ATG18E Antibody

What is ATG18E and what role does it play in autophagy?

ATG18E belongs to the PROPPIN family of proteins that function in autophagy regulation. Like other ATG18 proteins, it contains a WD40 repeat domain and binds specifically to phosphoinositides, particularly phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) . This phosphoinositide binding is critical for ATG18's functional roles in both selective and non-selective autophagy pathways. ATG18E forms a complex with ATG2 that is essential for autophagosome formation, and this complex is recruited to autophagic membranes through interaction with phospholipids . The protein participates in both macroautophagy (bulk degradation) and selective autophagy processes such as the Cvt (cytoplasm to vacuole targeting) pathway .

How should I select an appropriate ATG18E antibody for my research?

When selecting an ATG18E antibody, consider these key factors:

• Experimental application: Different applications (western blotting, immunoprecipitation, immunofluorescence) may require antibodies with different characteristics. For western blotting, prioritize antibodies validated for denatured proteins; for immunoprecipitation, choose antibodies that recognize native epitopes.

• Epitope targeting: Consider whether you need to detect specific regions or modifications of ATG18E. If studying phosphoinositide binding, select antibodies that don't interfere with the FRRG motif (critical for this function) .

• Species cross-reactivity: Ensure the antibody recognizes ATG18E in your model organism. ATG18 studies have been conducted in various organisms including yeast (S. cerevisiae) and mammalian systems .

• Phosphorylation status detection: If studying phosphoregulation of ATG18E, select antibodies that either specifically detect phosphorylated forms or are not affected by phosphorylation state .

• Validation data: Review manufacturer's validation data specifically for your intended application, including positive and negative controls.

What are the typical characteristics of commercially available ATG18E antibodies?

Commercial ATG18E antibodies typically share these characteristics:

The selection should be guided by the specific research question and experimental system in use .

How can I optimize ATG18E detection in western blotting experiments?

For optimal western blot detection of ATG18E:

• Sample preparation: Lyse cells in buffer containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease inhibitors. Include phosphatase inhibitors if studying phosphorylated forms .

• Gel parameters: Use 10-12% polyacrylamide gels for optimal separation.

• Membrane selection: PVDF membranes typically provide better results than nitrocellulose for ATG18E detection.

• Blocking conditions: Block with 5% non-fat dry milk in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature.

• Antibody incubation: Incubate with anti-ATG18E primary antibody (typically 1:1000-1:5000 dilution) overnight at 4°C, followed by appropriate HRP-conjugated secondary antibody.

• Detection system: Use enhanced chemiluminescence (ECL) systems for visualization, as demonstrated in studies with anti-Atg18 antibodies .

• Controls: Include appropriate positive controls (tissues/cells known to express ATG18E) and negative controls (ATG18E knockdown/knockout samples).

• Phosphorylation studies: For phosphorylation analysis, run parallel samples treated with phosphatase to distinguish between phosphorylated and non-phosphorylated forms .

What is the optimal protocol for immunoprecipitating ATG18E and its binding partners?

For effective ATG18E immunoprecipitation:

• Cell preparation: Treat cells with autophagy inducers (e.g., rapamycin) for 10 minutes before harvesting to enhance complex formation .

• Lysis conditions: Use a gentle lysis buffer (e.g., 50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100) with protease and phosphatase inhibitors. Maintain cells in buffers containing autophagy inducers during spheroplast generation if working with yeast models .

• Pre-clearing: Pre-clear lysates with protein A/G beads for 1 hour at 4°C.

• Antibody incubation: Incubate cleared lysates with anti-ATG18E antibody (typically 2-5 μg) overnight at 4°C with gentle rotation.

• Complex capture: Add protein A/G beads and incubate for 2-4 hours at 4°C.

• Washing: Wash beads 4-5 times with lysis buffer, maintaining consistent conditions with the immunoprecipitation process .

• Elution: Elute proteins by boiling in SDS sample buffer for western blot analysis.

• Controls: Include antibody-only and lysate-only controls to confirm specificity, as demonstrated in studies showing that ATG18 co-immunoprecipitates with ATG2 in an antibody-dependent and ATG2-dependent manner .

How can I effectively visualize ATG18E localization using immunofluorescence microscopy?

For optimal immunofluorescence visualization of ATG18E:

• Cell preparation: Culture cells on coverslips and induce autophagy with appropriate stimuli (e.g., rapamycin, starvation).

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature. Avoid methanol fixation as it may disrupt membrane structures critical for ATG18E localization.

• Permeabilization: Permeabilize with 0.1% Triton X-100 for 5 minutes.

• Blocking: Block with 5% normal serum (matching secondary antibody species) for 30 minutes.

• Primary antibody: Incubate with anti-ATG18E antibody (1:100-1:500) overnight at 4°C.

• Secondary antibody: Use appropriate fluorophore-conjugated secondary antibody (1:500-1:1000) for 1 hour at room temperature.

• Co-staining markers: Co-stain with markers for autophagic structures (LC3), PAS (pre-autophagosomal structure), or phosphoinositide markers.

• Imaging: Use a high-resolution fluorescence microscope with appropriate filter sets. For dynamics studies, consider time-lapse imaging with ATG18E-GFP constructs .

• Controls: Include cells with known ATG18E expression patterns and negative controls (primary antibody omission, non-specific IgG, or ATG18E-depleted cells).

Research shows that wild-type ATG18-GFP localizes to punctate structures under autophagy-inducing conditions, while the ATG18(FTTG) mutant shows diffuse localization due to impaired phosphoinositide binding .

How can I investigate the phosphoinositide binding properties of ATG18E using its antibody?

The phosphoinositide binding of ATG18E can be investigated through these methodologies:

• Lipid binding assays with purified protein:

  • PIP strip assay: Immobilize phosphoinositides on membranes and probe with purified ATG18E, detecting bound protein with anti-ATG18E antibody .

  • Liposome pull-down: Create liposomes containing specific phosphoinositides, incubate with ATG18E, and detect bound protein using anti-ATG18E antibody .

  • Surface plasmon resonance: Measure binding kinetics between ATG18E and immobilized phosphoinositides .

• Mutation analysis of binding sites:

  • Generate ATG18E variants with mutations in the FRRG motif (e.g., FTTG mutant) which disrupt phosphoinositide binding .

  • Compare binding properties of wild-type and mutant proteins using the above assays.

  • Assess the impact on autophagy using functional assays like the alkaline phosphatase (ALP) assay .

• Phosphorylation effects:

  • Treat purified ATG18E with phosphatase and compare phosphoinositide binding between phosphorylated and dephosphorylated forms .

  • Research shows phosphatase treatment dramatically increases binding activity toward PI(3,5)P2 compared with PI(3)P .

• Rescue experiments:

  • Create chimeric proteins (e.g., ATG18(FTTG)-2×FYVE) to restore phosphoinositide binding through alternative domains and assess functional rescue .

  • This approach has demonstrated that targeting ATG18(FTTG) to PtdIns(3)P-enriched sites restores both autophagy and Cvt pathway functions .

What approaches can distinguish between ATG18E's roles in selective versus non-selective autophagy?

To differentiate ATG18E's roles in different autophagy pathways:

• Pathway-specific mutants:

  • Utilize ATG18(FTTG) phosphoinositide-binding mutant, which shows differential effects: severely reduced non-selective autophagy and completely blocked Cvt pathway (selective autophagy) .

  • Compare the effects of these mutations using quantitative assays for each pathway.

• Quantitative autophagy assays:

  • For non-selective autophagy: Use the ALP assay to measure bulk autophagy flux quantitatively .

  • For selective autophagy (Cvt pathway): Monitor processing of prApe1 to mature Ape1 via immunoblotting .

  • Microscopy-based analysis: Observe autophagic body accumulation in the vacuole using phase-contrast microscopy under starvation conditions .

• Comparative analysis:

  • Induce autophagy using different conditions:

    • Starvation media lacking nitrogen and carbon sources (S (-NC) medium) for non-selective autophagy

    • Rapamycin treatment for general autophagy induction

    • Nutrient-rich conditions for Cvt pathway analysis

Data from experimental studies show that ATG18(FTTG) cells have severely reduced autophagic body accumulation under starvation conditions, with approximately 80% reduction in alkaline phosphatase activity compared to wild-type cells, while the Cvt pathway is completely blocked .

How does phosphorylation regulate ATG18E function and how can this be studied?

Phosphorylation plays a critical role in regulating ATG18E function through several mechanisms:

• Effect on phosphoinositide binding:

  • Phosphorylation significantly decreases ATG18's binding affinity for phosphoinositides, particularly PI(3,5)P2 .

  • Dephosphorylation dramatically increases binding activity toward PI(3,5)P2 compared to PI(3)P .

• Experimental approaches to study phosphoregulation:

  • Phosphatase treatment: Treat purified ATG18E with phosphatase and compare lipid binding properties .

  • Phosphomimetic mutations: Create phosphomimetic (S→D/E) and phosphodeficient (S→A) mutants of ATG18E at key phosphorylation sites.

  • Mass spectrometry: Identify specific phosphorylation sites under different cellular conditions.

  • Phospho-specific antibodies: Generate antibodies that specifically recognize phosphorylated forms of ATG18E.

• Functional consequences:

  • Membrane association: Monitor how phosphorylation affects ATG18E recruitment to membranes.

  • Organelle dynamics: Investigate how phosphorylation regulates ATG18E's role in controlling organellar dynamics .

  • Protein-protein interactions: Determine whether phosphorylation affects ATG18E's interaction with binding partners like ATG2.

Research indicates that phosphoregulation of ATG18 provides a mechanism to control organellar dynamics by modulating protein-phosphoinositide interactions, affecting both autophagy and vacuolar morphology regulation .

What methods can assess the interaction between ATG18E and ATG2?

The ATG18E-ATG2 complex can be studied using these approaches:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate with anti-ATG2 antibody and detect ATG18E in the precipitate, or vice versa .

  • Compare wild-type ATG18E with mutants like ATG18(FTTG) to determine domains essential for interaction.

  • Research shows that ATG18(FTTG) co-immunoprecipitates with ATG2 similarly to wild-type ATG18, indicating that phosphoinositide binding is not required for complex formation .

• Gel filtration analysis:

  • Subject cell lysates to gel filtration chromatography to separate protein complexes based on size.

  • Analyze fractions by immunoblotting with anti-ATG18E and anti-ATG2 antibodies.

  • Research has shown that approximately 20-30% of ATG18-HA-GFP elutes in fractions corresponding to ~500 kDa, likely representing the ATG18-ATG2 complex .

• PtdIns(3)P-independent complex formation:

  • Use vps34Δ cells expressing PtdIns 3-kinase-deficient enzyme (Vps34 N736K) to create a system lacking detectable PtdIns(3)P.

  • Demonstrate that the ATG18-ATG2 complex still forms in these conditions, confirming that complex formation is independent of PtdIns(3)P binding .

• Fluorescence microscopy:

  • Use fluorescently tagged proteins (e.g., ATG18-GFP and ATG2-RFP) to visualize co-localization.

  • Perform Fluorescence Resonance Energy Transfer (FRET) analysis to confirm direct interaction.

  • Analyze recruitment dynamics to autophagic structures under different conditions.

These methodologies provide complementary approaches to characterize the ATG18E-ATG2 complex and its functional significance in autophagy regulation.

What are common issues when using ATG18E antibodies and how can they be resolved?

When working with ATG18E antibodies, researchers may encounter these common issues:

• Low signal intensity:

  • Increase antibody concentration or incubation time

  • Use more sensitive detection methods (e.g., higher sensitivity ECL substrates)

  • Enrich target protein through immunoprecipitation before western blotting

  • Optimize protein extraction methods to improve yield

• High background:

  • Increase blocking time or concentration (5-10% blocking agent)

  • Dilute primary antibody further

  • Increase washing steps (number and duration)

  • Use alternative blocking agents (switch between milk and BSA)

  • Pre-adsorb antibody with cell/tissue lysate from negative control samples

• Multiple bands in western blot:

  • Verify with positive and negative controls

  • Use freshly prepared samples with protease inhibitors

  • Test antibody specificity using peptide competition assays

  • Consider that additional bands may represent isoforms, degradation products, or post-translationally modified variants

• Inconsistent results between applications:

  • Verify antibody is validated for each specific application

  • Adjust fixation/permeabilization conditions for immunofluorescence

  • Optimize lysis conditions to preserve native protein structure for immunoprecipitation

  • Consider epitope accessibility differences between applications

• Poor reproducibility:

  • Standardize lysate preparation procedures

  • Use consistent antibody lots when possible

  • Maintain consistent incubation times and temperatures

  • Document detailed protocols including all reagents and conditions

How can I differentiate between ATG18E's roles in autophagy versus vacuolar morphology regulation?

To distinguish between ATG18E's dual functions in autophagy and vacuolar morphology:

• Phosphoinositide binding specificity:

  • PtdIns(3)P binding primarily mediates autophagy functions

  • PI(3,5)P2 binding is essential for vacuolar morphology regulation

  • Use mutations that selectively disrupt binding to specific phosphoinositides

• Functional assays to separate roles:

  • Autophagy measurement: Monitor autophagosome formation, autophagic flux, and selective cargo degradation

  • Vacuolar morphology assessment: Observe vacuole size, number, and structure using microscopy with vacuolar membrane markers

• Domain-specific constructs:

  • Create chimeric proteins with specific domains to rescue individual functions

  • The ATG18(FTTG)-2×FYVE chimera can distinguish functions by restoring PtdIns(3)P binding through an alternative domain

• Comparative analysis:

  • Analyze autophagy and vacuolar morphology phenotypes across a panel of ATG18E mutants

  • Create a correlation matrix between specific mutations and functional outcomes

• Genetic complementation:

  • Express distinct ATG18E domains or mutants in ATG18-deficient cells

  • Determine which constructs rescue autophagy versus vacuolar morphology defects

Research has shown that the phosphoinositide-binding FRRG motif of ATG18 is essential for both the Cvt pathway and efficient non-selective autophagy, while having distinct effects on vacuolar morphology regulation through different phosphoinositide interactions .

What considerations are important when using ATG18E antibodies for energy-based optimization in antibody design approaches?

When applying ATG18E antibodies in energy-based optimization for antibody design:

• Structural optimization considerations:

  • Utilize residue-level decomposed energy preference approaches to optimize antibody-antigen binding

  • Consider both total energy (Etotal) and binding energy (ΔG) parameters when designing ATG18E-targeting antibodies

  • Employ gradient surgery techniques to address conflicts between various types of energy (attraction vs. repulsion)

• Technical parameters for optimization:

  • Integrate multiple energy metrics:

    • CDR Etotal: Total energy of the designed CDR

    • CDR-Ag ΔG: Difference in total energy between bound and unbound states

    • Repulsive (ERep) and non-repulsive (EnonRep) energy components

• Energy evaluation metrics:

  • Target antibody designs with energies resembling natural antibodies

  • Balance multiple objectives (low total energy and high binding affinity)

  • Rank designed antibodies using uniform strategies to identify optimal candidates

• Validation approaches:

  • Compare generated antibodies against natural antibodies using energy-based metrics

  • Assess ability to generate antibodies with fewer structural clashes

  • Evaluate proper relative spatial positioning toward antigens

Research utilizing direct energy-based preference optimization approaches has demonstrated effectiveness in generating antibodies with energies resembling natural antibodies while maintaining high binding affinity to targets .