GO:0080025 phosphatidylinositol-3,5-bisphosphate binding: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0080025 describes the molecular function of selectively binding phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), a low-abundance phosphoinositide phosphorylated at the 3' and 5' positions of the inositol ring.
• PtdIns(3,5)P2 is enriched on endosomal and lysosomal membranes, where it recruits effector proteins that control membrane trafficking, ion transport and organelle homeostasis.
• Direct PtdIns(3,5)P2-binding proteins include the two-pore channel TPC2, the neuronal protein NSG1/NEEP21 and the innate immune sensor STING.
• The PtdIns(3,5)P2-synthesizing kinase FAB1/PIKfyve and its machinery regulate neurite thickness, lysosomal hydrolysis and plant stress responses.
• Dysregulated PtdIns(3,5)P2 binding is linked to neurodegeneration, lysosomal storage defects, mitochondrial respiratory chain deficiency and immune signalling.
• CRISPR knockout, point-mutation knock-in and overexpression models are key tools for testing whether candidate PtdIns(3,5)P2-binding proteins are causally involved in these pathways.
Description
Phosphatidylinositol-3,5-bisphosphate binding (GO:0080025) is a molecular function that enables a protein to selectively recognize and interact with phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), a phosphoinositide phosphorylated at the 3' and 5' positions of the inositol ring. PtdIns(3,5)P2 is a low-abundance lipid that is concentrated on endosomal and lysosomal membranes, where it acts as a spatial and temporal signal for membrane trafficking and organelle function. Because the lipid is scarce and dynamically regulated, the proteins that bind it must achieve high specificity, and this binding event is often the decisive step that recruits an effector to the correct membrane. For researchers, GO:0080025 is important because it defines the molecular interface between a lipid signal and a cellular response. Proteins annotated with this function include ion channels such as TPC2, neuronal trafficking factors such as NSG1/NEEP21, and immune sensors such as STING. Perturbing these interactions alters lysosomal hydrolysis, neurite morphology and innate immune signalling, which connects the term to neurodegeneration, lysosomal disease and host defence. This article summarizes the QuickGO definition, the biological processes and cellular structures in which PtdIns(3,5)P2 binding operates, the key genes and proteins involved, and the experimental methods, including CRISPR-based models, that are used to study this function.
phosphatidylinositol-3,5-bisphosphate binding At A Glance
| GO ID | GO:0080025 |
|---|---|
| GO term | phosphatidylinositol-3,5-bisphosphate binding |
| Ontology | molecular_function |
| Synonym | PtdIns(3,5)P2 binding |
| Definition | Binding to phosphatidylinositol-3,5-bisphosphate, a derivative of phosphatidylinositol in which the inositol ring is phosphorylated at the 3' and 5' positions |
| Major function | Recruitment of effector proteins to endosomal and lysosomal membranes enriched in PtdIns(3,5)P2 |
| Representative binders | TPC2, NSG1/NEEP21, STING |
| Related lipid kinase | FAB1/PIKfyve, which synthesizes PtdIns(3,5)P2 |
| Cellular context | Endosomes, lysosomes and associated membrane domains |
What Is GO:0080025?
GO:0080025 phosphatidylinositol-3,5-bisphosphate binding is defined as the binding to phosphatidylinositol-3,5-bisphosphate, a derivative of phosphatidylinositol in which the inositol ring is phosphorylated at the 3' and 5' positions. In practical terms, it is the molecular function of a protein domain or surface that non-covalently and selectively interacts with PtdIns(3,5)P2, often within endosomal or lysosomal membrane microdomains. The synonym PtdIns(3,5)P2 binding is used interchangeably with the official term.
Why Is phosphatidylinositol-3,5-bisphosphate binding Important in Cell Biology?
GO:0080025 is important because it converts a rare lipid signal into specific cellular outputs. PtdIns(3,5)P2 is generated on endosomal and lysosomal membranes, and proteins that bind it are recruited to these compartments to control ion flux, membrane fusion, cargo sorting and immune sensing. Disrupting this binding function alters lysosomal hydrolysis and neurite morphology, and has been associated with neurodegenerative and lysosomal disorders. Thus, the term provides a mechanistic entry point for understanding how lipid identity is decoded by the proteome.
• Defines a specific lipid-protein interaction that targets effectors to endosomes and lysosomes.
• Controls ion transport through TPC2, a channel activated by PtdIns(3,5)P2 binding.
• Regulates neurite thickness through the neuron-specific endosomal protein NSG1/NEEP21.
• Participates in innate immune signalling as an endogenous ligand of STING.
• Links mitochondrial respiratory chain function to lysosomal hydrolysis.
• Is conserved in plants, where FAB1 and PtdIns(3,5)P2 have diverse physiological functions.
• Provides a mechanistic basis for Charcot-Marie-Tooth hereditary neuropathies and related neurodegeneration.
• Offers a target for CRISPR-based dissection of lipid-binding domains and disease variants.
Molecular Mechanism of phosphatidylinositol-3,5-bisphosphate binding
Lipid synthesis and membrane enrichment
In simple terms: The cell first makes PtdIns(3,5)P2 and concentrates it on specific membranes.
PtdIns(3,5)P2 is synthesized by phosphorylation of phosphatidylinositol 3-phosphate at the 5' position, a reaction catalyzed by the FAB1/PIKfyve kinase. This produces a lipid that is enriched on endosomal and lysosomal membranes, where it forms PtdIns(3,5)P2-rich membrane domains. The local concentration of the lipid determines which proteins can bind it, making synthesis and turnover the first layer of regulation.
Recognition by lipid-binding domains
In simple terms: Proteins use specialized surfaces to grab the lipid headgroup.
Proteins annotated with GO:0080025 bind the PtdIns(3,5)P2 headgroup through positively charged or structurally complementary surfaces. This interaction is non-covalent and selective, allowing the protein to distinguish PtdIns(3,5)P2 from other phosphoinositides. For TPC2, lipid binding induces conformational changes that lead to channel activation.
Recruitment to endosomal and lysosomal domains
In simple terms: Binding acts like a zip code that sends proteins to the right organelle.
Once bound, effector proteins are concentrated in PtdIns(3,5)P2-rich domains on endosomes and lysosomes. This recruitment is essential for processes such as membrane trafficking, cargo sorting and organelle homeostasis. NSG1/NEEP21, for example, depends on PtdIns(3,5)P2 machinery to regulate neurite thickness.
Downstream signalling and ion transport
In simple terms: After binding, the protein changes its activity and sends a signal.
PtdIns(3,5)P2 binding can directly activate ion channels such as TPC2, altering endolysosomal ion flux. It can also serve as an endogenous ligand for STING, linking lipid metabolism to innate immune signalling. In mitochondria-deficient cells, impaired lysosomal hydrolysis is connected to PtdIns(3,5)P2-dependent pathways.
Turnover and signal termination
In simple terms: The signal is switched off when the lipid is removed.
The PtdIns(3,5)P2 signal is terminated by lipid phosphatases that dephosphorylate the inositol ring, releasing bound proteins from the membrane. This turnover is necessary to reset the system and prevent constitutive activation of effectors. In plants, FAB1 and PtdIns(3,5)P2 levels are similarly dynamic and control diverse physiological responses.
Key Genes Involved in GO:0080025 phosphatidylinositol-3,5-bisphosphate binding
The following genes and proteins are representative binders, regulators or downstream effectors of phosphatidylinositol-3,5-bisphosphate binding (GO:0080025).
| Gene | Major Role | Research Relevance |
|---|---|---|
| TPC2 | PtdIns(3,5)P2-activated two-pore channel | Direct lipid-binding activation studied by electrophysiology and mutagenesis |
| NSG1/NEEP21 | Neuron-specific endosomal protein | Regulates neurite thickness through PtdIns(3,5)P2 machinery |
| STING | Innate immune sensor | PtdIns(3,5)P2 acts as an endogenous ligand in immune signalling |
| PIKfyve | Kinase that synthesizes PtdIns(3,5)P2 | Central regulator of endosomal/lysosomal lipid identity |
| FAB1 | Plant PtdIns(3,5)P2 kinase | Controls diverse physiological functions in plants |
| FIG4 | Phosphatase that turns over PtdIns(3,5)P2 | Regulates lipid levels and endolysosomal function |
| MTM1 | Myotubularin phosphatase | Modulates PtdIns(3,5)P2-dependent trafficking |
| VAC14 | Scaffold for PIKfyve complex | Required for PtdIns(3,5)P2 synthesis |
| TRPML1 | Endolysosomal cation channel | Often studied alongside PtdIns(3,5)P2-dependent transport |
| ATG9 | Trafficking factor | Linked to lysosomal hydrolysis and lipid-dependent pathways |
| LAMP1 | Lysosomal marker | Used to localize PtdIns(3,5)P2-rich domains |
| EEA1 | Early endosome marker | Helps define endosomal compartments enriched in PtdIns(3,5)P2 |
| RAB7 | Late endosome regulator | Coordinates with PtdIns(3,5)P2 in endolysosomal trafficking |
| CLN3 | Lysosomal protein | Associated with neurodegeneration and lipid-dependent pathways |
| MFN2 | Mitochondrial fusion protein | Linked to Charcot-Marie-Tooth neuropathy and organelle crosstalk |
| GDAP1 | Mitochondrial dynamics protein | Associated with hereditary neuropathy |
| MPZ | Myelin protein | Relevant to Charcot-Marie-Tooth neuropathies |
How Is phosphatidylinositol-3,5-bisphosphate binding Regulated?
PtdIns(3,5)P2 binding is regulated primarily by the availability of the lipid itself. The kinase PIKfyve/FAB1 synthesizes PtdIns(3,5)P2, while phosphatases such as FIG4 and MTM1 remove it, creating a dynamic cycle that controls effector recruitment. In cells with mitochondrial respiratory chain deficiency, lysosomal hydrolysis is inhibited, indirectly affecting PtdIns(3,5)P2-dependent pathways. In innate immunity, PtdIns(3,5)P2 serves as an endogenous ligand for STING, linking lipid levels to immune activation. Plant FAB1 activity further shows that this regulation is conserved across kingdoms.
phosphatidylinositol-3,5-bisphosphate binding and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| NSG1/NEEP21 | Neurite thickness and neuronal morphology | Knockout and tagged knock-in in neuronal cell lines |
| STING | Innate immune signalling | Point-mutation knock-in to test lipid binding |
| TPC2 | Endolysosomal ion transport | Overexpression and patch-clamp in HEK293 cells |
| FIG4 | PtdIns(3,5)P2 turnover and lysosomal function | Knockout with lipidomics readout |
| MFN2 | Charcot-Marie-Tooth neuropathy | Patient-derived fibroblasts and CRISPR correction |
Neurodegeneration and hereditary neuropathy
PtdIns(3,5)P2-binding proteins such as NSG1/NEEP21 regulate neurite thickness, and disruption of this machinery alters neuronal morphology. Charcot-Marie-Tooth hereditary neuropathies are a group of inherited disorders affecting peripheral nerves, and several associated genes overlap with endolysosomal and mitochondrial pathways. These connections make GO:0080025 relevant to understanding how lipid signalling contributes to axonal maintenance.
Lysosomal dysfunction and mitochondrial crosstalk
Mitochondrial respiratory chain deficiency inhibits lysosomal hydrolysis, a process that depends on endolysosomal lipid organization. Because PtdIns(3,5)P2-rich domains define lysosomal membrane identity, defects in binding or turnover can impair degradation and recycling. This links GO:0080025 to lysosomal storage-like phenotypes and organelle crosstalk.
Innate immunity and STING signalling
PtdIns(3,5)P2 has been identified as an endogenous ligand of STING, directly connecting this lipid to innate immune signalling. This finding expands the role of GO:0080025 beyond trafficking into host defence and inflammation. It also suggests that lipid metabolism can tune immune responses.
Plant physiology and stress responses
In plants, FAB1 and PtdIns(3,5)P2 have diverse physiological functions, including responses to environmental stress. Although not a human disease, this conservation highlights the fundamental importance of the binding function. It also provides a comparative framework for studying lipid signalling.
From phosphatidylinositol-3,5-bisphosphate binding-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does a candidate protein directly bind PtdIns(3,5)P2? | Recombinant protein-lipid overlay and point-mutation knock-in |
| What happens when the binding domain is lost? | CRISPR knockout of the lipid-binding domain |
| Does a disease variant alter lipid binding? | Point-mutation knock-in of the patient allele |
| Where is the protein localized? | Tagged knock-in with fluorescent reporter |
| Does overexpression change trafficking? | Stable overexpression in endolysosomal reporter lines |
| Is the pathway conserved? | Ortholog knockout in plant or invertebrate models |
How to Study the phosphatidylinositol-3,5-bisphosphate binding Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Protein-lipid overlay | Direct binding to PtdIns(3,5)P2 | Initial annotation of lipid-binding proteins |
| Liposome co-sedimentation | Affinity for lipid vesicles | Quantitative binding studies |
| Live-cell confocal imaging | Subcellular localization | Endosomal/lysosomal domain tracking |
| Patch-clamp electrophysiology | Channel activation by lipid | TPC2 functional studies |
| CRISPR knockout | Loss-of-function phenotype | Testing causal roles in trafficking |
| Point-mutation knock-in | Effect of specific residues | Disease variant modelling |
| Lipidomics | Cellular phosphoinositide levels | Pathway regulation studies |
| Proteomics | Protein recruitment changes | Identifying downstream effectors |
Lipid-protein interaction assays
Protein-lipid overlay assays and liposome co-sedimentation are used to test direct binding of candidate proteins to PtdIns(3,5)P2. These methods define whether a protein qualifies for GO:0080025 annotation. They can be combined with mutagenesis to map the binding surface.
Live-cell imaging of endosomal domains
Fluorescently tagged lipid-binding domains and organelle markers allow visualization of PtdIns(3,5)P2-rich membrane domains in living cells. This approach reveals recruitment dynamics and colocalization with endosomal or lysosomal markers. It is often paired with knockout or knockdown to test dependence on the lipid.
Electrophysiology of lipid-gated channels
Patch-clamp and planar lipid bilayer recordings measure activation of channels such as TPC2 by PtdIns(3,5)P2. These experiments provide direct functional evidence that lipid binding changes channel activity. They are useful for testing point mutations that affect lipid sensitivity.
CRISPR-based perturbation and omics
CRISPR knockout, knock-in and overexpression models are combined with transcriptomics, proteomics or lipidomics to determine downstream consequences of altered PtdIns(3,5)P2 binding. Such studies can link the molecular function to lysosomal hydrolysis, immune signalling or neuronal morphology.
How CRISPR Can Be Used to Study GO:0080025 phosphatidylinositol-3,5-bisphosphate binding
Knockout
CRISPR knockout of genes encoding PtdIns(3,5)P2-binding proteins or their regulators can reveal loss-of-function phenotypes in endosomal trafficking, lysosomal hydrolysis and neurite morphology. For example, knocking out NSG1/NEEP21 affects neurite thickness, linking the lipid machinery to neuronal structure. Knockout of lipid kinases or phosphatases alters the abundance of PtdIns(3,5)P2 and the recruitment of downstream effectors.
Point Mutation
Point-mutation knock-in allows precise testing of residues predicted to contact the PtdIns(3,5)P2 headgroup. Such models can distinguish lipid-binding defects from other functions of the same protein. They are also useful for modelling disease-associated variants in genes such as STING or TPC2.
Knock-in
Tagged knock-in of endogenous loci enables visualization of PtdIns(3,5)P2-binding proteins at their native expression levels. This avoids overexpression artifacts and provides physiological localization data. Knock-in of reporter domains can also be used to monitor lipid domain dynamics.
Overexpression
Overexpression of wild-type or mutant PtdIns(3,5)P2-binding proteins can amplify phenotypes and facilitate biochemical purification. It is commonly used in combination with imaging or electrophysiology to study lipid-gated channels. However, results should be interpreted with attention to potential artifacts of excess protein.
How EDITGENE Supports phosphatidylinositol-3,5-bisphosphate binding Research
Researchers studying phosphatidylinositol-3,5-bisphosphate binding-related genes often need to determine whether a candidate gene is causally involved in endolysosomal trafficking, immune signalling or neuronal morphology. Establishing causality requires precise genetic models that isolate lipid-binding function from other activities of the protein. EDITGENE provides CRISPR-based tools and bioinformatics support to build such models efficiently.
Contact EDITGENE today to design your custom CRISPR model for phosphatidylinositol-3,5-bisphosphate binding research.
Frequently Asked Questions About phosphatidylinositol-3,5-bisphosphate binding
What is phosphatidylinositol-3,5-bisphosphate binding?
It is the molecular function defined by GO:0080025, in which a protein selectively binds phosphatidylinositol-3,5-bisphosphate, a phosphoinositide phosphorylated at the 3' and 5' positions of the inositol ring.
What is the GO ID for phosphatidylinositol-3,5-bisphosphate binding?
The GO ID is GO:0080025, and the synonym is PtdIns(3,5)P2 binding.
What genes are involved in phosphatidylinositol-3,5-bisphosphate binding?
Representative genes include TPC2, NSG1/NEEP21, STING, PIKfyve, FAB1, FIG4, MTM1 and VAC14.
Where is phosphatidylinositol-3,5-bisphosphate found in the cell?
It is enriched on endosomal and lysosomal membranes, where it forms PtdIns(3,5)P2-rich membrane domains.
How does PtdIns(3,5)P2 activate TPC2?
Lipid binding induces conformational changes in TPC2 that lead to channel activation, as shown by functional studies.
Is PtdIns(3,5)P2 involved in immunity?
Yes, PtdIns(3,5)P2 has been identified as an endogenous ligand of STING in innate immune signalling.
What diseases are linked to PtdIns(3,5)P2 binding?
Links include neurodegeneration, Charcot-Marie-Tooth hereditary neuropathies, lysosomal dysfunction and immune signalling defects.
How is PtdIns(3,5)P2 synthesized and turned over?
It is synthesized by the kinase PIKfyve/FAB1 and turned over by phosphatases such as FIG4 and MTM1.
What methods are used to study PtdIns(3,5)P2 binding?
Common methods include protein-lipid overlay, liposome co-sedimentation, live-cell imaging, electrophysiology and CRISPR-based perturbation.
Can CRISPR be used to study PtdIns(3,5)P2 binding?
Yes, CRISPR knockout, point-mutation knock-in, tagged knock-in and overexpression models are widely used to test the function of PtdIns(3,5)P2-binding proteins.
Conclusion
GO:0080025 phosphatidylinositol-3,5-bisphosphate binding defines a precise lipid-protein interaction that recruits effectors to endosomal and lysosomal membranes. Through proteins such as TPC2, NSG1/NEEP21 and STING, this function connects lipid metabolism to ion transport, neuronal morphology and innate immunity. Its dysregulation has been linked to neurodegeneration, lysosomal dysfunction and immune signalling defects, making it a compelling area for mechanistic and translational research. CRISPR-based models, including knockout, point-mutation knock-in, tagged knock-in and overexpression, provide the tools needed to dissect this function in physiologically relevant systems. Combined with lipidomics, imaging and electrophysiology, these approaches can clarify how PtdIns(3,5)P2 binding shapes cellular behaviour and disease.
References
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