Recombinant Mouse V-Type Proton ATPase Subunit E1 (ATP6V0E1) is a bioengineered protein corresponding to the mouse homolog of the vacuolar ATPase (V-ATPase) subunit E1. This subunit is a critical component of the V₀ domain of V-ATPase, a proton pump essential for acidifying eukaryotic intracellular organelles such as lysosomes, endosomes, and synaptic vesicles . Its primary function involves proton translocation and maintaining organelle homeostasis, which is vital for processes like protein sorting, receptor-mediated endocytosis, and autophagy .
Recombinant ATP6V0E1 is produced via bacterial (e.g., E. coli) or yeast expression systems, with His-tagged purification for research applications . Key specifications include:
Role in V-ATPase Assembly
ATP6V0E1 interacts with subunits D (V₁) and d (V₀) to stabilize the V₁-V₀ interface, ensuring efficient proton pumping . Mutations in ATP6V0E1 or related subunits (e.g., ATP6V0A1) impair V-ATPase assembly, leading to lysosomal dysfunction .
Disease Relevance
While ATP6V0E1-specific disease associations are less documented, V-ATPase subunit defects (e.g., ATP6V0A1 mutations) are linked to neurodegenerative disorders like developmental and epileptic encephalopathies (DEE) and progressive myoclonus epilepsy (PME) . These mutations disrupt proton translocation, causing autophagosome accumulation and neuronal degeneration .
Subunit Interactions: ATP6V0E1 binds Ragulator and Rag GTPases in lysosomes, modulating mTORC1 signaling . Covalent inhibitors targeting ATP6V1A (a V₁ subunit) decouple mTORC1 from lysosomal membranes, inducing autophagy .
Tissue-Specific Expression: In renal intercalated cells, ATP6V0E1 is downregulated in B1-deficient mice, impairing acid-base regulation .
Vacuolar ATPases are responsible for acidifying various intracellular compartments in eukaryotic cells.
Mouse Atp6v0e1 is a subunit of the V0 domain of vacuolar-type proton pumping ATPases. V-ATPases consist of two main sectors: the membrane-bound V0 domain involved in proton translocation and the peripheral V1 domain that catalyzes ATP hydrolysis. Atp6v0e1 specifically contributes to the assembly and stability of the V-ATPase complex in vacuolar membranes .
The N-terminal domain of the E subunit, particularly the region between Lys26 and Val83, contains eight residues that are not conserved between the testis-specific E1 and ubiquitous E2 isoforms. This domain is crucial for the assembly and proper functioning of the V-ATPase complex . When studying recombinant Atp6v0e1, researchers should pay particular attention to this domain, as modifications may significantly alter its functionality.
While V-ATPases are present throughout eukaryotic cells, their subunit isoforms show tissue-specific expression patterns. The V-ATPase subunit E has two main isoforms in mice: E1 (testis-specific) and E2 (ubiquitous) . This differential expression allows for tissue-specific regulation of V-ATPase activity.
For researchers investigating tissue-specific roles, it's important to note that Atp6v0e1 (E1) shows highest expression in testicular tissue, while other tissues predominantly express the E2 isoform. When designing experiments with recombinant Atp6v0e1, consider:
Expression systems that can accommodate proper folding of membrane proteins
The addition of appropriate tags that don't interfere with the N-terminal domain function
Validation of expression using tissue-specific antibodies
Several methodological approaches have been developed to investigate Atp6v0e1 function:
Hybrid V-ATPase systems: Mouse Atp6v0e1 can be expressed alongside yeast V-ATPase components to study its specific properties. This approach allows for the assessment of proton transport activity at different temperatures (30°C vs. 37°C) and under various conditions .
CRISPR-Cas9 gene editing: Targeted knockdown or knockout of Atp6v0e1 in cell lines (e.g., HeLa cells) or animal models (e.g., zebrafish) provides valuable insights into its functional roles .
Proton transport assays: Measuring V-ATPase activity through proton pumping assays in isolated vacuoles or reconstituted systems helps quantify the functional impact of Atp6v0e1 modifications .
Immunochemical detection: Using antibodies against specific epitopes of Atp6v0e1 or other V-ATPase subunits to detect conformational changes and protein-protein interactions .
Recent research has revealed that Atp6v0e1 plays a crucial role in the regulation of Hypoxia-Inducible Factor 1-alpha (HIF-1α). CRISPR-Cas9 knockdown of ATP6V0E1 in HeLa cells resulted in increased HIF-1α levels, as measured by HIF1α-GFP ODD reporter activity . This effect appears to be mediated through iron metabolism, as supplementation with Fe(III) citrate reversed the elevated HIF-1α levels in ATP6V0E1-depleted cells.
The relationship between Atp6v0e1 and HIF-1α has significant implications for:
Cellular response to hypoxia: V-ATPase activity influences cellular adaptation to low oxygen conditions
Retinal degeneration: HIF-1α dysregulation is associated with photoreceptor degeneration in inherited retinal diseases
Cancer research: HIF-1α is a key regulator of tumor angiogenesis and metabolism
When designing experiments to study this relationship, researchers should consider:
Using iron chelators or supplementation to modulate the Atp6v0e1-HIF-1α axis
Monitoring cellular pH alongside HIF-1α levels
Examining downstream HIF-1α target genes to assess functional impact
Zebrafish models with atp6v0e1 knockout display distinct phenotypes that provide insights into its physiological roles:
Visual impairment: atp6v0e1-/- zebrafish exhibit profound visual defects as measured by optokinetic response (OKR) .
Retinal abnormalities: Histological examination of atp6v0e1-/- zebrafish retinas shows structural changes and photoreceptor defects .
Gene expression changes: Knockout models show altered expression of phototransduction genes, particularly at 6 days post-fertilization (dpf), with significantly reduced levels of transcripts for Hif-1α and phototransduction signaling genes .
Response to HDAC6 inhibitors: Treatment with selective HDAC6 inhibitors like Tubastatin A (TubA) can partially restore visual function and increase expression of photoreceptor-specific genes like opn1lw2 (red-sensitive opsin) .
Interestingly, supplementation with iron (Fe(III) citrate, ascorbic acid, and lactoferrin) failed to rescue visual function in atp6v0e1-/- zebrafish, despite normalizing HIF-1α reporter activity in vitro . This suggests that the visual phenotype may involve mechanisms beyond HIF-1α regulation.
The V-ATPase complex is essential for acidification of endosomes, lysosomes, and other intracellular compartments. Atp6v0e1, as part of the V0 domain, directly contributes to proton translocation across membranes. Recent studies in Caenorhabditis elegans have demonstrated that knockdown of the ATP6V0A1 ortholog (another V-ATPase subunit) results in:
Reduced protein clearance in lysosomes
Altered regulation of several components of the autophagic machinery in neurons
When investigating Atp6v0e1's role in autophagy, researchers should consider:
Monitoring lysosomal pH using pH-sensitive fluorescent probes
Tracking autophagy flux with markers like LC3-II and p62
Analyzing degradation of specific autophagy substrates
Examining the interaction between Atp6v0e1 and other V-ATPase subunits
The E subunit of V-ATPase (including Atp6v0e1) plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes. Key structural insights include:
N-terminal domain significance: The domain between Lys26 and Val83 of E1 contains eight residues not conserved between E1 and E2 isoforms and is responsible for the unique properties of E1/yeast hybrid V-ATPases .
Temperature sensitivity: E1/yeast hybrid V-ATPases show defective proton transport at 37°C but regain function when shifted to 30°C, suggesting conformational changes in the complex .
Subunit interactions: The E subunit influences the accessibility of the V₀ subunit a epitope to antibodies, indicating its role in maintaining proper V-ATPase conformation .
For researchers working with recombinant Atp6v0e1, these structural features suggest:
The importance of maintaining native N-terminal domain structure
The potential impact of temperature on experimental results
The need to consider protein-protein interactions within the V-ATPase complex
When producing recombinant Atp6v0e1 for research purposes, several factors should be considered:
Expression system selection: Yeast systems have been successfully used to study mouse Atp6v0e1 function, as demonstrated by hybrid V-ATPase experiments . Mammalian expression systems may provide more native post-translational modifications.
Purification strategy:
Use mild detergents to maintain membrane protein structure
Consider co-expression with other V-ATPase subunits for stability
Avoid harsh conditions that might disrupt the critical N-terminal domain
Functional validation: Assess proton transport activity using reconstituted proteoliposomes or cellular assays. The temperature-dependent activity of E1/yeast hybrid V-ATPases suggests testing function at multiple temperatures (30°C and 37°C) .
Storage conditions: Optimize buffer composition (pH, salt concentration, glycerol) to maintain protein stability. Consider flash-freezing aliquots to preserve activity for long-term storage.
Multiple complementary approaches can be employed to assess Atp6v0e1 function in cells:
V-ATPase activity assays:
Measure intracellular pH using ratiometric dyes or pH-sensitive fluorescent proteins
Assess lysosomal acidification using LysoTracker or acridine orange
Quantify proton pumping in isolated organelles using fluorescent quenching assays
Protein interaction studies:
Co-immunoprecipitation to identify binding partners
Proximity labeling approaches (BioID, APEX) to map the protein interaction network
FRET-based assays to detect conformational changes upon activation
Functional readouts:
Monitor autophagy flux using LC3-II/LC3-I ratios and p62 levels
Assess HIF-1α stabilization and target gene expression
Evaluate endosomal trafficking and lysosomal degradation
Genetic manipulation:
CRISPR-Cas9 knockout/knockdown followed by rescue with wild-type or mutant Atp6v0e1
Site-directed mutagenesis of key residues in the N-terminal domain
Domain swapping between E1 and E2 isoforms to identify functional regions
Several experimental models have proven valuable for investigating Atp6v0e1's role in disease:
Zebrafish models:
Cell culture systems:
Mouse models:
Conditional knockout approaches may circumvent embryonic lethality
Tissue-specific deletion can reveal functions in specific organs
Can be used to model human diseases associated with V-ATPase dysfunction
C. elegans models:
When selecting an experimental model, researchers should consider:
The specific research question and disease context
Available genetic tools and readouts
Evolutionary conservation of Atp6v0e1 function
Technical expertise and resources required
Researchers working with Atp6v0e1 often encounter several challenges:
Protein instability: As a membrane protein component, recombinant Atp6v0e1 may exhibit stability issues. Consider:
Using stabilizing additives like glycerol or specific lipids
Co-expression with other V-ATPase subunits
Optimizing buffer conditions for each experimental step
Functional assessment limitations: V-ATPase activity depends on multiple subunits, making it difficult to isolate Atp6v0e1-specific effects. Strategies include:
Comparing E1 vs. E2 isoforms in the same cellular context
Using hybrid systems (e.g., mouse Atp6v0e1 with yeast V-ATPase components)
Employing rescue experiments with wild-type and mutant Atp6v0e1
Compensatory mechanisms: Genetic knockout may trigger upregulation of other V-ATPase subunits. Address by:
Using inducible knockdown systems
Monitoring expression of other V-ATPase components
Combining genetic and pharmacological approaches
Environmental variables: V-ATPase function is sensitive to temperature, pH, and ionic conditions. Control by:
Standardizing experimental conditions
Including appropriate controls in each experiment
Testing function across a range of physiologically relevant conditions
The scientific literature may contain seemingly contradictory results regarding Atp6v0e1 function. These discrepancies often arise from:
Isoform differences: E1 (testis-specific) and E2 (ubiquitous) isoforms have distinct properties and expression patterns . When comparing studies, verify which isoform was investigated.
Model system variations: Results from zebrafish, mice, and cell culture may differ due to species-specific factors. Consider evolutionary conservation and model-specific limitations.
Methodological differences: Proton transport assays, pH measurements, and protein interaction studies use diverse techniques with varying sensitivities. Standardized protocols and multiple methodological approaches can help resolve discrepancies.
Contextual factors: V-ATPase function is influenced by cellular context, metabolic state, and environmental conditions. When integrating findings from different studies, consider:
Cell/tissue type and developmental stage
Metabolic conditions (glucose availability, oxygen levels)
Presence of compensatory mechanisms
To reconcile contradictory findings, researchers should:
Directly compare experimental conditions
Reproduce key experiments using standardized protocols
Consider multiple readouts of V-ATPase function
Acknowledge limitations and context-dependencies of each finding
Several cutting-edge technologies hold promise for deepening our understanding of Atp6v0e1:
Cryo-electron microscopy: Recent advances have enabled visualization of V-ATPase structure at unprecedented resolution, potentially revealing how Atp6v0e1 contributes to proton translocation .
Single-cell transcriptomics and proteomics: These approaches can identify cell-specific expression patterns and regulatory networks involving Atp6v0e1.
Optogenetic tools: Light-controlled activation or inhibition of V-ATPase function could enable precise temporal and spatial manipulation in living cells.
Organoid models: Three-dimensional tissue cultures may better recapitulate the physiological context of Atp6v0e1 function in specific organs.
AI-driven protein structure prediction: Tools like AlphaFold could help predict how Atp6v0e1 mutations affect structure and function, guiding experimental design.
Research on Atp6v0e1 and other V-ATPase components suggests several potential therapeutic applications:
Retinal degeneration: HDAC6 inhibitors like Tubastatin A (TubA) have shown promise in restoring visual function in zebrafish models with atp6v0e1 deficiency . These compounds could potentially be developed for treating inherited retinal diseases.
Neurological disorders: Given the link between ATP6V0A1 (another V-ATPase subunit) variants and progressive myoclonus epilepsy , modulating V-ATPase function might be therapeutic for certain neurological conditions.
Lysosomal storage diseases: Enhancing V-ATPase activity could potentially improve lysosomal function in disorders characterized by impaired protein degradation.
Cancer therapy: As V-ATPases regulate pH homeostasis and HIF-1α signaling, both critical for tumor growth, targeting specific isoforms might offer selective approaches to cancer treatment.
When exploring therapeutic applications, researchers should consider:
Isoform selectivity to minimize off-target effects
Tissue-specific delivery methods
Combination approaches targeting multiple aspects of the affected pathway
Potential compensatory mechanisms that might limit efficacy