ABSTRACT
Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.
Subject(s)
Mitochondria , Mitochondrial Dynamics , Peroxisomes , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Peroxisomes/metabolism , Mitochondrial Dynamics/physiology , Mitochondria/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Fatty Acids/metabolism , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/genetics , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Proteasome Endopeptidase Complex/metabolism , Citric Acid Cycle , Membrane Potential, Mitochondrial/physiology , Mitochondrial Membranes/metabolism , HumansABSTRACT
Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.
Subject(s)
Mitochondria , Peroxisomes , Endoplasmic Reticulum , Cell Movement , Cell Respiration , Saccharomyces cerevisiaeABSTRACT
Membrane-bound organelles play important, frequently essential, roles in cellular metabolism in eukaryotes. Hence, cells have evolved molecular mechanisms to closely monitor organelle dynamics and maintenance. The actin cytoskeleton plays a vital role in organelle transport and positioning across all eukaryotes. Studies in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) revealed that a block in actomyosin-dependent transport affects organelle inheritance to daughter cells. Indeed, class V Myosins, Myo2, and Myo4, and many of their organelle receptors, have been identified as key factors in organelle inheritance. However, the spatiotemporal regulation of yeast organelle transport remains poorly understood. Using peroxisome inheritance as a proxy to study actomyosin-based organelle transport, we performed an automated genome-wide genetic screen in S. cerevisiae. We report that the spindle position checkpoint (SPOC) kinase Kin4 and, to a lesser extent, its paralog Frk1, regulates peroxisome transport, independent of their role in the SPOC. We show that Kin4 requires its kinase activity to function and that both Kin4 and Frk1 protect Inp2, the peroxisomal Myo2 receptor, from degradation in mother cells. In addition, vacuole inheritance is also affected in kin4/frk1-deficient cells, suggesting a common regulatory mechanism for actin-based transport for these two organelles in yeast. More broadly our findings have implications for understanding actomyosin-based transport in cells.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Actomyosin/metabolism , Mitosis , Spindle Apparatus/metabolism , OrganellesABSTRACT
Lipid mobilization through fatty acid ß-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where ß-oxidation products enter mitochondria and fuel the tricarboxylic acid cycle. Little is known about the physical and metabolic cooperation between these organelles. Here we found that expression of fatty acid transporters and of the rate-limiting enzyme involved in ß-oxidation is decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. Consequently, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although ß-oxidation occurs in both mitochondria and peroxisomes in mammals, Arf1's role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact sites.
Subject(s)
Mitochondria , Peroxisomes , Animals , Mitochondria/metabolism , Peroxisomes/metabolism , Fatty Acids/metabolism , Oxidation-Reduction , Lipid Metabolism/genetics , Homeostasis , Mammals/metabolismABSTRACT
The mechanism behind peroxisomal membrane protein targeting is still poorly understood, with only two yeast proteins believed to be involved and no consensus targeting sequence. Pex19 is thought to bind peroxisomal membrane proteins in the cytosol, and is subsequently recruited by Pex3 at the peroxisomal surface, followed by protein insertion via a mechanism that is as-yet-unknown. However, some peroxisomal membrane proteins still correctly sort in the absence of Pex3 or Pex19, suggesting that multiple sorting pathways exist. Here, we studied sorting of yeast peroxisomal ABC transporter Pxa1. Co-localisation analysis of Pxa1-GFP in a collection of 86 peroxisome-related deletion strains revealed that Pxa1 sorting requires Pex3 and Pex19, while none of the other 84 proteins tested were essential. To identify regions with peroxisomal targeting information in Pxa1, we developed a novel in vivo re-targeting assay, using a reporter consisting of the mitochondrial ABC transporter Mdl1 lacking its N-terminal mitochondrial targeting signal. Using this assay, we showed that the N-terminal 95 residues of Pxa1 are sufficient for retargeting this reporter to peroxisomes. Interestingly, truncated Pxa1 lacking residues 1-95 still localised to peroxisomes. This was confirmed via localisation of various Pxa1 truncation and deletion constructs. However, localisation of Pxa1 lacking residues 1-95 depended on the presence of its interaction partner Pxa2, indicating that this truncated protein does not contain a true targeting signal.
Subject(s)
ATP-Binding Cassette Transporters , Saccharomyces cerevisiae Proteins , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Peroxisomes/genetics , Peroxisomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Membrane Proteins/genetics , Membrane Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Peroxins/genetics , Peroxins/metabolismABSTRACT
Peroxisomes are organelles with vital functions in metabolism and their dysfunction is associated with human diseases. To fulfill their multiple roles, peroxisomes import nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p2D) show decreased import of GFP with a PTS1. We show that the binding affinity between a PTS1-protein and Pex5p2D is reduced. An in vivo analysis of the effect of the phospho-mimicking mutant on PTS1-proteins revealed that import of most, but not all, cargos is affected. The physiological effect of the phosphomimetic mutations correlates with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes. In a broader view, this suggests that posttranslational modifications can function in fine-tuning the peroxisomal protein composition and, thus, cellular metabolism.
Subject(s)
Peroxisomes , Receptors, Cytoplasmic and Nuclear , Humans , Phosphorylation , Peroxisomes/metabolism , Peroxisome-Targeting Signal 1 Receptor/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Carrier Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Protein TransportABSTRACT
Accurate and regulated protein targeting is crucial for cellular function and proteostasis. In the yeast Saccharomyces cerevisiae, peroxisomal matrix proteins, which harboring a Peroxisomal Targeting Signal 1 (PTS1), can utilize two paralog targeting factors, Pex5 and Pex9, to target correctly. While both proteins are similar and recognize PTS1 signals, Pex9 targets only a subset of Pex5 cargo proteins. However, what defines this substrate selectivity remains uncovered. Here, we used unbiased screens alongside directed experiments to identify the properties underlying Pex9 targeting specificity. We find that the specificity of Pex9 is largely determined by the hydrophobic nature of the amino acid preceding the PTS1 tripeptide of its cargos. This is explained by structural modeling of the PTS1-binding cavities of the two factors showing differences in their surface hydrophobicity. Our work outlines the mechanism by which targeting specificity is achieved, enabling dynamic rewiring of the peroxisomal proteome in changing metabolic needs.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Peroxisome-Targeting Signal 1 Receptor/metabolism , Saccharomyces cerevisiae/metabolism , Protein Transport , Saccharomyces cerevisiae Proteins/metabolism , Peroxisomes/metabolismABSTRACT
Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi-ome, remains undefined. Uncovering the peroxi-ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover peroxisomal proteins in the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi-ome by ~40% and paved the way for performing systematic, whole-organellar proteome assays. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.
Subject(s)
Peroxisomes , Proteome , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Peroxisomes/metabolism , Proteome/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Metabolism is emerging as a central influencer of multiple disease states in humans. Peroxisomes are central metabolic organelles whose decreased function gives rise to severe peroxisomal diseases. Recently, it is becoming clear that, beyond such rare inborn errors, the deterioration of peroxisomal functions contributes to multiple and prevalent diseases such as cancer, viral infection, diabetes, and neurodegeneration. Despite the clear importance of peroxisomes in common pathophysiological processes, research on the mechanisms underlying their contributions is still sparse. Here, we highlight the timeliness of focusing on peroxisomes in current research on central, abundant, and society-impacting human pathologies. As peroxisomes are now coming into the spotlight, it is clear that intensive research into these important organelles will enable a better understanding of their contribution to human health, serving as the basis to develop new diagnostic and therapeutic approaches to prevent and treat human diseases.
Subject(s)
Peroxisomal Disorders , Humans , Peroxisomal Disorders/diagnosis , Peroxisomal Disorders/genetics , Peroxisomal Disorders/metabolism , Peroxisomes/metabolismABSTRACT
Peroxisomes host essential metabolic enzymes and are crucial for human health and survival. Although peroxisomes were first described over 60 years ago, their entire proteome has not yet been identified. As a basis for understanding the variety of peroxisomal functions, we used a high-throughput screen to discover peroxisomal proteins in yeast. To visualize low abundance proteins, we utilized a collection of strains containing a peroxisomal marker in which each protein is expressed from the constitutive and strong TEF2 promoter. Using this approach, we uncovered 18 proteins that were not observed in peroxisomes before and could show their metabolic and targeting factor dependence for peroxisomal localization. We focus on one newly identified and uncharacterized matrix protein, Ynl097c-b, and show that it localizes to peroxisomes upon lysine deprivation and that its localization to peroxisomes depends on the lysine biosynthesis enzyme, Lys1. We demonstrate that Ynl097c-b affects the abundance of Lys1 and the lysine biosynthesis pathway. We have therefore renamed this protein Pls1 for Peroxisomal Lys1 Stabilizing 1. Our work uncovers an additional layer of regulation on the central lysine biosynthesis pathway. More generally it highlights how the discovery of peroxisomal proteins can expand our understanding of cellular metabolism.
Subject(s)
Peroxisomes , Saccharomyces cerevisiae Proteins , Humans , Lysine/metabolism , Peroxisomes/metabolism , Proteome/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Crucial metabolic functions of peroxisomes rely on a variety of peroxisomal membrane proteins (PMPs). While mRNA transcripts of PMPs were shown to be colocalized with peroxisomes, the process by which PMPs efficiently couple translation with targeting to the peroxisomal membrane remained elusive. Here, we combine quantitative electron microscopy with proximity-specific ribosome profiling and reveal that translation of specific PMPs occurs on the surface of peroxisomes in the yeast Saccharomyces cerevisiae. This places peroxisomes alongside chloroplasts, mitochondria, and the endoplasmic reticulum as organelles that use localized translation for ensuring correct insertion of hydrophobic proteins into their membranes. Moreover, the correct targeting of these transcripts to peroxisomes is crucial for peroxisomal and cellular function, emphasizing the importance of localized translation for cellular physiology.
ABSTRACT
Eukaryotic cells have evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge of molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore crucial for understanding eukaryotic metabolism. Here, we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. This dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Amino Acid Sequence , Cytosol/metabolism , Glyoxylates , Malate Dehydrogenase/genetics , Malate Dehydrogenase/metabolism , Peroxisomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and non-phosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes in oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Overexpression of Cit2p rescued the growth phenotype of yeast cells expressing Pex14p-S266D in oleic acid. Our data indicate that phosphorylation of Pex14p at S266 provides a mechanism for controlling the peroxisomal import of Cit2p, which helps S. cerevisiae cells to adjust their carbohydrate metabolism according to the nutritional conditions.
ABSTRACT
Approximately half of eukaryotic proteins reside in organelles. To reach their correct destination, such proteins harbor targeting signals recognized by dedicated targeting pathways. It has been shown that differences in targeting signals alter the efficiency in which proteins are recognized and targeted. Since multiple proteins compete for any single pathway, such differences can affect the priority for which a protein is catered. However, to date the entire repertoire of proteins with targeting priority, and the mechanisms underlying it, have not been explored for any pathway. Here we developed a systematic tool to study targeting priority and used the Pex5-mediated targeting to yeast peroxisomes as a model. We titrated Pex5 out by expressing high levels of a Pex5-cargo protein and examined how the localization of each peroxisomal protein is affected. We found that while most known Pex5 cargo proteins were outcompeted, several cargo proteins were not affected, implying that they have high targeting priority. This priority group was dependent on metabolic conditions. We dissected the mechanism of priority for these proteins and suggest that targeting priority is governed by different parameters, including binding affinity of the targeting signal to the cargo factor, the number of binding interfaces to the cargo factor, and more. This approach can be modified to study targeting priority in various organelles, cell types, and organisms.
Subject(s)
Peroxisomal Targeting Signals , Peroxisome-Targeting Signal 1 Receptor/metabolism , Peroxisomes/metabolism , Proof of Concept Study , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolismSubject(s)
Communicable Diseases/metabolism , Health , Peroxisomes/metabolism , Protein Transport , Signal Transduction , Autophagy , Humans , Tropical MedicineABSTRACT
Fatty acyl-CoA reductase 1 (Far1) is a ubiquitously expressed peroxisomal membrane protein that generates the fatty alcohols required for the biosynthesis of ether lipids. Lipid droplet localization of exogenously expressed and endogenous human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization, protease sensitivity and N-glycosylation tagging suggested that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively. Two closely spaced hydrophobic domains are contained within the C-terminal region. When analyzed separately, the second domain was sufficient for the localization of a fluorescent reporter to lipid droplets. Targeting of Far1 to lipid droplets was not impaired in either Pex19 or ASNA1 (also known as TRC40) CRISPR/Cas9 knockout cells. In conclusion, our data suggest that Far1 is a novel member of the rather exclusive group of dual topology membrane proteins. At the same time, Far1 shows lipid metabolism-dependent differential subcellular localizations to peroxisomes and lipid droplets.
Subject(s)
Aldehyde Oxidoreductases/metabolism , Intracellular Membranes/ultrastructure , Lipid Droplets/ultrastructure , Peroxisomes/ultrastructure , Animals , Humans , Intracellular Membranes/chemistry , Intracellular Membranes/metabolism , Lipid Droplets/metabolism , Lipid Metabolism , Membrane Proteins/metabolism , Microscopy, Confocal , Microscopy, Electron, Transmission/methods , Peroxisomes/metabolismABSTRACT
The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.
ABSTRACT
The current view on peroxisomes has changed dramatically from being human cell oddities to vital organelles that host several key metabolic pathways. To fulfil over 50 different enzymatic functions, human peroxisomes host either unique peroxisomal proteins or dual-localized proteins. The identification and characterization of the complete peroxisomal proteome in humans is important for diagnosis and treatment of patients with peroxisomal disorders as well as for uncovering novel peroxisomal functions and regulatory modules. Hence, here we compiled a comprehensive list of mammalian peroxisomal and peroxisome-associated proteins by curating results of several quantitative and non-quantitative proteomic studies together with entries in the UniProtKB and Compartments knowledge channel databases. Our analysis gives a holistic view on the mammalian peroxisomal proteome and brings to light potential new peroxisomal and peroxisome-associated proteins. We believe that this dataset, represents a valuable surrogate map of the human peroxisomal proteome.
Subject(s)
Peroxisomes/metabolism , Proteome/analysis , Proteome/metabolism , Proteomics , Animals , Humans , Metabolic Networks and Pathways , Peroxisomal Disorders/metabolismABSTRACT
Malate dehydrogenases (Mdhs) reversibly convert malate to oxaloacetate and serve as important enzymes in several metabolic pathways. In the yeast Saccharomyces cerevisiae there are three Mdh isozymes, localized to different compartments in the cell. In order to identify specifically the Mdh2 isozyme, GenScript USA produced three different antibodies that we further tested by western blot. All three antibodies recognized the S. cerevisiae Mdh2 with different background and specificity properties. One of the antibodies had a relatively low background and high specificity and thus can be used for specific identification of Mdh2 in various experimental settings.
Subject(s)
Antibodies/immunology , Antibodies/metabolism , Blotting, Western/methods , Malate Dehydrogenase/immunology , Malate Dehydrogenase/metabolism , Saccharomyces cerevisiae Proteins/immunology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Antibodies/isolation & purification , IsoenzymesABSTRACT
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 Pex5â»PTS1 interaction is functional in the coccidia and indicate that a nonconventional peroxisomal import mechanism may operate in the absence of Pex13 and Pex14.