GO:0006625 protein targeting to peroxisome: Protein Import Pathway, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0006625 (protein targeting to peroxisome) describes the directed transport of proteins into peroxisomes, typically guided by intrinsic targeting signals such as PTS1 or PTS2.
• Peroxisomal matrix protein import is a complex, multi-step process involving receptor recognition, docking, translocation, and receptor recycling.
• Recent studies reveal that phase separation and a nuclear pore-like phase facilitate the import of folded proteins across the peroxisomal membrane.
• Defects in peroxisomal protein targeting cause severe human diseases, including Zellweger spectrum disorders and X-linked adrenoleukodystrophy.
• Key genes include PEX5, PEX7, PEX13, PEX14, and PEX39, which are essential for receptor-mediated import and have been characterized across species.
• CRISPR-based models (knockout, point mutation, knock-in, overexpression) enable precise dissection of PEX gene functions and disease mechanisms.
Description
Protein targeting to peroxisome (GO:0006625) is the biological process that directs proteins to the peroxisome, a ubiquitous organelle involved in fatty acid oxidation, reactive oxygen species metabolism, and plasmalogen synthesis. This process is essential for peroxisomal biogenesis and function, as most peroxisomal matrix proteins are synthesized in the cytosol and must be imported post-translationally. The targeting is usually mediated by peroxisomal targeting signals (PTS1 or PTS2) contained within the cargo proteins, which are recognized by cytosolic receptors. Understanding protein targeting to peroxisome is critical because defects in this pathway lead to peroxisome biogenesis disorders (PBDs), a group of often lethal inherited diseases with diverse clinical manifestations, including neurological dysfunction, developmental delay, and metabolic abnormalities. Moreover, recent discoveries have unveiled unexpected mechanisms, such as phase separation and a nuclear pore-like phase, that govern the import of folded proteins across the peroxisomal membrane. These findings have reshaped our view of peroxisomal protein import and opened new avenues for therapeutic intervention. This article provides a comprehensive overview of GO:0006625, covering its definition, molecular mechanisms, key genes, associated diseases, and state-of-the-art research methods. It is intended for researchers, clinicians, and students seeking a authoritative, citation-backed resource on peroxisomal protein targeting.
protein targeting to peroxisome At A Glance
| GO ID | GO:0006625 |
|---|---|
| GO term | protein targeting to peroxisome |
| Ontology | biological_process |
| Synonym | protein-peroxisome targeting |
| Definition | The process of directing proteins towards the peroxisome, usually using signals contained within the protein. |
| Major function | Post-translational import of matrix proteins into peroxisomes, essential for peroxisome biogenesis and metabolic functions. |
| Key targeting signals | PTS1 (C-terminal tripeptide) and PTS2 (N-terminal nonapeptide). |
| Major receptors | PEX5 (PTS1 receptor), PEX7 (PTS2 receptor). |
| Docking complex | PEX13, PEX14, PEX17. |
| Translocation machinery | PEX2, PEX10, PEX12 (RING finger proteins). |
| Receptor recycling | PEX1, PEX6, PEX26 (AAA-ATPases). |
What Is GO:0006625?
According to the Gene Ontology, GO:0006625 (protein targeting to peroxisome) is defined as the process of directing proteins towards the peroxisome, usually using signals contained within the protein [QuickGO]. This encompasses the recognition of peroxisomal targeting signals (PTS) by cytosolic receptors, the docking of receptor-cargo complexes at the peroxisomal membrane, translocation of cargo into the matrix, and recycling of receptors for further rounds of import.
Why Is protein targeting to peroxisome Important in Cell Biology?
Protein targeting to peroxisome is fundamental to cellular metabolism and human health. Peroxisomes house over 50 enzymes that participate in fatty acid beta-oxidation, detoxification of hydrogen peroxide, and synthesis of plasmalogens and bile acids. Without proper targeting, these enzymes fail to reach the peroxisome, leading to organelle dysfunction and severe disease. Mutations in PEX genes that mediate targeting cause peroxisome biogenesis disorders, including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, which are characterized by neurological impairment, liver dysfunction, and early death. Thus, deciphering the molecular details of GO:0006625 is essential for understanding disease pathogenesis and developing targeted therapies.
• Peroxisomal protein targeting is required for the import of enzymes involved in fatty acid oxidation, plasmalogen synthesis, and ROS detoxification.
• Defects in targeting cause Zellweger spectrum disorders, a continuum of lethal multisystem diseases.
• The process is evolutionarily conserved from plants to humans, with PEX genes identified across species.
• Recent discovery of phase separation in peroxisome biogenesis highlights novel biophysical principles.
• A nuclear pore-like phase for protein import suggests mechanistic parallels with nuclear transport.
• PEX39 was recently identified as a facilitator of PTS2 protein import, expanding the list of targeting factors.
• Understanding targeting mechanisms can inform therapies for peroxisomal disorders and metabolic diseases.
• Research on peroxisomal targeting provides insights into organelle biogenesis and protein sorting in general.
What Happens During protein targeting to peroxisome?
Recognition of Peroxisomal Targeting Signals (PTS)
In simple terms: Proteins destined for the peroxisome carry a molecular 'zip code' that is recognized by receptor proteins in the cytosol.
Most peroxisomal matrix proteins contain either a C-terminal PTS1 (Ser-Lys-Leu or variants) or an N-terminal PTS2 (Arg-Leu-X5-His-Leu). PTS1 is recognized by the cytosolic receptor PEX5, while PTS2 is recognized by PEX7. In plants, PEX7 and PEX5 cooperate for PTS2 import. The recognition step is highly specific and ensures that only proteins with the appropriate signal are targeted to the peroxisome.
Docking at the Peroxisomal Membrane
In simple terms: The receptor-cargo complex binds to a docking station on the peroxisome surface.
The cargo-loaded receptors (PEX5 and/or PEX7) dock at the peroxisomal membrane by interacting with the docking complex, which includes PEX13, PEX14, and in some organisms PEX17. This interaction is essential for subsequent translocation. Recent studies suggest that the docking step may involve phase separation, forming a distinct import-competent phase.
Translocation of Cargo into the Peroxisomal Matrix
In simple terms: The protein is moved across the peroxisomal membrane into the interior of the organelle.
Translocation of folded or oligomeric proteins occurs through a dynamic pore-like structure formed by the RING finger proteins PEX2, PEX10, and PEX12, along with PEX5. A recent study revealed that protein import into peroxisomes occurs through a nuclear pore-like phase, suggesting a unique mechanism for transporting large cargo. PEX39 was identified as a novel component that facilitates the import of PTS2-containing proteins.
Receptor Recycling and Ubiquitination
In simple terms: After delivering the cargo, the receptor is extracted from the membrane and reused.
Following cargo release, PEX5 is monoubiquitinated at a conserved cysteine residue by the E3 ligases PEX2, PEX10, and PEX12. This modification allows the AAA-ATPases PEX1 and PEX6, anchored by PEX26, to extract PEX5 from the membrane for recycling. In plants, similar recycling mechanisms exist, though with some variations. Defects in recycling lead to impaired import and peroxisome dysfunction.
Phase Separation in Peroxisome Biogenesis
In simple terms: Some proteins spontaneously cluster into droplets to help build new peroxisomes.
A recent study demonstrated that peroxisome biogenesis is initiated by protein phase separation, where PEX proteins condense into liquid-like droplets that mature into new peroxisomes. This phase separation is driven by multivalent interactions and may represent a general principle for organelle formation. The discovery adds a new layer to our understanding of GO:0006625, linking targeting to organelle biogenesis.
Key Genes Involved in GO:0006625 protein targeting to peroxisome
The following genes encode proteins that are directly involved in peroxisomal protein targeting and import, as established by genetic and biochemical studies across model organisms.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PEX5 | Cytosolic receptor for PTS1-containing proteins; shuttles cargo to peroxisome and is recycled | Mutations cause Zellweger spectrum disorders; key target for studying import cycle |
| PEX7 | Cytosolic receptor for PTS2-containing proteins | Defects cause rhizomelic chondrodysplasia punctata type 1; used to study PTS2 pathway |
| PEX13 | Docking complex component; interacts with PEX5 and PEX14 | Mutations linked to Zellweger syndrome; important for membrane docking |
| PEX14 | Docking complex component; binds PEX5 and PEX7 | Essential for import; knockout models show severe peroxisomal defects |
| PEX2 | RING finger protein; E3 ligase for PEX5 ubiquitination | Mutations cause Zellweger; involved in receptor recycling |
| PEX10 | RING finger protein; E3 ligase for PEX5 ubiquitination | Mutations cause Zellweger; part of translocation machinery |
| PEX12 | RING finger protein; E3 ligase for PEX5 ubiquitination | Mutations cause Zellweger; essential for import |
| PEX1 | AAA-ATPase; extracts ubiquitinated PEX5 from membrane | Mutations cause Zellweger; required for receptor recycling |
| PEX6 | AAA-ATPase; works with PEX1 in receptor extraction | Mutations cause Zellweger; involved in recycling |
| PEX26 | Membrane anchor for PEX1/PEX6 complex | Mutations cause Zellweger; targets ATPases to peroxisome |
| PEX39 | Facilitates peroxisomal import of PTS2-containing proteins | Novel component; recent discovery expands import machinery |
| PEX3 | Peroxisomal membrane protein; involved in peroxisome biogenesis | Key for early steps of peroxisome formation; phase separation studies |
| PEX19 | Chaperone and import receptor for peroxisomal membrane proteins | Essential for membrane protein targeting; mutations cause Zellweger |
| PEX16 | Peroxisomal membrane protein; involved in peroxisome biogenesis | Mutations cause Zellweger; role in membrane protein import |
| PEX11 | Peroxisomal membrane protein; regulates peroxisome proliferation | Not directly in matrix protein targeting but affects organelle dynamics |
| PEX15 | Plant homolog of PEX26; involved in receptor recycling | Studied in plants for peroxisome biogenesis mechanisms |
| PEX4 | Ubiquitin-conjugating enzyme in plants; involved in PEX5 recycling | Plant-specific factor for peroxisomal import |
| PEX22 | Anchors PEX4 in plants; involved in receptor recycling | Plant peroxisome biogenesis factor |
How Is protein targeting to peroxisome Regulated?
The process of protein targeting to peroxisome is regulated at multiple levels. Transcriptional regulation of PEX genes occurs in response to metabolic cues, such as fatty acid levels, via transcription factors like PPARα. Post-translational modifications, particularly ubiquitination of PEX5, control the receptor cycle and import efficiency. Additionally, the AAA-ATPases PEX1 and PEX6 are regulated by their association with PEX26 and ATP hydrolysis. Recent evidence suggests that phase separation of PEX proteins may be regulated by concentration and valency of interactions, providing a biophysical layer of control. In plants, PEX gene expression is modulated during development and in response to stress.
protein targeting to peroxisome and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PEX5 | Zellweger spectrum disorder | Knockout in human fibroblasts or HEK293; point mutations to mimic patient alleles |
| PEX7 | Rhizomelic chondrodysplasia punctata type 1 | Knockout in chondrocytes or patient-derived iPSCs; knock-in of patient mutations |
| PEX1 | Zellweger spectrum disorder | Knockout in HeLa or patient fibroblasts; overexpression of wild-type vs. mutant |
| PEX13 | Zellweger spectrum disorder | Knockout in mouse models; knock-in of patient mutations |
| PEX26 | Zellweger spectrum disorder | Knockout in HEK293; point mutation to disrupt PEX1/PEX6 anchoring |
Peroxisome Biogenesis Disorders (Zellweger Spectrum)
Mutations in PEX genes that mediate protein targeting cause Zellweger spectrum disorders (ZSD), a group of autosomal recessive diseases characterized by severe neurological impairment, hypotonia, seizures, liver dysfunction, and characteristic facial features. ZSD includes Zellweger syndrome (most severe), neonatal adrenoleukodystrophy, and infantile Refsum disease. Defects in PEX5, PEX7, PEX13, PEX14, PEX2, PEX10, PEX12, PEX1, PEX6, and PEX26 have all been linked to ZSD. The severity correlates with the extent of import deficiency.
X-linked Adrenoleukodystrophy (X-ALD)
X-ALD is caused by mutations in ABCD1, a peroxisomal membrane transporter for very long-chain fatty acids. While ABCD1 is not directly involved in protein targeting, the disease highlights the metabolic importance of peroxisomes. However, some cases of X-ALD-like phenotypes can result from defects in peroxisomal protein import, underscoring the interplay between targeting and metabolism.
Rhizomelic Chondrodysplasia Punctata (RCDP)
RCDP is a rare disorder caused by mutations in PEX7 or enzymes involved in plasmalogen synthesis. PEX7 deficiency impairs PTS2-mediated import, leading to skeletal abnormalities, cataracts, and severe growth retardation. This condition exemplifies how a specific targeting defect can cause a distinct clinical phenotype.
Neurodegeneration and Aging
Emerging evidence links peroxisomal dysfunction to age-related neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Impaired protein targeting may contribute to oxidative stress and lipid metabolism defects observed in these conditions. Further research is needed to establish causal relationships.
From protein targeting to peroxisome-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of PEX5 abolish PTS1 import? | PEX5 knockout cell line (e.g., HEK293) via CRISPR |
| How does a patient mutation in PEX7 affect PTS2 import? | Point mutation knock-in of the specific allele in a cell line |
| Can wild-type PEX1 rescue import in patient cells? | Knock-in or overexpression of PEX1 in patient-derived fibroblasts |
| Where is PEX14 localized during import? | Tagged knock-in of PEX14 with fluorescent protein (e.g., GFP) for live imaging |
| What is the role of PEX39 in PTS2 import? | PEX39 knockout and overexpression in cell lines, followed by import assays |
| Does phase separation of PEX proteins drive peroxisome biogenesis? | Overexpression of PEX proteins with phase separation tags; knockout of key PEX genes |
How to Study the protein targeting to peroxisome Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Fluorescence microscopy | Localization and dynamics of tagged proteins | Visualizing PEX5 or cargo import in live cells |
| In vitro import assay | Efficiency of protein import into isolated peroxisomes | Testing patient mutations in PEX genes |
| Immunoblotting | Processing and abundance of peroxisomal proteins | Confirming knockout or knockdown efficiency |
| AP-MS | Protein-protein interactions | Identifying novel import components like PEX39 |
| Proximity labeling (BioID) | Spatial interactome of PEX proteins | Mapping the import machinery in living cells |
| CRISPR screen | Genes required for peroxisomal targeting | Discovery of new PEX genes or modifiers |
| RNA-seq | Transcriptional changes upon import defects | Assessing cellular response to peroxisome dysfunction |
| Yeast two-hybrid | Binary interactions between PEX proteins | Validating docking complex interactions |
Fluorescence Microscopy and Live Imaging
Fluorescence microscopy is widely used to study peroxisomal protein targeting. By tagging cargo proteins or PEX components with fluorescent proteins (e.g., GFP, mCherry), researchers can visualize their localization and dynamics in real time. Co-localization with peroxisomal markers (e.g., catalase, PEX14) confirms targeting. Advanced techniques like super-resolution microscopy and live-cell imaging have revealed the phase separation behavior of PEX proteins.
Biochemical Import Assays
In vitro import assays using purified peroxisomes and radiolabeled or fluorescently labeled cargo proteins allow quantitative measurement of import efficiency. These assays can be combined with immunoblotting to detect processed or unprocessed forms of cargo. They are essential for dissecting the roles of individual PEX proteins and for testing patient mutations.
Proteomics and Interaction Studies
Affinity purification coupled with mass spectrometry (AP-MS) has been used to identify novel components of the peroxisomal import machinery, such as PEX39. Proximity labeling (BioID) and yeast two-hybrid assays map interactions among PEX proteins and cargo. These methods provide a systems-level view of the targeting pathway.
CRISPR-Based Genetic Screens
Genome-wide CRISPR knockout screens can identify genes required for peroxisomal protein targeting. By using a reporter that fluoresces only when targeted to peroxisomes, researchers can sort cells with defects and identify enriched sgRNAs. This approach has been used in plants and mammalian cells to uncover new PEX genes and modifiers.
How CRISPR Can Be Used to Study GO:0006625 protein targeting to peroxisome
Knockout
CRISPR knockout of PEX genes (e.g., PEX5, PEX7, PEX13) in cell lines such as HEK293 or patient fibroblasts abolishes peroxisomal protein import, providing a clean background to study the function of individual components. Knockout models are also used to assess the requirement of specific genes for peroxisome biogenesis and metabolism.
Point Mutation
Introducing patient-specific point mutations (e.g., in PEX1 or PEX7) via CRISPR knock-in allows researchers to study the molecular consequences of disease-causing alleles in an isogenic background. This approach is invaluable for understanding genotype-phenotype correlations and for testing pharmacological chaperones.
Knock-in
Knock-in of tagged versions of PEX proteins (e.g., GFP-PEX14) enables live-cell imaging and proteomic studies without overexpression artifacts. Knock-in of wild-type PEX genes into patient cells can rescue import defects and serve as a proof-of-concept for gene therapy.
Overexpression
Overexpression of PEX proteins or cargo with targeting signals is used to study dominant-negative effects, phase separation, and saturation of the import machinery. For example, overexpression of PEX5 mutants can disrupt import in a dominant-negative manner.
How EDITGENE Supports protein targeting to peroxisome Research
Researchers studying protein targeting to peroxisome-related genes often need to determine whether a candidate gene is causally involved in peroxisomal import, how specific mutations affect function, and whether restoring gene activity can rescue defects. EDITGENE provides a comprehensive suite of CRISPR-based services to address these questions with precision and reproducibility.
Contact EDITGENE today to design your custom CRISPR model for protein targeting to peroxisome research.
Frequently Asked Questions About protein targeting to peroxisome
What is protein targeting to peroxisome?
Protein targeting to peroxisome (GO:0006625) is the process of directing proteins towards the peroxisome, usually using signals contained within the protein, such as PTS1 or PTS2.
What genes are involved in protein targeting to peroxisome?
Key genes include PEX5, PEX7, PEX13, PEX14, PEX2, PEX10, PEX12, PEX1, PEX6, PEX26, and PEX39, among others.
What diseases are associated with defects in peroxisomal protein targeting?
Defects cause peroxisome biogenesis disorders, including Zellweger spectrum disorders and rhizomelic chondrodysplasia punctata.
How does PEX5 recognize PTS1?
PEX5 binds the C-terminal PTS1 tripeptide (e.g., SKL) of cargo proteins in the cytosol, forming a receptor-cargo complex that docks at the peroxisome.
What is the role of PEX7?
PEX7 is the cytosolic receptor for PTS2-containing proteins, which typically have an N-terminal nonapeptide signal.
How are peroxisomal proteins imported?
They are imported post-translationally through a dynamic pore-like structure formed by PEX2, PEX10, PEX12, and PEX5, often as folded or oligomeric proteins.
What is the role of phase separation in peroxisome biogenesis?
Phase separation of PEX proteins initiates peroxisome biogenesis by forming liquid-like droplets that mature into new organelles.
What is PEX39?
PEX39 is a recently identified protein that facilitates the peroxisomal import of PTS2-containing proteins.
How can I study protein targeting to peroxisome in the lab?
Common methods include fluorescence microscopy, in vitro import assays, proteomics, and CRISPR-based screens.
What CRISPR models are available for studying peroxisomal targeting?
EDITGENE offers knockout, point mutation, knock-in, and overexpression models for PEX genes and related factors, as well as CRISPR library screening.
Conclusion
Protein targeting to peroxisome (GO:0006625) is a fundamental cellular process that ensures the correct localization of enzymes and other proteins to peroxisomes. Decades of research have elucidated the key components and steps, from signal recognition to receptor recycling, and recent discoveries have revealed unexpected mechanisms such as phase separation and a nuclear pore-like import phase. Defects in this pathway cause devastating human diseases, underscoring its clinical importance. Continued research using advanced CRISPR models and screening technologies will further illuminate the molecular details and may lead to new therapeutic strategies for peroxisomal disorders.
References
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- 3. Skowyra ML et al.. 2024. Towards solving the mystery of peroxisomal matrix protein import.. Trends Cell Biol 34(5):388-405 PMID: 37743160
- 4. Cross LL et al.. 2016. Peroxisome biogenesis, protein targeting mechanisms and PEX gene functions in plants.. Biochim Biophys Acta 1863(5):850-62 PMID: 26408938
- 5. Purdue PE et al.. 2001. Peroxisome biogenesis.. Annu Rev Cell Dev Biol 17:701-52 PMID: 11687502
- 6. Chen WW et al.. 2025. PEX39 facilitates the peroxisomal import of PTS2-containing proteins.. Nat Cell Biol 27(8):1256-1271 PMID: 40739340
- 7. Gao Y et al.. 2022. Protein import into peroxisomes occurs through a nuclear pore-like phase.. Science 378(6625):eadf3971 PMID: 36520918
- 8. Baker A et al.. 2016. Peroxisome protein import: a complex journey.. Biochem Soc Trans 44(3):783-9 PMID: 27284042
