Recombinant Human V-type proton ATPase subunit e 1 (ATP6V0E1)

What is the basic structure and function of ATP6V0E1 within the V-ATPase complex?

ATP6V0E1 is a component of the vacuolar ATPase (V-ATPase), a multisubunit enzyme that mediates acidification of eukaryotic intracellular organelles. V-ATPase is composed of two domains:

• V1 domain (cytosolic): Contains eight subunits (A-H) that catalyze ATP hydrolysis

• V0 domain (transmembrane): Contains five different subunits (a, c, c', c", and d) involved in proton translocation

ATP6V0E1 encodes the e1 subunit of the V0 domain. The protein is relatively small at approximately 9.4 kDa with 81 amino acid residues in the canonical form in humans .

V-ATPase-dependent organelle acidification is necessary for crucial cellular processes including protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation . The e subunit has been shown to be essential for normal V-ATPase function, with studies demonstrating that knockdown of the e subunit reduces V-ATPase efficiency .

How does ATP6V0E1 differ from other V-ATPase subunits, and what techniques are most reliable for studying its specific functions?

ATP6V0E1 differs from other V-ATPase subunits in several key ways:

• Size and location: It is one of the smaller subunits within the V0 domain (9.4 kDa)

• Isoform variation: ATP6V0E1 has a paralog called ATP6V0E2, with distinct expression patterns

• Functional specificity: While many V-ATPase subunits have tissue-specific expression, ATP6V0E1 is more ubiquitously expressed across many tissue types

For studying ATP6V0E1 specifically:

Recommended techniques:

• RNA interference (siRNA): Effective for knockdown studies to assess functional impact, as demonstrated in neuroblastoma research

• Immunofluorescence microscopy: For subcellular localization studies

• Co-immunoprecipitation: To identify interaction partners

• CRISPR-Cas9 gene editing: For generating knockout cell lines

Methodological considerations:

• When performing knockdown experiments, include ATP6V0E2 controls to account for potential compensatory mechanisms

• Use multiple antibodies targeting different epitopes for validation due to the small size of the protein

• Consider V-ATPase activity assays (measuring proton transport or ATP hydrolysis) to assess functional impact of ATP6V0E1 manipulation

How can researchers accurately determine ATP6V0E1 expression patterns across different tissues and cell types?

To accurately determine ATP6V0E1 expression patterns:

Transcriptional analysis approaches:

• RT-qPCR: Design primers specific to ATP6V0E1 that do not amplify ATP6V0E2 or pseudogenes

• RNA-Seq: For comprehensive transcriptome analysis and splice variant identification

• In situ hybridization: For spatial expression analysis in tissue sections

Protein-level approaches:

• Western blotting: Using validated ATP6V0E1-specific antibodies

• Mass spectrometry: For unbiased proteomic profiling

• Immunohistochemistry: For spatial localization in tissues

Important methodological considerations:

• Validate antibody specificity against recombinant ATP6V0E1 protein

• Include ATP6V0E2 controls to ensure isoform specificity

• Consider post-translational modifications like glycosylation that may affect detection

• Use subcellular fractionation to confirm organelle-specific localization

Studies have shown that ATP6V0E1 is ubiquitously expressed across many tissue types but may show variable expression levels. Proper controls and validation steps are essential for accurate expression profiling.

What approaches can be used to study the specific subcellular localization and trafficking of ATP6V0E1?

Recommended approaches for subcellular localization studies:

• Fluorescently-tagged fusion proteins:

  • GFP-ATP6V0E1 fusion constructs for live-cell imaging

  • Consider both N-terminal and C-terminal tags to determine which preserves function

  • Validate with co-localization studies using organelle markers

• Immunofluorescence microscopy:

  • Use validated ATP6V0E1 antibodies

  • Co-stain with organelle markers (LAMP1 for lysosomes, EEA1 for early endosomes)

  • Super-resolution microscopy for precise localization

• Biochemical fractionation:

  • Differential centrifugation to isolate organelle fractions

  • Density gradient separation of organelles

  • Western blotting of fractions to detect ATP6V0E1

• Proximity labeling methods:

  • BioID or TurboID fusion proteins to identify proximal proteins

  • APEX2 for electron microscopy visualization

Important considerations:

• The small size of ATP6V0E1 (9.4 kDa) may make certain fusion proteins challenging

• Trafficking studies should include inhibitors of specific trafficking pathways

• Consider temporal dynamics using live-cell imaging

• Verify functional incorporation into the V-ATPase complex

Research on other V-ATPase subunits has shown remarkable organelle specificity. For example, studies in Paramecium identified 17 genes encoding a-subunit isoforms, with representatives showing highly specific targeting to at least seven different compartments . This suggests ATP6V0E1 may also have specific targeting mechanisms worth investigating.

How does ATP6V0E1 contribute to lysosomal acidification and autophagy, and what methods can quantify these effects?

ATP6V0E1, as part of the V0 domain, is critical for V-ATPase function in lysosomal acidification, which directly impacts autophagy:

Role in lysosomal acidification:

• ATP6V0E1 is essential for normal V-ATPase function and proton translocation

• Knockdown studies suggest reduced ATP6V0E1 decreases V-ATPase efficiency and impacts lysosomal pH

Impact on autophagy:

• V-ATPase activity is required for autophagosome-lysosome fusion and degradation of autophagic cargo

• Research has shown that targeting V-ATPase subunits can activate autophagy through mTORC1 inhibition

Methods to quantify lysosomal acidification:

• pH-sensitive fluorescent probes:

  • LysoSensor probes that exhibit pH-dependent changes in fluorescence intensity

  • LysoTracker dyes that accumulate in acidic compartments

  • Ratiometric measurements using pH-sensitive fluorophores like pHrodo

• Direct measurement of lysosomal pH:

  • FITC-dextran pulse-chase followed by ratio imaging

  • Organelle-targeted pH-sensitive GFP variants

Methods to assess autophagy:

• LC3 processing and localization:

  • Western blotting for LC3-I to LC3-II conversion

  • GFP-LC3 puncta formation by microscopy

  • Tandem mRFP-GFP-LC3 to distinguish autophagosomes from autolysosomes

• Autophagy flux assays:

  • Treatment with bafilomycin A1 (V-ATPase inhibitor) to block autophagy completion

  • Monitoring degradation of autophagy substrates (p62/SQSTM1)

• Transmission electron microscopy:

  • Visualization of autophagic structures

  • Quantification of autophagic vacuoles

Research has shown that covalent targeting of the ATP6V1A subunit of V-ATPase activates autophagy via mTORC1 inhibition and increases lysosomal acidification . Similar approaches could be applied to study ATP6V0E1's specific role.

What is the relationship between ATP6V0E1 and other cellular processes like endocytosis and exocytosis?

ATP6V0E1, as part of the V-ATPase complex, plays important roles in various membrane trafficking processes:

Role in endocytosis:

• V-ATPase acidifies early endosomes, which is crucial for receptor-ligand dissociation

• Proper sorting in the endocytic pathway depends on pH gradients established by V-ATPase

• ATP6V0E1 knockdown may impair endosomal trafficking due to altered acidification

Role in exocytosis:

• V-ATPase acidifies secretory vesicles, necessary for proper protein sorting and processing

• In neurons, V-ATPase generates the proton gradient that drives neurotransmitter loading into synaptic vesicles

Experimental approaches to study these relationships:

• Receptor-mediated endocytosis assays:

  • Fluorescently-labeled transferrin uptake and recycling

  • EGFR internalization and degradation kinetics

  • Quantitative analysis of endocytic rate using flow cytometry

• Exocytosis measurement techniques:

  • Total internal reflection fluorescence (TIRF) microscopy of labeled secretory vesicles

  • Capacitance measurements (for electrophysiological detection of exocytosis)

  • Secreted protein quantification (ELISA or Western blotting)

• Live cell imaging approaches:

  • pH-sensitive cargo to track acidification during trafficking

  • Dual-color imaging of ATP6V0E1 and endocytic/exocytic markers

  • Super-resolution microscopy to visualize V-ATPase distribution on vesicles

Research has shown that ATP6V0E1 knockdown in neuroblastoma cells reduced cell viability but was not sufficient to induce neural cell differentiation, suggesting complex roles beyond simple pH regulation . Additionally, some V-ATPase subunits have been implicated in membrane fusion events independent of their role in acidification, which may be worth investigating for ATP6V0E1.

How are mutations or altered expression of ATP6V0E1 associated with human diseases, and what experimental models best recapitulate these conditions?

While ATP6V0E1-specific mutations have not been extensively characterized, research on V-ATPase subunits has revealed important disease associations:

Disease associations of V-ATPase subunits:

ATP6V0E1-specific research findings:

• In neuroblastoma, ATP6V0E1 is directly targeted by microRNA-506-3p, which functions as a tumor suppressor

• Knockdown of ATP6V0E1 reduced neuroblastoma cell proliferation and viability

• ATP6V0E1 appears essential for cancer cell survival, suggesting its potential relevance in cancer biology

Recommended experimental models:

• Cellular models:

  • Patient-derived fibroblasts or iPSCs

  • CRISPR-engineered cell lines with ATP6V0E1 mutations

  • Conditional knockdown systems (inducible shRNA)

• Animal models:

  • Conditional knockout mice (since complete knockout may be lethal)

  • Zebrafish models for high-throughput phenotypic analysis

  • Drosophila models for genetic interaction studies

• Organoid models:

  • Brain organoids for neurological disease modeling

  • Tumor organoids for cancer studies

Important considerations:

• Due to the essential nature of V-ATPase function, complete loss of ATP6V0E1 may be lethal, necessitating conditional approaches

• Compensatory mechanisms involving ATP6V0E2 should be considered

• Tissue-specific phenotypes should be evaluated when designing disease models

Research on other V-ATPase subunits has demonstrated that even subtle functional changes can lead to tissue-specific disease manifestations, suggesting that careful phenotypic analysis across multiple systems is necessary.

What are the current approaches for targeting ATP6V0E1 in therapeutic development, and how can research methods be optimized for drug screening?

While ATP6V0E1-specific therapeutics are not yet well-established, several approaches for targeting V-ATPase components show promise:

Current therapeutic approaches for V-ATPase targets:

• Small molecule inhibitors:

  • Bafilomycin A1 and concanamycin A (general V-ATPase inhibitors)

  • Archazolid (V-ATPase inhibitor with anti-cancer properties)

  • EN6 (covalently targets ATP6V1A subunit, activates autophagy)

• RNA-based therapeutics:

  • siRNA/shRNA for knockdown

  • Antisense oligonucleotides

  • microRNA mimics or inhibitors (e.g., miR-506-3p that targets ATP6V0E1)

• Structure-based drug design:

  • Targeting specific interfaces between V-ATPase subunits

  • Allosteric modulators of V-ATPase assembly/disassembly

Optimized methods for ATP6V0E1-focused drug screening:

• High-throughput screening approaches:

  • Cell-based assays measuring lysosomal pH (LysoSensor-based fluorescence)

  • ATP hydrolysis assays using purified V-ATPase complexes

  • Thermal shift assays to identify compounds binding directly to ATP6V0E1

• Target validation methods:

  • CETSA (Cellular Thermal Shift Assay) to confirm target engagement

  • Chemoproteomics approaches like activity-based protein profiling (ABPP)

  • Mutational analysis of binding sites

• Functional readouts for screening:

  • Autophagy modulation (LC3-II/LC3-I ratio)

  • Endolysosomal acidification (pH-sensitive fluorescent probes)

  • Cell type-specific functional assays (e.g., neuroblastoma cell viability)

Emerging directions:

• Research on colorectal cancer has shown that ATP6V0A1-dependent cholesterol absorption triggers immunosuppressive signaling, suggesting similar mechanisms might exist for ATP6V0E1

• Targeting specific V-ATPase subunit isoforms may allow more precise modulation of compartment-specific functions

• Developing compounds that modify V-ATPase assembly/disassembly rather than blocking activity completely may offer therapeutic advantages

Research has demonstrated that the V-ATPase regulates mTORC1 via the Rag GTPases, and that covalent targeting of ATP6V1A can decouple the V-ATPase from the Rags, leading to mTORC1 inhibition and autophagy activation . Similar target-specific approaches could be developed for ATP6V0E1.

How does ATP6V0E1 interact with other V-ATPase subunits to regulate assembly, disassembly, and activity of the complete complex?

The regulation of V-ATPase through assembly/disassembly is a sophisticated process that ATP6V0E1 likely contributes to:

V-ATPase assembly/disassembly regulation:

• V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and V0 proton channel subcomplexes

• The TLDc protein Oxr1p has been shown to induce V-ATPase disassembly in vitro and is essential for efficient disassembly in cells

• ATP hydrolysis is needed for release of Oxr1p so that free V1 can adopt the autoinhibited conformation

Methods to study ATP6V0E1's role in V-ATPase assembly:

• Biochemical approaches:

  • Co-immunoprecipitation with other V-ATPase subunits

  • Blue native PAGE to analyze intact complexes

  • Chemical crosslinking followed by mass spectrometry

  • In vitro reconstitution assays with purified components

• Structural biology methods:

  • Cryo-electron microscopy of assembled complexes

  • X-ray crystallography of subdomains

  • Hydrogen-deuterium exchange mass spectrometry to map interaction sites

• Dynamic interaction studies:

  • FRET/BRET pairs between ATP6V0E1 and other subunits

  • Split-GFP complementation assays

  • Single-molecule tracking in live cells

Important considerations:

• The assembly state of V-ATPase changes in response to nutrient conditions

• Different cellular compartments may have different assembly regulation mechanisms

• ATP6V0E1 may interact with chaperones or assembly factors before incorporation into the complete complex

Research on yeast V-ATPase has shown that the protein Oxr1p is essential for efficient V-ATPase disassembly in vivo, with its absence resulting in ~40% more V1 on vacuoles compared to wild-type . This suggests that regulating assembly/disassembly is a critical control point for V-ATPase function, where ATP6V0E1 likely plays an important role.

What are the current challenges and emerging techniques in studying ATP6V0E1's role in different cellular compartments and specialized cell types?

Current challenges in ATP6V0E1 research:

• Technical limitations:

  • Small size of ATP6V0E1 (9.4 kDa) making it difficult to detect

  • Potential functional redundancy with ATP6V0E2

  • Limited availability of highly specific antibodies

  • Challenges in reconstituting functional V-ATPase complexes in vitro

• Biological complexity:

  • Compartment-specific roles of V-ATPase complexes

  • Cell type-specific functions and regulation

  • Integration with other pH regulatory mechanisms

  • Distinguishing between direct effects of ATP6V0E1 versus indirect effects of altered pH

Emerging techniques and solutions:

• Advanced imaging approaches:

  • Expansion microscopy for improved resolution of small proteins

  • Live-cell super-resolution microscopy (PALM/STORM)

  • Cryo-electron tomography of intact cellular compartments

  • Correlative light and electron microscopy (CLEM)

• Genomic and proteomic technologies:

  • Proximity labeling (BioID, APEX) to map compartment-specific interactomes

  • Single-cell transcriptomics to identify cell type-specific expression patterns

  • Targeted proteomics for accurate quantification of ATP6V0E1 and interacting proteins

  • CRISPR screening to identify genetic interactions

• Functional analysis innovations:

  • Optogenetic control of ATP6V0E1 activity or localization

  • Genetically encoded pH sensors targeted to specific compartments

  • Organelle-specific isolation techniques for biochemical analysis

  • Microfluidic approaches for real-time measurement of ion transport

Research frontiers to explore:

• Specialized cell types of particular interest:

  • Neurons (role in synaptic vesicle acidification)

  • Immune cells (antigen processing, phagocytosis)

  • Cancer cells (metabolic adaptation, drug resistance)

  • Renal tubular cells (acid-base homeostasis)

• Emerging biological contexts:

  • Integration with metabolic pathways

  • Roles in signaling beyond pH regulation

  • Connections to membrane contact sites between organelles

  • Contributions to organelle identity and maturation

Research in Paramecium has revealed 17 genes encoding a-subunit isoforms with highly specific targeting to different compartments, demonstrating remarkable specialization within the V-ATPase family . This suggests that ATP6V0E1 may have similarly specialized functions that remain to be fully elucidated using these emerging techniques.

What are the optimal conditions and methods for producing recombinant ATP6V0E1 protein for structural and functional studies?

Challenges in ATP6V0E1 recombinant production:

• Small protein size (9.4 kDa)

• Membrane protein requiring proper folding environment

• Potential need for other V-ATPase subunits for stability

Recommended expression systems:

Optimization strategies:

• Fusion tags and constructs:

  • Solubility-enhancing tags (MBP, SUMO, Trx)

  • Affinity tags (His, GST, FLAG) for purification

  • Fluorescent protein fusions for functional validation

  • Consider both N- and C-terminal tag positions

• Membrane protein considerations:

  • Inclusion of detergents (DDM, CHAPS, Triton X-100)

  • Reconstitution in lipid nanodiscs or liposomes

  • Co-expression with partner subunits from V0 domain

  • Bicelle or amphipol formulations for structural studies

• Purification approach:

  • Two-step affinity purification (e.g., His tag followed by second affinity tag)

  • Size exclusion chromatography for final polishing

  • On-column refolding for proteins expressed in inclusion bodies

  • Blue native PAGE to verify complex assembly

Validation methods:

• Circular dichroism spectroscopy for secondary structure assessment

• Thermal shift assays for stability analysis

• Limited proteolysis to verify proper folding

• Functional reconstitution assays measuring proton transport

How can researchers assess the functional integrity of purified ATP6V0E1 and its incorporation into the V-ATPase complex?

Functional integrity assessment techniques:

• Biochemical activity assays:

  • ATP hydrolysis measurements (colorimetric phosphate release assays)

  • Proton transport assays using pH-sensitive fluorescent dyes in reconstituted vesicles

  • ATPase coupled enzyme assays (e.g., pyruvate kinase/lactate dehydrogenase coupled system)

• Structural integrity verification:

  • Limited proteolysis resistance compared to unfolded protein

  • Antibody recognition of conformational epitopes

  • Thermal stability measurement using differential scanning fluorimetry

  • Native mass spectrometry to assess complex formation

• Interaction assays:

  • Surface plasmon resonance (SPR) to measure binding kinetics with other V-ATPase subunits

  • Microscale thermophoresis (MST) for protein-protein interaction studies

  • Isotherm titration calorimetry (ITC) for thermodynamic binding parameters

  • Pull-down assays with other purified V-ATPase components

V-ATPase complex assembly assessment:

• Biochemical approaches:

  • Blue native PAGE to visualize intact complexes

  • Gradient ultracentrifugation to separate assembled complexes from individual components

  • Chemical crosslinking followed by SDS-PAGE or mass spectrometry

  • Co-immunoprecipitation with antibodies against other V-ATPase subunits

• Structural analysis methods:

  • Negative stain electron microscopy for complex visualization

  • Cryo-electron microscopy for higher resolution structural analysis

  • Small-angle X-ray scattering (SAXS) for solution structure analysis

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Functional reconstitution:

  • Proteoliposome reconstitution with purified components

  • Activity measurements of reconstituted V-ATPase

  • Comparison of activities with and without ATP6V0E1

  • Patch-clamp electrophysiology of reconstituted complexes

Research on V-ATPase regulation has shown that ATP hydrolysis is crucial for the proper disassembly of the complex . Therefore, testing both assembly and disassembly dynamics in reconstituted systems would provide valuable insights into ATP6V0E1's role in V-ATPase function.

What genetic approaches are most effective for studying ATP6V0E1 function, and how can phenotypic effects be comprehensively assessed?

Genetic modification approaches:

• Gene knockout technologies:

  • CRISPR-Cas9 for complete gene knockout

  • Conditional knockout systems (Cre-loxP, FLP-FRT)

  • Inducible degradation systems (auxin-inducible, dTAG)

• Gene knockdown methods:

  • siRNA for transient knockdown

  • shRNA for stable knockdown

  • Antisense oligonucleotides for splice modulation

  • CRISPRi for transcriptional repression

• Modification strategies:

  • Knock-in of point mutations to study specific residues

  • Tagging at endogenous loci (FLAG, HA, fluorescent proteins)

  • Domain swapping with ATP6V0E2 to identify functional regions

  • Reporter gene fusion for expression monitoring

Phenotypic assessment approaches:

• Cellular phenotypes:

  • Organelle pH measurement with ratiometric probes

  • Lysosomal function assays (enzyme activity, cargo degradation)

  • Endocytosis and exocytosis rates

  • Autophagy flux analysis

  • Cell viability and proliferation

• Molecular phenotypes:

  • Transcriptomic analysis to identify compensatory responses

  • Proteomic profiling of V-ATPase complex components

  • Metabolomic analysis for changes in pH-dependent pathways

  • Lipidomic analysis for membrane composition changes

• Tissue and organism phenotypes:

  • Tissue-specific conditional knockout phenotyping

  • Developmental analysis in model organisms

  • Histological assessment for morphological changes

  • Physiological measurements (e.g., renal acid secretion)

Important considerations:

• Complete knockout may be lethal, so inducible or cell type-specific approaches may be necessary

• Compensatory upregulation of ATP6V0E2 should be monitored

• Phenotypes may manifest differently across cell types and tissues

• Combined approaches (e.g., knockdown plus rescue experiments) provide stronger evidence

Research in neuroblastoma cells demonstrated that siRNA knockdown of ATP6V0E1 reduced cell proliferation and viability, suggesting essential functions for cell survival . This highlights the importance of carefully titrated genetic modification approaches and comprehensive phenotypic analysis.

How can researchers design experiments to distinguish ATP6V0E1-specific functions from general V-ATPase perturbations?

Experimental design strategies:

• Comparative subunit analysis:

  • Parallel knockout/knockdown of different V-ATPase subunits

  • Comparison with general V-ATPase inhibitors (bafilomycin A1, concanamycin A)

  • Analysis of ATP6V0E1 vs. ATP6V0E2 (paralog) knockdown effects

  • Domain-swap chimeras between ATP6V0E1 and other subunits

• Targeted rescue experiments:

  • Rescue of ATP6V0E1 knockout with wildtype vs. mutant versions

  • Rescue with ATP6V0E2 to test functional redundancy

  • Rescue with orthologous ATP6V0E1 from other species

  • Rescue with minimal domains to identify functional regions

• Structure-function analysis:

  • Point mutations of conserved residues vs. isoform-specific residues

  • Identification of post-translational modification sites specific to ATP6V0E1

  • Analysis of protein-protein interaction interfaces

  • Compartment-specific targeting signals

Technical approaches for distinguishing specific effects:

• Temporal analysis:

  • Acute vs. chronic depletion to separate direct vs. compensatory effects

  • Time-resolved phenotypic analysis after perturbation

  • Pulse-chase experiments for dynamic processes

• Spatial analysis:

  • Organelle-specific pH measurements

  • Subcellular fractionation followed by biochemical analysis

  • High-resolution imaging of specific compartments

  • Targeted mass spectrometry of isolated organelles

• Context-dependent analysis:

  • Cell type-specific effects

  • Stress conditions that may reveal specialized functions

  • Developmental stage-specific phenotypes

  • Disease-relevant contexts

Case study examples:

• Research on V-ATPase a-subunit isoforms in Paramecium revealed 17 genes with highly specific targeting to at least seven different compartments, confirming functional specialization

• Studies on ATP6V0A1 showed that it contributes to endolysosomal acidification and its knockdown leads to specific effects on autophagy and mitochondrial function

• Research on dominant ATP6V1C1/ATP6V1B2 variants demonstrated gain-of-function effects leading to increased lysosomal acidification and disrupted lysosomal morphology

These approaches help distinguish between general effects of disrupting the V-ATPase complex versus specific roles of individual subunits like ATP6V0E1 in particular cellular contexts.

How can researchers integrate ATP6V0E1 studies into broader cellular pathways and networks?

Systems biology approaches for ATP6V0E1:

• Network analysis methods:

  • Protein-protein interaction mapping (Y2H, AP-MS, BioID)

  • Genetic interaction screens (CRISPR screens, synthetic lethality)

  • Transcriptomic analysis after ATP6V0E1 perturbation

  • Pathway enrichment analysis integrating multi-omics data

• Mathematical modeling approaches:

  • Kinetic modeling of V-ATPase assembly/disassembly dynamics

  • pH regulation network models including V-ATPase function

  • Integration of ATP6V0E1 into cellular metabolism models

  • Agent-based models of organelle pH maintenance

• Multi-omics integration:

  • Combined analysis of transcriptomics, proteomics, and metabolomics data

  • Correlation of ATP6V0E1 expression with other genes across cell types

  • Mapping ATP6V0E1 to known pH-responsive pathways

  • Network visualization tools for data integration

Relevant biological pathways to investigate:

• mTORC1 signaling pathway:

  • V-ATPase has been shown to regulate mTORC1 via Rag GTPases

  • Analyze how ATP6V0E1 perturbation affects mTOR localization and activity

  • Map interactions between ATP6V0E1 and components of amino acid sensing machinery

• Autophagy-lysosome system:

  • V-ATPase activity is critical for autophagy progression

  • Investigate ATP6V0E1's role in autophagic flux

  • Study connections to lysosomal biogenesis pathways (CLEAR network)

• Endocytic trafficking:

  • Map ATP6V0E1 to Rab GTPase networks

  • Analyze effects on receptor recycling vs. degradation pathways

  • Investigate connections to ESCRT machinery

• Metabolic networks:

  • Study how ATP6V0E1 integrates with cellular energy metabolism

  • Investigate connections to lipid metabolism (especially cholesterol)

  • Analyze pH-dependent metabolic enzymes affected by ATP6V0E1 function

Research has shown that V-ATPase subunits interact with key regulators like RABGEF1, which affects endosome maturation and is crucial for processes like cholesterol absorption . Similarly, covalent targeting of ATP6V1A revealed connections between the V-ATPase and mTORC1 signaling . These findings highlight the importance of investigating ATP6V0E1 within broader cellular networks.

What computational approaches can help predict and validate new functions or interactions of ATP6V0E1?

Computational prediction approaches:

• Structural bioinformatics:

  • Homology modeling of ATP6V0E1 structure

  • Molecular docking to predict protein-protein interactions

  • Molecular dynamics simulations to study conformational changes

  • Identification of functional domains and motifs

• Sequence-based predictions:

  • Evolutionary conservation analysis to identify critical residues

  • Co-evolution analysis to predict interaction partners

  • Post-translational modification site prediction

  • Subcellular localization signal prediction

• Advanced machine learning methods:

  • Deep learning for protein function prediction

  • Network-based function prediction algorithms

  • Text mining of scientific literature for association discovery

  • Integration of multi-omics data for functional prediction

Validation approaches:

• Structure-guided experimental design:

  • Site-directed mutagenesis based on computational predictions

  • Domain deletion/swapping guided by structural models

  • Design of peptide inhibitors targeting predicted interfaces

  • Structure-based drug design for ATP6V0E1 modulation

• Network-based validation:

  • Testing predicted genetic interactions via CRISPR screens

  • Validation of physical interactions using co-IP or proximity labeling

  • Testing predicted pathway connections with targeted inhibitors

  • Perturbation studies of computationally identified hubs

• Integrative data analysis:

  • Correlation analysis across large-scale datasets

  • Patient data mining for clinical correlations

  • Meta-analysis of ATP6V0E1-related studies

  • Multivariate statistical approaches to identify patterns

Emerging computational tools:

• AlphaFold2 and RoseTTAFold for accurate protein structure prediction

• Molecular dynamics platforms (GROMACS, NAMD) for studying dynamic processes

• Network analysis tools (Cytoscape, STRING) for visualizing interactions

• Omics data integration platforms (Galaxy, Bioconductor) for multi-level analysis

• Pathway enrichment tools (GSEA, Metascape) for biological context

Research on V-ATPase has benefited from computational approaches, with structural studies revealing mechanisms of assembly/disassembly regulation and interaction networks identifying connections to cellular processes like cholesterol absorption and immune signaling . Similar approaches applied to ATP6V0E1 could reveal new functions and therapeutic targets.