GO:0043813 phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0043813 describes the enzymatic removal of the 5-phosphate from phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), yielding phosphatidylinositol-3-phosphate (PI3P) and free phosphate.
• The reaction is catalyzed by the Sac3/FIG4 phosphatase, which opposes the lipid kinase PIKfyve that synthesizes PI(3,5)P2.
• PI(3,5)P2 homeostasis is essential for endolysosomal membrane trafficking, lysosome reformation, and autophagy.
• Deficiency of FIG4 causes Charcot-Marie-Tooth disease type 4J and related neurodevelopmental phenotypes.
• PI(3,5)P2 machinery also influences neurite thickness through NSG1/NEEP21 and is required for efficient Ebola virus entry.
• CRISPR knockout, point-mutation, and knock-in models enable precise interrogation of FIG4 and PIKfyve function in disease-relevant cell types.
Description
Phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity (GO:0043813) is a molecular function that catalyzes the hydrolysis of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) to phosphatidylinositol-3-phosphate (PI3P) and phosphate. This activity is critical for maintaining the cellular pools of these signaling lipids, which control endosomal and lysosomal membrane dynamics. The principal enzyme responsible for this reaction in mammalian cells is the Sac3/FIG4 phosphatase, a member of the myotubularin-related protein family. Because PI(3,5)P2 regulates organelle homeostasis, autophagy, and membrane trafficking, its precise turnover is essential for neuronal and muscular function. Dysregulation of GO:0043813 has been linked to severe human disorders, including Charcot-Marie-Tooth disease type 4J, a peripheral neuropathy caused by loss-of-function mutations in FIG4. Recent studies have also implicated PI(3,5)P2 metabolism in neurodevelopmental and neurodegenerative conditions, as well as in host-pathogen interactions such as Ebola virus entry. Understanding the molecular mechanism, regulation, and disease relevance of this phosphatase activity is therefore a high-priority research area. This article provides a research-grade overview of GO:0043813, covering its definition, biological roles, key genes, regulatory mechanisms, disease associations, and state-of-the-art methods for studying it. It is designed for researchers, drug developers, and AI-driven knowledge retrieval systems seeking authoritative, citation-backed information.
phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity At A Glance
| GO ID | GO:0043813 |
|---|---|
| GO term | phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity |
| Ontology | molecular_function |
| Synonym | phosphatidylinositol 3,5-bisphosphate 5-phosphatase activity |
| Definition | Catalysis of the reaction: a 1,2-diacyl-sn-glycero-3-phospho-(1D-myo-inositol-3,5-bisphosphate) + H2O = a 1,2-diacyl-sn-glycero-3-phospho-(1D-myo-inositol-3-phosphate) + phosphate. |
| Major function | Hydrolyzes PI(3,5)P2 to PI3P, regulating endolysosomal lipid homeostasis. |
| Key enzyme | FIG4 (Sac3) phosphatase |
| Substrate | Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) |
| Product | Phosphatidylinositol-3-phosphate (PI3P) and phosphate |
| Cellular process | Endosomal trafficking, lysosome reformation, autophagy |
What Is GO:0043813?
GO:0043813, phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity, is defined as the catalysis of the reaction: a 1,2-diacyl-sn-glycero-3-phospho-(1D-myo-inositol-3,5-bisphosphate) + H2O = a 1,2-diacyl-sn-glycero-3-phospho-(1D-myo-inositol-3-phosphate) + phosphate. In simpler terms, it is the enzymatic removal of the phosphate group at the 5-position of the inositol ring of PI(3,5)P2, converting it to PI3P and inorganic phosphate.
Why Is phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity Important in Cell Biology?
GO:0043813 is essential for cellular lipid homeostasis because it directly controls the levels of PI(3,5)P2, a low-abundance phosphoinositide that orchestrates endolysosomal membrane dynamics, autophagy, and ion transport. Imbalances in this activity lead to severe human diseases, including Charcot-Marie-Tooth neuropathy and potentially other neurodegenerative conditions. Moreover, the PI(3,5)P2 pathway is exploited by pathogens such as Ebola virus, making this activity a potential therapeutic target.
• Maintains PI(3,5)P2 and PI3P pools critical for endosomal and lysosomal function.
• Regulates lysosome reformation from endolysosomes, a process required for sustained autophagy.
• Mutations in FIG4, the enzyme responsible for this activity, cause Charcot-Marie-Tooth disease type 4J.
• FIG4-related leukoencephalopathy presents with characteristic neuroimaging findings such as T2 olivary nuclei hyperintensities.
• PI(3,5)P2 machinery, including this phosphatase, modulates neurite thickness via NSG1/NEEP21.
• Ebola virus entry depends on PI(3,5)P2 production, highlighting a role in viral pathogenesis.
• Defective autophagy linked to PI(3,5)P2 dysregulation contributes to neurodevelopmental and neurodegenerative disorders.
• The activity is a potential drug target for neuropathy and viral infections.
• CRISPR-based models allow precise dissection of FIG4 and PIKfyve functions in disease.
• Understanding this activity aids in interpreting genetic variants of uncertain significance in FIG4.
Mechanism, Genes and Research Methods
Substrate recognition and binding
In simple terms: The enzyme grabs PI(3,5)P2 from the membrane.
The FIG4 phosphatase specifically recognizes phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) embedded in endosomal membranes. Structural and biochemical studies of the PIKfyve lipid kinase complex, which includes FIG4, reveal that the phosphatase domain of FIG4 binds the inositol headgroup and the 3,5-bisphosphate moiety with high specificity. This binding positions the 5-phosphate for nucleophilic attack by a water molecule.
Catalytic hydrolysis of the 5-phosphate
In simple terms: The enzyme cuts off the phosphate at the 5-position.
The catalytic mechanism involves a conserved cysteine residue in the CX5R motif of FIG4, which performs a nucleophilic attack on the 5-phosphate of PI(3,5)P2, forming a covalent enzyme-phosphate intermediate. Subsequent hydrolysis by water releases inorganic phosphate and yields phosphatidylinositol-3-phosphate (PI3P). This reaction is essential for terminating PI(3,5)P2 signaling and recycling PI3P for further rounds of trafficking.
Regulation by PIKfyve and the lipid kinase complex
In simple terms: The enzyme works in a team with a kinase that makes its substrate.
FIG4 functions in a complex with the lipid kinase PIKfyve, which synthesizes PI(3,5)P2 from PI3P. The opposing activities of PIKfyve and FIG4 maintain steady-state levels of PI(3,5)P2. Structural analysis of the PIKfyve complex shows that FIG4's phosphatase activity is tightly coupled to PIKfyve's kinase activity, ensuring rapid turnover of the lipid. Loss of FIG4 leads to accumulation of PI(3,5)P2 and impaired lysosome reformation.
Downstream effects on endolysosomal trafficking and autophagy
In simple terms: The reaction controls how cells recycle their internal compartments.
By converting PI(3,5)P2 to PI3P, GO:0043813 activity regulates the recruitment of effector proteins that mediate endosome-to-lysosome trafficking, lysosome reformation, and autophagosome clearance. Studies in FIG4-deficient cells show enlarged endolysosomes and defective terminal storage lysosome reformation, leading to impaired autophagy. These defects contribute to neuronal dysfunction and neurodegeneration.
Role in neurite outgrowth and viral entry
In simple terms: The enzyme also affects nerve cell shape and virus infection.
PI(3,5)P2 machinery, including the 5-phosphatase activity, regulates neurite thickness through the neuron-specific endosomal protein NSG1/NEEP21. Additionally, efficient Ebola virus entry requires PI(3,5)P2 production, and perturbation of this pathway reduces viral infection. These findings highlight diverse physiological and pathological roles of GO:0043813.
Key Genes Involved in GO:0043813 phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity
The following genes encode proteins directly involved in phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity, its regulation, or its downstream effects.
| Gene | Major Role | Research Relevance |
|---|---|---|
| FIG4 | PI(3,5)P2 5-phosphatase; catalyzes the reaction | Mutations cause Charcot-Marie-Tooth 4J; key for neuropathy research |
| PIKfyve | PI(3,5)P2 5-kinase; synthesizes substrate | Opposes FIG4; essential for endolysosomal homeostasis |
| Vac14 | Scaffold protein in PIKfyve-FIG4 complex | Regulates complex assembly and lipid turnover |
| Vac7 | Accessory protein in yeast PIKfyve complex | Model for studying complex regulation |
| NSG1/NEEP21 | Neuron-specific endosomal protein | Mediates neurite thickness via PI(3,5)P2 |
| MTMR2 | Related myotubularin phosphatase | Comparative studies of phosphoinositide phosphatases |
| MTMR13 | Pseudophosphatase partner of MTMR2 | Regulates MTMR2 stability and function |
| ATG5 | Autophagy machinery | Links PI(3,5)P2 defects to autophagy impairment |
| ATG7 | Autophagy machinery | Used in autophagy flux assays |
| LAMP1 | Lysosomal marker | Assesses lysosome reformation defects |
| LAMP2 | Lysosomal marker | Evaluates endolysosomal trafficking |
| EEA1 | Early endosome marker | Monitors endosomal recruitment |
| RAB7 | Late endosome marker | Tracks endolysosomal maturation |
| SNX1 | Retromer component | PI3P effector; downstream of FIG4 activity |
| WIPI2 | PI3P-binding autophagy protein | Links PI3P production to autophagosome formation |
| NPC1 | Cholesterol trafficking | Interacts with PI(3,5)P2 pathway |
| Ebola GP | Viral glycoprotein | Requires PI(3,5)P2 for entry |
How Is phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity Regulated?
The activity of phosphatidylinositol-3,5-bisphosphate 5-phosphatase is primarily regulated by its incorporation into a multiprotein complex with the lipid kinase PIKfyve and the scaffold protein Vac14. This complex ensures coordinated synthesis and degradation of PI(3,5)P2. Additionally, cellular stress conditions such as nutrient deprivation can modulate autophagy, indirectly affecting the demand for this phosphatase activity. Mutations in FIG4 that impair its catalytic activity or complex formation lead to disease, underscoring the importance of tight regulation.
phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| FIG4 | Charcot-Marie-Tooth 4J; leukoencephalopathy | Patient iPSC-derived neurons; Fig4 KO mouse |
| PIKfyve | Endolysosomal dysfunction; viral entry | PIKfyve KO cell lines; Ebola pseudovirus entry assay |
| NSG1/NEEP21 | Neurite thickness regulation | NSG1 KO primary neurons; overexpression |
| ATG5/ATG7 | Autophagy impairment | CRISPR KO in neuronal cells; LC3 flux assays |
| MTMR2/MTMR13 | Peripheral neuropathy | KO models; lipid phosphatase assays |
Charcot-Marie-Tooth disease type 4J
Biallelic loss-of-function mutations in FIG4, the enzyme responsible for GO:0043813, cause Charcot-Marie-Tooth disease type 4J, a severe peripheral neuropathy characterized by demyelination and axonal loss. Patients homozygous for the FIG4 p.Ile41Thr mutation exhibit early-onset motor and sensory deficits. Studies in patient-derived cells show altered phosphoinositide profiles, confirming the link between phosphatase deficiency and disease.
FIG4-related leukoencephalopathy
FIG4 mutations also cause a leukoencephalopathy with distinct neuroimaging features, including T2 hyperintensities in the olivary nuclei. This condition expands the phenotypic spectrum of FIG4-related disorders beyond peripheral neuropathy. The underlying mechanism involves impaired endolysosomal function and autophagy in neurons.
Neurodevelopmental and neurodegenerative disorders
Defective autophagy due to PI(3,5)P2 dysregulation contributes to a range of neurodevelopmental, neuromuscular, and neurodegenerative disorders. Mutations in genes controlling this pathway, including FIG4, have been associated with developmental delay, seizures, and progressive neurodegeneration. The PI(3,5)P2 machinery also regulates neurite thickness, providing a cellular basis for neurodevelopmental phenotypes.
Viral infection
Ebola virus requires PI(3,5)P2 production for efficient entry into host cells. Perturbation of the PI(3,5)P2 pathway, including the 5-phosphatase activity, reduces viral entry, suggesting that modulating GO:0043813 could be an antiviral strategy.
From phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of FIG4 phosphatase activity cause endolysosomal defects? | FIG4 knockout cell lines (HeLa, HEK293) |
| How does the FIG4 p.Ile41Thr mutation affect lipid levels? | Point-mutation knock-in via CRISPR in patient iPSCs |
| Can wild-type FIG4 rescue disease phenotypes? | Knock-in of tagged FIG4 (e.g., GFP-FIG4) in KO background |
| What is the effect of FIG4 overexpression on PI(3,5)P2 levels? | Overexpression of FIG4 in neuronal cell lines |
| How does PIKfyve-FIG4 complex assembly regulate activity? | Knock-in of tagged PIKfyve and FIG4 for co-IP |
| Does FIG4 deficiency alter neurite outgrowth? | Primary neurons from Fig4 KO mice; NSG1 co-KO |
How to Study the phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Lipidomics (LC-MS/MS) | PI(3,5)P2 and PI3P levels | Quantify lipid changes in FIG4 KO cells |
| Fluorescence microscopy | Endolysosome morphology, lysosome reformation | Assess FIG4 KO phenotypes |
| CRISPR knockout screening | Genes affecting PI(3,5)P2 homeostasis | Identify modifiers of FIG4 pathway |
| In vitro phosphatase assay | Enzymatic activity of FIG4 | Validate mutations and inhibitors |
| Co-immunoprecipitation | PIKfyve-FIG4 complex assembly | Study regulation by Vac14 |
| Neurite outgrowth assay | Neurite thickness and length | Evaluate NSG1/NEEP21 role |
| Viral entry assay | Ebola pseudovirus infection | Test PI(3,5)P2 requirement |
| Autophagy flux assay (LC3-II) | Autophagosome turnover | Link FIG4 to autophagy |
Lipidomics and phosphoinositide profiling
Mass spectrometry-based lipidomics enables quantification of PI(3,5)P2 and PI3P levels in cells with modulated GO:0043813 activity. This approach has been used to demonstrate altered phosphoinositide profiles in FIG4-deficient patient cells. Isotope labeling and HPLC can further resolve individual species.
Fluorescence microscopy and live-cell imaging
Fluorescently tagged lipid-binding domains (e.g., GFP-2xFYVE for PI3P) and organelle markers (LAMP1, EEA1) allow visualization of endolysosomal dynamics. Live-cell imaging of lysosome reformation after FIG4 knockout reveals enlarged endolysosomes and defective tubulation. Neurite thickness can be measured using phase-contrast or fluorescence microscopy.
CRISPR screening and functional genomics
Genome-wide CRISPR knockout screens can identify modifiers of PI(3,5)P2 homeostasis and FIG4-dependent phenotypes. Such screens have been instrumental in mapping the autophagy and endolysosomal networks linked to this activity. Bioinformatics analysis of screening data reveals enriched pathways and potential drug targets.
Biochemical assays for phosphatase activity
In vitro phosphatase assays using synthetic PI(3,5)P2 substrates and recombinant FIG4 protein measure catalytic activity directly. Malachite green or fluorescent phosphate release assays quantify the 5-phosphatase reaction. These assays are used to validate disease-associated mutations.
How CRISPR Can Be Used to Study GO:0043813 phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity
Knockout
CRISPR-Cas9 knockout of FIG4 or PIKfyve in cell lines (e.g., HeLa, HEK293, iPSC-derived neurons) abolishes GO:0043813 activity, leading to PI(3,5)P2 accumulation and endolysosomal defects. These models are used to study lysosome reformation, autophagy, and neuronal survival. Knockout of NSG1 in neurons reveals its role in neurite thickness downstream of PI(3,5)P2.
Point Mutation
CRISPR-mediated knock-in of disease-associated point mutations, such as FIG4 p.Ile41Thr, recapitulates patient-specific lipid abnormalities and cellular phenotypes. These models are valuable for testing genotype-phenotype correlations and drug responses. Point mutations in the catalytic CX5R motif can abolish phosphatase activity and serve as negative controls.
Knock-in
Knock-in of epitope-tagged FIG4 (e.g., GFP or HA) at the endogenous locus enables real-time tracking of protein localization and interaction with PIKfyve and Vac14. This approach preserves native expression levels and regulation, providing insights into complex assembly and dynamics.
Overexpression
Overexpression of wild-type or mutant FIG4 in neuronal cell lines or primary neurons can enhance or disrupt PI(3,5)P2 turnover. Overexpression studies have shown effects on neurite outgrowth and endosomal trafficking. Inducible systems allow temporal control of expression to avoid compensatory adaptations.
How EDITGENE Supports phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity Research
Researchers studying phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity-related genes often need to determine whether a candidate gene is causally involved in endolysosomal dysfunction, autophagy, or neuropathy. EDITGENE provides a comprehensive suite of CRISPR-based services to generate precisely engineered cell models, enabling rigorous functional validation of GO:0043813 components.
Contact EDITGENE today to design your custom CRISPR model for phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity research.
Frequently Asked Questions About phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity
What is phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity?
It is the enzymatic activity (GO:0043813) that removes the 5-phosphate from PI(3,5)P2 to produce PI3P and phosphate, catalyzed primarily by the FIG4 phosphatase.
What genes are involved in phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity?
The main gene is FIG4 (also known as SAC3), which encodes the 5-phosphatase. It functions with PIKfyve, Vac14, and other proteins in the PI(3,5)P2 regulatory complex.
What diseases are associated with GO:0043813?
Mutations in FIG4 cause Charcot-Marie-Tooth disease type 4J, FIG4-related leukoencephalopathy, and contribute to neurodevelopmental and neurodegenerative disorders.
How is phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity regulated?
It is regulated by incorporation into a complex with PIKfyve and Vac14, which coordinates PI(3,5)P2 synthesis and degradation. Cellular stress and autophagy demand also modulate its activity.
What is the substrate of phosphatidylinositol-3,5-bisphosphate 5-phosphatase?
The substrate is phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), a low-abundance phosphoinositide found on endosomal membranes.
What is the product of the 5-phosphatase reaction?
The products are phosphatidylinositol-3-phosphate (PI3P) and inorganic phosphate.
How can I study phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity in the lab?
Common methods include lipidomics, fluorescence microscopy with PI3P probes, in vitro phosphatase assays, and CRISPR knockout of FIG4 followed by phenotypic analysis.
What cell models are available for FIG4 research?
EDITGENE provides FIG4 knockout, point-mutation knock-in, tagged knock-in, and overexpression models in various cell types, including iPSC-derived neurons.
Does phosphatidylinositol-3,5-bisphosphate 5-phosphatase play a role in viral infection?
Yes, PI(3,5)P2 production is required for efficient Ebola virus entry, and modulating this pathway can affect viral infection.
What is the relationship between FIG4 and autophagy?
FIG4 deficiency impairs lysosome reformation and autophagosome clearance, linking GO:0043813 to autophagy and neurodegenerative disorders.
Conclusion
Phosphatidylinositol-3,5-bisphosphate 5-phosphatase activity (GO:0043813) is a fundamental enzymatic function that controls PI(3,5)P2 turnover and endolysosomal homeostasis. Its dysregulation causes Charcot-Marie-Tooth disease and contributes to broader neurodevelopmental and neurodegenerative pathologies. The activity also plays a role in viral entry and neurite outgrowth, underscoring its diverse physiological importance. Advances in CRISPR-based models, lipidomics, and imaging are accelerating our understanding of this activity and its therapeutic potential. EDITGENE's comprehensive services empower researchers to generate precise cell models and uncover new insights into GO:0043813-related biology.
References
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- 3. Qiu S et al.. 2018. Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry.. Virology 513:17-28 PMID: 29031163
- 4. Lafontaine M et al.. 2021. Clinical features of homozygous FIG4-p.Ile41Thr Charcot-Marie-Tooth 4J patients.. Ann Clin Transl Neurol 8(2):471-476 PMID: 33405357
- 5. Bissig C et al.. 2017. PIKfyve activity regulates reformation of terminal storage lysosomes from endolysosomes.. Traffic 18(11):747-757 PMID: 28857423
- 6. Lees JA et al.. 2020. Insights into Lysosomal PI(3,5)P(2) Homeostasis from a Structural-Biochemical Analysis of the PIKfyve Lipid Kinase Complex.. Mol Cell 80(4):736-743.e4 PMID: 33098764
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- 8. Sait H et al.. 2023. T2 olivary nuclei hyperintensities: A characteristic neuroimaging finding in FIG4-related leukoencephalopathy.. Am J Med Genet A 191(3):864-869 PMID: 36529678
