Recombinant Human ATP synthase subunit f, mitochondrial (ATP5J2)

What is the structure and function of ATP synthase subunit f in mitochondria?

ATP synthase subunit f is a component of the mitochondrial F-type ATPase complex, specifically functioning within the F₀ domain. This subunit plays a critical role in the interaction between the F₁ catalytic domain and the F₀ membrane domain of ATP synthase . The F₁ component contains the solvent-exposed catalytic sites, while the F₀ component spans the inner mitochondrial membrane.

Structurally, ATP synthase comprises multiple subunits arranged in two linked multi-subunit complexes:

• F₁ complex: Contains 5 different subunits (α, β, γ, δ, and ε) in a 3:3:1:1:1 ratio

• F₀ complex: Contains nine subunits (a, b, c, d, e, f, g, F6, and 8)

The F subunit helps maintain the structural integrity of ATP synthase and is critical for the proper functioning of the enzyme during oxidative phosphorylation, where it contributes to ATP production by harnessing the proton gradient across the inner mitochondrial membrane .

How does ATP synthase subunit f contribute to mitochondrial function in neurons?

In neuronal cells, ATP synthase subunit f contributes significantly to mitochondrial function through several mechanisms:

• Energy production: As part of the ATP synthase complex, it facilitates ATP synthesis by contributing to the rotary mechanism that couples proton translocation to ATP synthesis

• Synaptic function: Neuronal activity requires substantial energy resources, and ATP synthase dysfunction can lead to decreased extracellular ATP, disrupting synaptic plasticity and long-term potentiation (LTP)

• Neuroinflammatory regulation: Research has shown that ATP synthase subunits, including subunit f, can regulate microglial activation and neuroinflammatory pathways

• Neuroprotection: Proper functioning of ATP synthase is essential for maintaining neuronal viability, with dysfunction linked to neurodegenerative conditions like Alzheimer's disease

Experimental models have demonstrated that alterations in ATP synthase subunit expression correlate with cognitive deficits in neurodegenerative disease models, suggesting a critical role in maintaining neuronal health .

What experimental models are most suitable for studying recombinant ATP5J2 function?

Several experimental models have proven effective for studying recombinant ATP5J2 function:

For in vitro studies, BV2 microglial cells stimulated with oxyhemoglobin have been used to model mitochondrial dysfunction associated with ATP synthase alterations . For in vivo assessment, transgenic mouse models expressing mutations in mitochondrial proteins or amyloid precursor protein have been valuable for studying ATP synthase function in disease contexts .

The choice of model should align with specific research questions, with neuronal or microglial cell cultures appropriate for mechanistic studies and animal models better suited for understanding systemic effects.

What are the key differences between ATP5J and ATP5J2 in terms of structure and function?

While the search results primarily address ATP5J (now known as ATP5PF), understanding the distinctions between ATP synthase subunits is important for research specificity:

ATP5J/ATP5PF (ATP Synthase Peripheral Stalk Subunit F6):

• Functions as part of the peripheral stalk (stator) of ATP synthase

• Required for F₁ and F₀ complex interactions

• Involved in restoring oligomycin-sensitive ATPase activity to depleted F₁-F₀ complexes

• Previously known by multiple names including ATP5A, ATPM, and CF6

• Located on chromosome 21 in humans

ATP5J2 (ATP Synthase Subunit f):

• Component of the F₀ domain of ATP synthase

• Contributes to the structural stability of the ATP synthase complex

• Has distinct genetic encoding and regulation compared to ATP5J/ATP5PF

The functional differences between these subunits reflect their distinct roles within the ATP synthase complex, with ATP5J/ATP5PF participating in the peripheral stalk structure, while ATP5J2 contributes to the F₀ domain architecture.

When designing experiments targeting specific subunits, researchers should verify gene and protein sequences to ensure targeting specificity and avoid cross-reactivity between related subunits.

How is ATP5J2 expression regulated during cellular stress responses?

ATP synthase subunit expression undergoes significant regulation during cellular stress responses:

• Oxidative stress: Increased oxidative stress can lead to post-translational modifications of ATP synthase subunits, including 3-nitrotyrosine (3-NT) modifications and 4-hydroxy-2-nonenal (4-HNE) attachments

• Energy demand adaptation: Expression of ATP synthase subunits, including f, can increase significantly (up to 12.2-fold) in response to cellular stress as an adaptive mechanism to maintain energy production

• Disease-specific regulation: In pathological conditions like Alzheimer's disease or intracerebral hemorrhage, ATP synthase subunit expression changes correlate with disease progression

• Post-translational modifications: O-GlcNAcylation (a glycosylation process) of ATP synthase subunits is reduced in Alzheimer's disease brains, contributing to reduced ATP levels

• Inflammatory signaling: During neuroinflammation, ATP5J upregulation in microglia corresponds with increased pro-inflammatory cytokine production and microglial activation

These regulatory mechanisms highlight the dynamic nature of ATP synthase expression and modification in response to cellular stress, suggesting potential targets for therapeutic intervention in conditions involving mitochondrial dysfunction.

What methodologies are most effective for studying ATP5J2 interactions with other mitochondrial proteins?

Advanced investigation of ATP5J2 protein interactions requires sophisticated methodological approaches:

• Proximity-based labeling techniques:

  • BioID or TurboID: Fusion of a biotin ligase to ATP5J2 allows identification of proximal proteins through biotinylation

  • APEX2: Provides high temporal resolution for capturing dynamic interactions in the mitochondrial environment

• Crosslinking mass spectrometry (XL-MS):

  • Enables identification of direct protein-protein contact sites

  • Particularly valuable for membrane protein complexes like ATP synthase

  • Can be combined with cryo-EM for structural validation

• Co-immunoprecipitation with quantitative proteomics:

  • Apply SILAC or TMT labeling to quantify differential interactions under varying conditions

  • Requires validated antibodies specific to ATP5J2 to avoid cross-reactivity with related subunits

• Mitochondrial fractionation protocols:

  • Separation of inner membrane, outer membrane, and matrix fractions

  • Blue Native PAGE for studying intact protein complexes

  • Sequential extraction approaches to distinguish peripheral versus integral membrane associations

• Live-cell imaging techniques:

  • FRET or BRET assays for dynamic interaction studies

  • Split-GFP complementation to visualize specific protein associations

These methods can be applied to study how ATP5J2 interacts with other ATP synthase subunits, mitochondrial fission/fusion proteins (like Drp1 and Fis1), and components of the respiratory electron transport chain to better understand its role in mitochondrial function and disease pathology .

How do post-translational modifications affect ATP5J2 function in different cellular contexts?

Post-translational modifications (PTMs) significantly impact ATP synthase subunit function through diverse mechanisms:

Research methodologies to study these modifications include:

• Mass spectrometry-based approaches for comprehensive PTM mapping

• Site-directed mutagenesis of key modification sites to establish functional significance

• Development of modification-specific antibodies for immunodetection

• Enzymatic assays to measure ATP synthase activity before and after specific PTM modulation

• In situ proximity ligation assays to detect modified forms within cellular contexts

Studies have shown that the α-subunit of ATP synthase undergoes 4-HNE modification in hippocampal tissue from mild cognitive impairment patients, correlating with a 35% decrease in ATP synthase activity compared to controls . Similarly, reduced O-GlcNAcylation of the α-subunit at Thr432 in Alzheimer's disease brains and Aβ-treated cell cultures results in decreased ATP production .

What is the relationship between ATP5J2 dysfunction and mitochondrial dynamics in neurodegenerative diseases?

ATP synthase dysfunction and altered mitochondrial dynamics are intricately linked in neurodegenerative pathology:

• Mitochondrial fission/fusion balance: ATP synthase subunit alterations affect the expression and activity of mitochondrial dynamics regulators, including Drp1 (dynamin-related protein 1) and Fis1 (mitochondrial fission 1 protein)

• Mitochondrial membrane potential: Dysfunction in ATP synthase components disrupts membrane potential maintenance, triggering excessive mitochondrial fission and permeability transition pore opening

• Reactive oxygen species (ROS) production: Impaired ATP synthase function increases ROS generation, which further damages mitochondrial DNA and proteins, creating a pathological feedback loop

• Cristae remodeling: ATP synthase, particularly its dimers at cristae tips, helps maintain cristae structure; dysfunction leads to abnormal cristae morphology observed in neurodegenerative conditions

• Calcium homeostasis: ATP synthase components contribute to mitochondrial calcium handling; dysfunction disrupts neuronal calcium homeostasis

Experimental evidence from both in vitro and in vivo models demonstrates that alterations in ATP synthase subunits correlate with excessive mitochondrial fragmentation and dysfunction. In microglial cells, ATP5J knockdown reversed the upregulation of Drp1 and Fis1 induced by oxyhemoglobin, reducing mitochondrial overdivision and restoring normal mitochondrial ridge morphology .

These findings suggest that targeting ATP synthase subunits may represent a therapeutic strategy to normalize mitochondrial dynamics in neurodegenerative conditions.

How can recombinant ATP5J2 be used to study mitochondrial bioenergetics in neuroinflammatory conditions?

Recombinant ATP5J2 serves as a valuable tool for investigating mitochondrial bioenergetics in neuroinflammatory contexts:

• Overexpression studies: Viral vector-mediated ATP synthase subunit overexpression can be used to examine the consequences of increased expression on mitochondrial function, as demonstrated in studies where AAV9-mediated ATP5J overexpression worsened neurobehavioral deficits and increased neuroinflammation in intracerebral hemorrhage models

• Knockdown/knockout approaches: RNA interference or CRISPR-based targeting of ATP synthase subunits helps establish their necessity in maintaining normal mitochondrial function during inflammatory challenges

• Mitochondrial respiration analysis: Utilizing recombinant proteins to:

  • Reconstruct partial or complete ATP synthase complexes in liposomes

  • Measure proton flux across membranes using pH-sensitive fluorophores

  • Analyze oxygen consumption rates in response to specific inhibitors

• Structure-function studies: Site-directed mutagenesis of recombinant ATP5J2 allows investigation of how specific residues contribute to:

  • Complex assembly and stability

  • Catalytic efficiency

  • Interaction with regulatory factors

  • Susceptibility to inflammatory damage

• Integrated bioenergetic assessment: Combining recombinant protein studies with:

  • Seahorse XF analysis of cellular respiratory parameters

  • Live-cell ATP measurements using genetically encoded sensors

  • Mitochondrial membrane potential dynamics using potential-sensitive dyes

Research has shown that modulating ATP synthase subunit expression affects microglial activation states, proliferation, migration, and inflammatory cytokine production in neuroinflammatory conditions . These methodologies enable mechanistic understanding of how ATP synthase components influence cellular energetics during inflammation.

What experimental contradictions exist in the literature regarding ATP synthase subunit function, and how can these be reconciled?

Several notable contradictions exist in the ATP synthase research literature:

• Activity in Alzheimer's disease:

  • Contradiction: Early studies found no significant decrease in ATP synthase catalytic activity in isolated mitochondria from AD patient hippocampal tissue , while later research demonstrated a 35% decrease in activity in mild cognitive impairment patients

  • Reconciliation approach: Standardize tissue preparation methods, account for disease stage variations, and measure activity using multiple complementary assays

• Expression patterns in disease models:

  • Contradiction: Some studies report decreased expression of ATP synthase complexes in AD models , while others show increased expression of specific subunits (12.2-fold increase in α-subunit in J20 Tg mice)

  • Reconciliation approach: Distinguish between whole complex versus individual subunit expression, account for compensatory mechanisms, and specify brain region and disease stage

• Oxidative modification status:

  • Contradiction: Evidence for oxidative modifications of ATP synthase subunits varies between studies, with some reporting significant 4-HNE modifications while others found no 3-NT modifications

  • Reconciliation approach: Utilize multiple oxidative stress markers, control for tissue specificity, and consider temporal dynamics of different modifications

• Primary role in disease pathogenesis:

  • Contradiction: Debates exist about whether ATP synthase dysfunction is a primary cause or secondary consequence of pathology

  • Reconciliation approach: Use temporal studies with precise disease staging, develop inducible genetic models, and apply systems biology approaches

These contradictions can be addressed through:

• Methodological standardization: Developing consensus protocols for tissue processing, activity measurements, and protein analysis

• Comprehensive profiling: Analyzing multiple modifications simultaneously rather than focusing on single PTMs

• Temporal resolution: Examining changes across disease progression from earliest stages

• Spatial resolution: Accounting for cell-type and brain-region specificity

• Multi-omics integration: Combining proteomic, transcriptomic, and metabolomic data for systems-level understanding

What are the optimal conditions for expressing and purifying recombinant human ATP5J2?

Successful expression and purification of recombinant human ATP5J2 requires careful optimization:

Expression systems comparison:

Optimization parameters for bacterial expression:

• Construct design:

  • Include a cleavable N-terminal tag (His6, GST, or MBP) to enhance solubility

  • Consider codon optimization for E. coli expression

  • Remove mitochondrial targeting sequence for improved expression

• Culture conditions:

  • Induction at lower temperatures (16-18°C) overnight improves folding

  • Use minimal media supplemented with glucose to reduce metabolic burden

  • Add 0.1-0.5% glucose to reduce leaky expression pre-induction

• Purification protocol:

  • Two-step purification combining affinity chromatography and size exclusion

  • Include mild detergents (0.1% DDM or 0.5% CHAPS) in buffers to maintain solubility

  • Use reducing agents (2-5 mM β-mercaptoethanol) to prevent oxidation

  • Optimize salt concentration (typically 150-300 mM NaCl) to reduce aggregation

• Quality control measures:

  • Circular dichroism to verify secondary structure

  • Dynamic light scattering to assess monodispersity

  • Activity assays to confirm functional integrity

  • Mass spectrometry to verify protein identity and modifications

For functional studies, co-expression with other ATP synthase subunits may be necessary to achieve proper folding and activity. Storage should include flash-freezing in small aliquots with 10% glycerol to maintain long-term stability.

How can researchers effectively measure ATP5J2 incorporation into functional ATP synthase complexes?

Assessing ATP5J2 incorporation into functional ATP synthase complexes requires multifaceted approaches:

• Blue Native PAGE analysis:

  • Preserves native protein-protein interactions

  • Allows visualization of intact ATP synthase complexes

  • Can be combined with western blotting using ATP5J2-specific antibodies

  • Enables comparison of complex assembly between experimental conditions

• Sucrose gradient ultracentrifugation:

  • Separates protein complexes based on size and density

  • Fractions can be analyzed by western blotting to track ATP5J2 distribution

  • Provides quantitative assessment of incorporation efficiency

• Immunoprecipitation-based approaches:

  • Pull-down with antibodies against other ATP synthase subunits

  • Western blot analysis for co-precipitation of ATP5J2

  • Can include crosslinking to stabilize transient interactions

• Functional reconstitution assays:

  • Measure ATP synthesis activity in isolated mitochondria or reconstituted liposomes

  • Compare activity with and without ATP5J2 incorporation

  • Assess proton translocation using pH-sensitive fluorescent dyes

• Structural biology techniques:

  • Cryo-electron microscopy to visualize ATP5J2 within the complex

  • Hydrogen-deuterium exchange mass spectrometry to monitor structural changes

  • Crosslinking mass spectrometry to identify physical proximity to other subunits

• Fluorescence-based approaches:

  • FRET pairs between ATP5J2 and other complex components

  • Fluorescence correlation spectroscopy to assess complex formation

  • Single-particle tracking to monitor complex dynamics

Decreased expression of the whole ATP synthase complex has been observed in hippocampal tissue of Alzheimer's disease patients through Blue Native PAGE analysis , demonstrating the utility of this approach for assessing complex integrity in disease states.

What techniques are most reliable for assessing ATP5J2 contribution to mitochondrial function in primary neurons?

Evaluating ATP5J2's contribution to mitochondrial function in primary neurons requires sensitive and specific methodologies:

• Genetic manipulation approaches:

  • AAV-mediated gene delivery for overexpression or shRNA knockdown

  • CRISPR-Cas9 for knockout or precise mutation introduction

  • Inducible expression systems to control timing of manipulation

• Live-cell imaging techniques:

  • Real-time ATP monitoring using genetically encoded sensors (e.g., ATeam)

  • Mitochondrial membrane potential using TMRM or JC-1 dyes

  • Mitochondrial calcium dynamics with mt-GCaMP

  • Mitochondrial morphology with mito-DsRed or mito-GFP

• Bioenergetic analysis:

  • Microplate-based respirometry for oxygen consumption rate (OCR)

  • Extracellular acidification rate (ECAR) measurement

  • ATP production rate calculation from OCR and ECAR data

  • Substrate-specific respiration with selective inhibitors

• Mitochondrial isolation and biochemical analysis:

  • Enzymatic activity assays for ATP synthase

  • Blue Native PAGE for complex assembly assessment

  • Proteomic analysis of purified mitochondria

  • Super-resolution microscopy of isolated mitochondria

• Functional readouts in intact neurons:

  • Electrophysiological recording of neuronal activity

  • Synaptic vesicle recycling with FM dyes or pHluorin

  • Calcium imaging during neuronal activation

  • Assessment of neurite outgrowth and spine morphology

Research has shown that decreased ATP synthase activity correlates with reduced extracellular ATP levels, disrupting synaptic plasticity and long-term potentiation . These techniques allow researchers to establish causal relationships between ATP5J2 function and neuronal physiology, particularly in the context of neurodegenerative disease models.

What protocols best measure the impact of ATP5J2 mutations on mitochondrial bioenergetics?

Comprehensive assessment of ATP5J2 mutations requires integrated bioenergetic protocols:

• Site-directed mutagenesis workflow:

  • Design mutations based on evolutionary conservation or disease-associated variants

  • Generate stable cell lines expressing wild-type or mutant ATP5J2

  • Verify expression levels and subcellular localization before functional analysis

• High-resolution respirometry protocols:

  • Substrate-uncoupler-inhibitor titration (SUIT) protocols to assess:

    • OXPHOS capacity (P)

    • Electron transfer system capacity (E)

    • Leak respiration (L)

    • Respiratory control ratio (P/L)

  • Substrate-specific protocols to evaluate complex-specific defects

  • Coupling control protocols to assess efficiency of ATP production

• ATP synthesis measurement approaches:

  • Luciferase-based ATP detection in isolated mitochondria

  • 31P-NMR spectroscopy for non-invasive ATP measurement

  • HPLC-based nucleotide quantification

  • Real-time ATP monitoring with genetically encoded sensors

• Proton motive force assessment:

  • Membrane potential measurement using potentiometric dyes

  • ΔpH measurement with pH-sensitive probes

  • Combined Δψ and ΔpH for complete pmf determination

• Mitochondrial structural analysis:

  • Electron microscopy for cristae morphology assessment

  • Super-resolution microscopy for ATP synthase organization

  • Tomographic analysis of ATP synthase dimer rows

• Complementation testing:

  • Rescue experiments in ATP5J2 knockout backgrounds

  • Competition assays between wild-type and mutant forms

  • Dominant-negative effect assessment in heterozygous models

These protocols have revealed that oxidative modifications to ATP synthase subunits can result in up to 35% decrease in enzymatic activity , while dysregulation of ATP synthase can impair oxidative phosphorylation and trigger compensatory responses in disease models .

How can researchers effectively study ATP5J2 interactions with mitochondrial dynamics proteins in neuroinflammatory conditions?

Investigating ATP5J2 interactions with mitochondrial dynamics proteins requires specialized approaches:

• Co-immunoprecipitation strategies:

  • Bidirectional pull-downs with ATP5J2 and dynamics proteins (Drp1, Fis1)

  • Use of membrane-permeable crosslinkers to stabilize transient interactions

  • Sequential immunoprecipitation to isolate specific subcomplexes

  • Mass spectrometry analysis of immunoprecipitated complexes

• Proximity labeling methods:

  • BioID fusion to ATP5J2 for identifying proximal proteins

  • APEX2-based labeling for temporal resolution of interaction changes

  • Split-BioID for detecting specific protein-protein interactions

  • Quantitative proteomics to measure interaction dynamics during inflammation

• Live-cell imaging protocols:

  • Dual-color tracking of fluorescently tagged proteins

  • FRET-based interaction sensors for real-time monitoring

  • Photoactivation or photoconversion for tracking subpopulations

  • High-content imaging for population-level interaction analysis

• Reconstitution systems:

  • Liposome-based reconstruction of protein interactions

  • GUV (Giant Unilamellar Vesicle) systems with purified components

  • Cell-free expression systems for direct interaction assessment

• Functional correlation analyses:

  • Mitochondrial morphology quantification following genetic manipulation

  • Fission/fusion event frequency measurement

  • Correlation of ATP production with dynamics protein activity

  • Assessment of respiratory chain activity during altered dynamics

Research has demonstrated that ATP5J knockdown reversed the upregulation of mitochondrial fission proteins Drp1 and Fis1 in microglial cells following oxyhemoglobin exposure . This intervention reduced excessive mitochondrial division, prevented mitochondrial permeability transition pore opening, decreased reactive oxygen species production, and restored normal mitochondrial ridge morphology .

These findings suggest a regulatory relationship between ATP synthase components and mitochondrial dynamics proteins that can be therapeutically targeted in neuroinflammatory conditions.