Recombinant Rat V-type proton ATPase subunit e 2 (Atp6v0e2)

What is ATP6V0E2 and what is its role in cellular function?

ATP6V0E2 is a protein-coding gene that encodes an isoform of the H⁺-ATPase V0 e subunit, which forms an essential component of the vacuolar-type ATPase (V-ATPase) proton pump. This multisubunit enzyme complex is responsible for acidifying various intracellular compartments by translocating protons from the cytosol into organelle lumen . The V-ATPase complex consists of two major sectors: the V1 sector that hydrolyzes ATP to provide energy for proton transport, and the V0 sector (where ATP6V0E2 is located) that forms an integral membrane domain through which protons are transported . In neurons specifically, V-ATPase activity is critical for the accumulation of neurotransmitters into secretory vesicles and their subsequent release at synapses .

How is ATP6V0E2 structurally organized within the V-ATPase complex?

ATP6V0E2 functions as a subunit of the membrane-integral V0 complex within the larger V-ATPase enzyme. Recent cryoEM analysis of mammalian brain V-ATPase reveals that the V0 region contains multiple subunits arranged in a specific architecture that enables proton translocation . The V0 complex includes a c-ring structure composed of multiple protomers, each containing conserved glutamate residues capable of carrying protons during translocation . ATP6V0E2 integrates into this complex to support the structural integrity and functional capacity of the proton pump. The ATP:H⁺ ratio for the mammalian brain V-ATPase has been determined to be 3:10, meaning three ATP molecules are hydrolyzed for every ten protons translocated .

What are the recommended methods for expressing and purifying recombinant rat ATP6V0E2?

For optimal expression and purification of recombinant rat ATP6V0E2, a methodological approach involving bacterial or mammalian expression systems is recommended. When using bacterial expression systems (e.g., E. coli), the following protocol has proven effective:

• Clone the ATP6V0E2 coding sequence into a pET vector with an N-terminal His-tag

• Transform into BL21(DE3) E. coli cells

• Induce expression with 0.5-1.0 mM IPTG at 16°C overnight

• Lyse cells in buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% Triton X-100

• Purify using nickel affinity chromatography followed by size exclusion chromatography

For mammalian expression which may better preserve post-translational modifications:

• Clone ATP6V0E2 into a mammalian expression vector (e.g., pcDNA3.1)

• Transfect HEK293 cells and select stable cell lines

• Extract membrane fractions using differential centrifugation

• Solubilize membrane proteins with appropriate detergents (e.g., DDM or CHAPS)

• Purify using affinity chromatography

Verification of purified protein can be performed through Western blotting with antibodies specific to ATP6V0E2 or the affinity tag .

How can researchers effectively knock down or knock out ATP6V0E2 for functional studies?

For effective manipulation of ATP6V0E2 expression, researchers can employ several complementary approaches:

siRNA/shRNA-mediated knockdown:

• Design at least 3-4 siRNA sequences targeting different regions of ATP6V0E2 mRNA

• Transfect cells with siRNAs using lipofection or electroporation

• Validate knockdown efficiency by qRT-PCR and Western blotting

• Assess phenotypic changes 48-72 hours post-transfection

CRISPR-Cas9 gene editing for knockout:

• Design guide RNAs targeting exons of ATP6V0E2 (preferably early exons)

• Clone guide RNAs into a Cas9-expressing vector

• Transfect cells and select for edited clones

• Validate knockout by sequencing, Western blotting, and functional assays

Mouse models:
Following the approach described in the literature for other V-ATPase subunits, gene targeting can be used to generate ATP6V0E2-null mice . When designing such experiments, it's important to consider potential compensatory mechanisms, as observed with the G2 subunit where G1 was upregulated in knockout mice . This may require additional experiments to identify and characterize compensatory pathways.

What are the best methods to study ATP6V0E2 protein-protein interactions within the V-ATPase complex?

To effectively study ATP6V0E2 interactions within the V-ATPase complex, researchers should consider these complementary methodological approaches:

Co-immunoprecipitation (Co-IP):

• Generate cell lysates under non-denaturing conditions

• Use antibodies against ATP6V0E2 or epitope tags for immunoprecipitation

• Analyze co-precipitated proteins by Western blotting or mass spectrometry

• Include appropriate controls (e.g., IgG control, lysates from knockout cells)

Proximity labeling approaches:

• Generate fusion proteins of ATP6V0E2 with BioID or APEX2 enzymes

• Express in relevant cell types and activate the enzyme to label proximal proteins

• Purify biotinylated proteins using streptavidin beads

• Identify binding partners via mass spectrometry

Structural biology approaches:
Recent advances in cryoEM have enabled detailed structural analysis of the V-ATPase complex. Similar approaches can be applied to study ATP6V0E2:

• Isolate intact V-ATPase complexes from rat brain or recombinant sources

• Use innovative approaches such as interaction with SidK (a Legionella pneumophila effector protein) to obtain homogeneous preparations

• Perform cryoEM analysis to determine structural arrangements

• Generate atomic models to define precise interaction interfaces

How does ATP6V0E2 contribute to neurotransmitter loading and release in synaptic vesicles?

ATP6V0E2, as part of the V-ATPase complex, plays a critical role in synaptic vesicle acidification which is essential for neurotransmitter loading and subsequent release. The proton gradient established by V-ATPase provides the driving force for neurotransmitter uptake via vesicular neurotransmitter transporters.

Mass spectrometry of purified rat brain synaptic vesicles has detected multiple V-ATPase subunits, including ATP6AP1/Ac45 and ATP6AP2/PRR, which facilitate assembly of the enzyme's catalytic and membrane regions . The specific contribution of ATP6V0E2 can be investigated through the following methodological approaches:

• Selective knockdown of ATP6V0E2 in neuronal cultures followed by measurement of:

  • Vesicular pH using pH-sensitive fluorescent probes

  • Neurotransmitter uptake using radiolabeled substrates

  • Vesicle release kinetics via electrophysiological recordings

• Generation of ATP6V0E2 conditional knockout mice with neuron-specific deletion to assess:

  • Synaptic transmission defects

  • Behavioral phenotypes

  • Compensatory mechanisms similar to those observed with G2 subunit knockout

Current evidence suggests that V-ATPase in neurons operates with an ATP:H⁺ ratio of 3:10, allowing it to establish a proton motive force equivalent to approximately 3 pH units or 180 mV . This gradient is sufficient to drive the concentrative uptake of neurotransmitters against their concentration gradient.

What is the relationship between ATP6V0E2 and signaling pathways such as Notch and Wnt?

ATP6V0E2, as a component of V-ATPase, contributes to the acidification of endocytic compartments which is crucial for proper signaling pathway function. Research has established important connections between V-ATPase activity and both Notch and Wnt signaling pathways:

Notch signaling:
V-ATPase activity is required for proper Notch signaling, with studies demonstrating that mutations in V-ATPase subunits phenocopy defects seen in Notch pathway components . The acidification of endocytic compartments appears crucial for Notch signal transduction in receiving cells . Methodologically, this connection can be investigated by:

• Examining Notch target gene expression after ATP6V0E2 knockdown

• Tracking Notch receptor trafficking and processing in cells with altered ATP6V0E2 levels

• Assessing genetic interactions between ATP6V0E2 and Notch pathway components

Wnt signaling:
V-ATPase components, particularly ATP6AP2/PRR (Pro-renin receptor), have been implicated in Wnt signaling pathways important for stem cell function and embryo development . Research methodologies to explore ATP6V0E2's potential role in Wnt signaling include:

• TOP/FOP reporter assays to measure canonical Wnt signaling activity in cells with ATP6V0E2 manipulation

• Analysis of Wnt receptor endocytosis and recycling using fluorescently tagged receptors

• Investigation of ATP6V0E2 interactions with known Wnt pathway components

The table below summarizes key signaling pathways influenced by V-ATPase activity:

How does ATP6V0E2 function differ across tissue types and developmental stages?

ATP6V0E2 expression and function vary across different tissues and developmental stages, contributing to tissue-specific V-ATPase roles. While comprehensive tissue-specific data for ATP6V0E2 specifically is limited in the provided search results, V-ATPase complexes show substantial tissue and developmental specificity through the expression of different subunit isoforms.

Tissue-specific expression patterns:
Mammals express multiple isoforms of V-ATPase subunits in a tissue-dependent and cellular-compartment-dependent manner, including "two isoforms of subunit B, two of C, two of E, three of G, four of a, two of d, and two of e" . These expression patterns likely reflect specialized functions:

• In neurons: V-ATPase is enriched in synaptic vesicles, with mass spectrometry detecting subunits A, B1, B2, C1, D, E1, F, G1, G2, a1, a4, c, d1, ATP6AP1/Ac45, and ATP6AP2/PRR

• In osteoclasts: V-ATPase is critical for bone resorption, consistent with the association of ATP6V0E2 with osteopetrosis

• In kidney intercalated cells: V-ATPase contributes to acid secretion and regulation of blood pH

• In tumor cells: V-ATPase may support acid secretion and contribute to cancer progression

Developmental regulation:
V-ATPase function is developmentally regulated, with complete loss of activity resulting in embryonic lethality . This suggests essential roles during early development. Methodological approaches to study developmental regulation include:

• Temporal expression analysis using qRT-PCR and Western blotting across developmental stages

• Conditional knockout models with temporally controlled deletion

• In situ hybridization to map expression patterns during embryogenesis

• Functional assays in embryonic versus adult tissues to compare activity levels

What are common challenges in generating functional recombinant ATP6V0E2 and how can they be overcome?

Researchers often encounter several challenges when working with recombinant V-ATPase subunits like ATP6V0E2, primarily due to its membrane protein nature and its function as part of a complex. These challenges and potential solutions include:

Protein solubility issues:

• Challenge: ATP6V0E2 is a membrane protein component and may show poor solubility.
  Solution: Use mild detergents (DDM, CHAPS) for extraction; consider fusion partners like MBP or SUMO to enhance solubility.

Proper folding concerns:

• Challenge: Bacterial expression systems may not provide appropriate folding environment.
  Solution: Express in eukaryotic systems (insect cells or mammalian cells); lower expression temperature (16-18°C); use molecular chaperones as co-expression partners.

Functional activity assessment:

• Challenge: Individual subunits may not show activity outside the V-ATPase complex.
  Solution: Co-express with other V0 subunits; establish functional assays that measure complex assembly rather than isolated activity.

Antibody specificity:

• Challenge: Limited availability of specific antibodies against ATP6V0E2.
  Solution: Use epitope tags (His, FLAG); validate antibodies using knockout/knockdown samples as negative controls.

How can researchers accurately measure V-ATPase activity in the context of ATP6V0E2 studies?

To accurately assess V-ATPase activity in studies focusing on ATP6V0E2, researchers should employ multiple complementary approaches:

pH-sensitive fluorescent probes:

• Acridine orange or BCECF to measure organelle acidification

• LysoSensor probes for lysosomal pH measurements

• pHluorin-tagged proteins for measuring synaptic vesicle pH

ATP hydrolysis assays:

• Malachite green assay to measure inorganic phosphate release

• Coupled enzyme assays linking ATP hydrolysis to NADH oxidation

• ATPase activity measurements in presence/absence of specific V-ATPase inhibitors (bafilomycin A1, concanamycin A)

Proton translocation measurements:

• ACMA (9-amino-6-chloro-2-methoxyacridine) fluorescence quenching assay

• Measurement of proton flux across reconstituted proteoliposomes

• Patch-clamp electrophysiology of isolated vesicles or reconstituted systems

The table below summarizes key V-ATPase activity assays:

What are the considerations for studying compensatory mechanisms when ATP6V0E2 is depleted?

When studying ATP6V0E2 depletion, researchers must carefully consider potential compensatory mechanisms, similar to those observed with the G2 subunit of V-ATPase where G1 was upregulated in knockout mice . Methodological considerations include:

Comprehensive isoform analysis:

• Quantify mRNA and protein levels of all V-ATPase subunits after ATP6V0E2 depletion

• Pay particular attention to ATP6V0E1, the important paralog of ATP6V0E2

• Use both qRT-PCR and Western blotting, as compensatory responses may occur at protein level without mRNA changes (as seen with G1 upregulation in G2-null mice)

Functional redundancy assessment:

• Generate single and double knockouts of ATP6V0E1 and ATP6V0E2

• Compare phenotypic severity between single and double knockouts

• Perform rescue experiments with each isoform to determine functional equivalence

Temporal analysis of compensation:

• Use inducible knockout systems to track the timeline of compensatory responses

• Distinguish between immediate/acute responses and long-term adaptations

• Consider developmental stage-specific differences in compensatory capacity

Pathway analysis:

• Perform transcriptomic/proteomic analysis at different timepoints after ATP6V0E2 depletion

• Use pathway enrichment analyses to identify regulatory networks involved in compensation

• Validate key regulatory factors using targeted approaches

What are the emerging areas of research regarding ATP6V0E2 in disease models?

Based on current knowledge of V-ATPase biology, several promising research directions regarding ATP6V0E2 in disease models warrant investigation:

Neurodegenerative disorders:
Given the importance of V-ATPase in synaptic vesicle acidification and neurotransmitter loading , ATP6V0E2's role in neurodegeneration represents an important research avenue. Methodological approaches include:

• Analysis of ATP6V0E2 expression in patient-derived samples from various neurodegenerative diseases

• Generation of conditional knockout models to assess contribution to disease progression

• Investigation of ATP6V0E2 polymorphisms as potential disease risk factors

Bone disorders:
The association of ATP6V0E2 with osteopetrosis suggests further investigation into broader bone pathologies:

• Detailed characterization of bone phenotypes in ATP6V0E2-deficient models

• Analysis of ATP6V0E2 expression and function in various bone cell types

• Testing therapeutic approaches targeting ATP6V0E2 for bone disorders

Cancer biology:
V-ATPases are implicated in tumor cell acid secretion , suggesting ATP6V0E2 may have relevance in cancer research:

• Analysis of ATP6V0E2 expression across cancer types and correlation with patient outcomes

• Investigation of its role in cancer cell metabolism, invasion, and drug resistance

• Evaluation as a potential therapeutic target for specific cancer subtypes

How might structural biology approaches advance our understanding of ATP6V0E2 function?

Recent advances in structural biology, particularly cryoEM, have revolutionized our understanding of V-ATPase structure and function . Future research using structural approaches could:

• Determine high-resolution structures of V-ATPase complexes with different subunit isoform compositions, including ATP6V0E2

• Characterize conformational changes during the catalytic cycle through time-resolved cryo-EM

• Identify binding sites for regulators and inhibitors to enable structure-based drug design

• Elucidate the structural basis for tissue-specific functions through comparative structural analysis

The recent cryoEM structure of mammalian brain V-ATPase has already provided valuable insights, showing how ATP6AP1/Ac45 and ATP6AP2/PRR enable assembly of the enzyme's catalytic and membrane regions . Similar approaches could reveal ATP6V0E2's precise structural role.

What technological innovations might advance ATP6V0E2 research in the coming years?

Several emerging technologies have the potential to significantly advance ATP6V0E2 research:

CRISPR-based screening approaches:

• Genome-wide CRISPR screens to identify genetic interactors of ATP6V0E2

• CRISPRi/CRISPRa libraries to modulate expression in different cellular contexts

• Base editing and prime editing for precise modification of regulatory elements

Advanced imaging techniques:

• Super-resolution microscopy to visualize ATP6V0E2 localization and dynamics

• Correlative light and electron microscopy to link function to ultrastructure

• Expansion microscopy to reveal nanoscale organization within complexes

Proteomics approaches:

• Proximity labeling (BioID, APEX) to map ATP6V0E2 interaction networks

• Crosslinking mass spectrometry to identify direct binding interfaces

• Targeted proteomics to quantify stoichiometry in different cellular contexts

Computational modeling:

• Molecular dynamics simulations to understand conformational dynamics

• Systems biology approaches to model compensatory network responses

• AI-based prediction of structure-function relationships