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

What is ATP6V0E1 and what is its role in cellular physiology?

ATP6V0E1 is a component of the vacuolar ATPase (V-ATPase), a multisubunit enzyme that mediates acidification of eukaryotic intracellular organelles. This protein is part of the V0 domain, which is the membrane-bound component involved in proton translocation across membranes . The V-ATPase-dependent organelle acidification is necessary for critical intracellular processes including protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation . ATP6V0E1 specifically contributes to the structural integrity and functional activity of the V0 domain, facilitating the movement of protons across membranes to maintain pH homeostasis within cellular compartments.

Methodologically, researchers investigating ATP6V0E1's role often employ pH-sensitive fluorescent probes to visualize organelle acidification in living cells, coupled with ATP6V0E1 knockdown or overexpression experiments to establish direct functional relationships.

What is the molecular structure of ATP6V0E1 from Pongo abelii?

Pongo abelii ATP6V0E1 (Sumatran orangutan) is a protein with 81 amino acids in its mature form. The amino acid sequence is: AYHGLTVPLIVMSVFWGFVGFLVPWFIPKGPNRGVIITMVTCSVCCYLFWLIAILAQLN PLFGPQLKNETIWYLKYHWP . The protein contains transmembrane regions that allow it to be incorporated into the V0 domain of the V-ATPase complex.

For structural analysis, researchers typically employ techniques such as X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy, often using recombinant protein preparations. Homology modeling based on related proteins with known structures may also provide insights when direct structural determination proves challenging.

How does recombinant ATP6V0E1 differ from native protein in experimental applications?

Recombinant ATP6V0E1 from Pongo abelii is typically produced in expression systems such as E. coli and may include additional modifications such as affinity tags to facilitate purification . While the core sequence matches the native protein, these modifications can influence protein folding, activity, and interaction properties. The recombinant protein is typically stored in a buffer containing 50% glycerol with Tris-based components to maintain stability .

When designing experiments, researchers should account for potential differences by including appropriate controls and validation steps. Native protein extraction methods generally yield lower quantities but may preserve post-translational modifications and conformational states that could be altered in recombinant systems.

What are the optimal storage conditions for recombinant ATP6V0E1?

Recombinant ATP6V0E1 is typically stored at -20°C for standard storage or -80°C for extended preservation . The protein is generally supplied in a storage buffer containing Tris-based components and 50% glycerol, which helps prevent freeze-thaw damage . Working aliquots can be maintained at 4°C for up to one week, but repeated freezing and thawing should be avoided as it can lead to protein denaturation and loss of activity .

For researchers planning long-term studies, it is advisable to divide the stock into single-use aliquots upon receipt to minimize freeze-thaw cycles. When reconstituting lyophilized protein, a recommended approach is to use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL, followed by the addition of glycerol to a final concentration of 5-50% for long-term storage .

How can ATP6V0E1 function be assessed in experimental systems?

Assessment of ATP6V0E1 function requires multi-parameter approaches that evaluate both its direct activity and broader cellular impacts. The primary functional readout is measurement of proton translocation efficiency, which can be quantified using pH-sensitive fluorescent dyes in reconstituted liposomes containing recombinant ATP6V0E1 within the V-ATPase complex.

Advanced methodologies include:

• Bafilomycin-sensitive ATPase activity assays to measure ATP hydrolysis rates

• Proton flux measurements using pH-sensitive fluorophores (e.g., ACMA or LysoSensor)

• Organelle-specific acidification assays in cellular systems with manipulated ATP6V0E1 expression

• Electrophysiological approaches to directly measure proton currents across membranes

Researchers should be mindful that ATP6V0E1 functions as part of the larger V-ATPase complex, so isolated protein studies must be complemented with investigations of assembled complexes to fully understand physiological relevance.

What approaches are used to study ATP6V0E1 interactions with other V-ATPase subunits?

Understanding the interactions between ATP6V0E1 and other V-ATPase subunits is crucial for elucidating the assembly and function of the complete enzyme complex. The V-ATPase is composed of a cytosolic V1 domain and a transmembrane V0 domain, with ATP6V0E1 being part of the V0 domain .

Advanced methodological approaches include:

When designing interaction studies, it's important to consider that V-ATPase assembly is dynamically regulated, and interactions may be tissue-specific or condition-dependent.

What are the implications of ATP6V0E1 dysfunction in cellular pathology?

While ATP6V0E1-specific pathologies have not been extensively documented in the provided search results, insights can be gained from related V-ATPase subunits. Variants in ATP6V0A1, another component of the V0 domain, have been associated with progressive myoclonus epilepsy, ataxia, and developmental epileptic encephalopathy . These conditions result from impaired endolysosomal acidification and subsequent disruption of autophagy and lysosomal function .

By extension, ATP6V0E1 dysfunction would likely affect similar cellular pathways, potentially impacting:

• Autophagy flux and clearance of cellular debris

• Endocytic trafficking and receptor recycling

• Lysosomal enzyme activation and protein degradation

• Neurotransmitter loading into synaptic vesicles

Research methodologies to investigate ATP6V0E1 dysfunction should include:

• Gene editing (CRISPR/Cas9) to introduce disease-relevant mutations

• Lysosomal function assays (DQ-BSA degradation, cathepsin activity)

• Autophagy flux monitoring (LC3-II/I ratio, p62 accumulation)

• Live-cell imaging of vesicular trafficking and organelle dynamics

How do species-specific variations in ATP6V0E1 affect functional studies?

The ATP6V0E1 protein from Pongo abelii (Sumatran orangutan) shows evolutionary conservation with human ATP6V0E1, but species-specific variations can influence experimental outcomes when using recombinant proteins across species boundaries. Understanding these differences is crucial for translational research.

Methodological considerations include:

• Comparative sequence analysis between Pongo abelii ATP6V0E1 (UniProt Q5RAV0) and human ATP6V0E1 (UniProt O15342) to identify conserved and variable regions

• Domain-specific functional assays to determine if species variations affect:

  • Proton translocation efficiency

  • Complex assembly kinetics

  • Regulatory protein interactions

  • Response to inhibitors

• Cell-based complementation studies, where the endogenous ATP6V0E1 is depleted and replaced with the Pongo abelii ortholog to assess functional equivalence

When using recombinant Pongo abelii ATP6V0E1 as a model for human studies, researchers should validate key findings with human protein where possible, particularly when investigating potential therapeutic targets or disease mechanisms.

What novel methodologies are being developed for ATP6V0E1 research?

Emerging methodologies for ATP6V0E1 research leverage advances in structural biology, single-molecule techniques, and genetic engineering:

• Single-particle cryo-EM analysis: Provides high-resolution structural information about ATP6V0E1 in the context of the assembled V-ATPase complex, revealing mechanism of proton translocation

• Optogenetic control of V-ATPase activity: Using light-sensitive domains fused to ATP6V0E1 allows temporal and spatial control of V-ATPase function in living cells

• Nanobody-based detection systems: Developing ATP6V0E1-specific nanobodies enables real-time tracking of protein localization and conformational changes

• Reconstitution in artificial membrane systems: Incorporation of purified ATP6V0E1 into synthetic membranes with defined lipid composition allows precise biophysical characterization

• CRISPR-based screening: Identification of genetic modifiers of ATP6V0E1 function through genome-wide loss-of-function screens

These advanced methodologies enable more precise investigation of ATP6V0E1's role in the V-ATPase complex and its contribution to cellular physiology and pathology.

What controls should be included when working with recombinant ATP6V0E1?

Rigorous experimental design for studies involving recombinant Pongo abelii ATP6V0E1 requires appropriate controls to ensure reliable and interpretable results:

• Protein quality controls:

  • SDS-PAGE analysis to confirm purity (>85% purity is generally acceptable)

  • Western blot verification of identity

  • Activity assays to confirm functional integrity

  • Thermal stability assessment (thermal shift assay)

• Experimental controls:

  • Heat-inactivated protein control to distinguish between specific and non-specific effects

  • Buffer-only control to account for vehicle effects

  • Wild-type vs. mutated protein comparisons

  • Species-matched controls when performing cross-species studies

• System-specific controls:

  • Specific V-ATPase inhibitors (e.g., bafilomycin A1) as positive controls for loss of function

  • ATP-depleted conditions to confirm ATP-dependency

  • pH-insensitive variants for acidification studies

Proper implementation of these controls helps distinguish between ATP6V0E1-specific effects and experimental artifacts, particularly when investigating complex cellular processes influenced by V-ATPase activity.

How can recombinant ATP6V0E1 be functionally validated?

Functional validation of recombinant ATP6V0E1 is essential to ensure that experimental observations reflect physiologically relevant activities. A comprehensive validation approach includes:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure

  • Limited proteolysis to verify proper folding

  • Size exclusion chromatography to assess oligomeric state

• Functional assays:

  • Incorporation into liposomes to measure proton pumping activity

  • Reconstitution with other V-ATPase subunits to form functional complexes

  • Membrane binding assays to confirm proper insertion

• Cellular complementation:

  • Rescue experiments in ATP6V0E1-depleted cells

  • Restoration of organelle acidification

  • Normalization of downstream pathways dependent on V-ATPase function

Validation should be performed under conditions that mimic the intended experimental environment, as buffer composition, pH, temperature, and the presence of other proteins can significantly impact ATP6V0E1 function.

How should conflicting results in ATP6V0E1 research be approached?

Contradictory findings in ATP6V0E1 studies may arise from several sources, requiring systematic investigation:

• Protein source variations:

  • Different expression systems (E. coli vs. mammalian cells)

  • Presence/absence of post-translational modifications

  • Variations in purification methods affecting protein conformation

• Experimental condition differences:

  • Buffer composition effects on protein stability and activity

  • Temperature and pH variances between studies

  • Repeated freeze-thaw cycles compromising protein integrity

• Methodological approach:

  • Direct vs. indirect measurement techniques

  • Isolated protein vs. complex-integrated studies

  • In vitro vs. cellular system discrepancies

When encountering conflicting results, researchers should:

• Directly compare experimental protocols and identify key differences

• Perform side-by-side validation experiments under standardized conditions

• Consider collaborating with groups reporting contradictory findings to resolve discrepancies

What are common pitfalls in ATP6V0E1 experimental design and how can they be avoided?

Research involving ATP6V0E1 presents several technical challenges that should be anticipated and addressed:

Researchers should document all experimental conditions meticulously and consider how variations in protein handling might affect experimental outcomes, particularly for sensitive functional assays.

How can structural insights from ATP6V0E1 inform therapeutic development?

Understanding the structure-function relationship of ATP6V0E1 within the V-ATPase complex provides opportunities for therapeutic targeting in V-ATPase-related disorders:

• Structure-guided drug design:

  • Identification of druggable pockets at subunit interfaces

  • Design of small molecules that selectively modulate ATP6V0E1 function

  • Development of peptide mimetics that disrupt pathological interactions

• Insights from disease-associated variants:

  • Analysis of variants in related V-ATPase subunits (like ATP6V0A1) provides insights into critical functional residues

  • The R740Q mutation in ATP6V0A1, for instance, disrupts proton translocation by impairing formation of a salt bridge necessary for proton release

  • Similar mechanistic insights from ATP6V0E1 can guide precision medicine approaches

• Subunit-specific targeting strategies:

  • Exploitation of tissue-specific isoform expression patterns

  • Design of bivalent compounds that target adjacent subunits

  • Development of conformation-specific modulators

These structural approaches enable more selective targeting of V-ATPase function in specific tissues or subcellular compartments, potentially reducing side effects compared to current pan-V-ATPase inhibitors.