is able to rescue peroxisomal import assay in yeast mutant ( 38 ). Using Alphafold and a 3D crystal structure of a bubble gum domain-containing ACS found in Mycobacterium tuberculosis ( 47 ), which shares 51% of sequence identity with Tg ACS1, we thus generated a 3D model prediction ( Fig.…”

“…Very interestingly, two studies independently identified strong homologs of PEX in T. gondii , notably Tg PEX5, all bearing putative PTS and most likely allowing peroxisome biogenesis and protein import. Indeed, PEX5 can recognize and bind to the proteins bearing a PTS1, allowing the transport of any soluble proteins destined to peroxisomal lumen ( 37 , 38 ). It was further demonstrated that the PTS1-binding domain of Tg PEX5, which is usually enabling peroxisomal protein import in eukaryotes, was interacting with the PTS1 of Tg SCP2 in vitro ( 38 ).…”

“…Indeed, PEX5 can recognize and bind to the proteins bearing a PTS1, allowing the transport of any soluble proteins destined to peroxisomal lumen ( 37 , 38 ). It was further demonstrated that the PTS1-binding domain of Tg PEX5, which is usually enabling peroxisomal protein import in eukaryotes, was interacting with the PTS1 of Tg SCP2 in vitro ( 38 ). Furthermore, Tg PEX5 was able to rescue a PEX-KO in yeast, and the PTS1 from the yeast PEX5 was able to direct a GFP fusion to distinct cytosolic/punctate localization similar to those of Tg SCP2 in T. gondii ( 38 ).…”

“…It was further demonstrated that the PTS1-binding domain of Tg PEX5, which is usually enabling peroxisomal protein import in eukaryotes, was interacting with the PTS1 of Tg SCP2 in vitro ( 38 ). Furthermore, Tg PEX5 was able to rescue a PEX-KO in yeast, and the PTS1 from the yeast PEX5 was able to direct a GFP fusion to distinct cytosolic/punctate localization similar to those of Tg SCP2 in T. gondii ( 38 ). Finally, bioinformatic analyses of the PTS1 proteome in T. gondii revealed the presence of the complete set of enzymes putatively responsible for β-oxidation in the parasite ( 37 ).…”

Evolutionary Maintenance of the PTS2 Protein Import Pathway in the Stramenopile Alga Nannochloropsis

“…By contrast, chromalveolates and particularly stramenopiles are amazingly heterogeneous with respect to both the presence of peroxisomes and the PTS2 import pathway. Non-coccidian apicomplexa (e.g., Plasmodium) lack peroxisomes entirely, while coccidian apicomplexa (e.g., Toxoplasma) possess solely the PTS1 import pathway (Schluter et al, 2006;Mbekeani et al, 2018), similar to nowadays red algae (Matsuzaki et al, 2004;Shinozaki et al, 2009). As shown by this study, some stramenopiles have maintained both pathways (Eustigmatophyceae, brown algae), while diatoms lost the PTS2 pathway entirely and transferred classical PTS2 cargo proteins (e.g., thiolase) to the PTS1 import pathway (Figure 6; Gonzalez et al, 2011).…”