Recombinant Mouse ATP synthase lipid-binding protein, mitochondrial (Atp5g2)

What is ATP synthase lipid-binding protein (ATP5G2) and what is its function?

ATP5G2 is one of three nuclear-encoded genes (along with ATP5G1 and ATP5G3) that encode the c-subunit of the mitochondrial ATP synthase complex. These three genes produce identical mature proteins but differ in their mitochondrial import sequences . The c-subunit forms the c-ring in the F₀ portion of ATP synthase, which is embedded in the inner mitochondrial membrane and functions as a proton channel. This channel allows hydrogen ions to move across the membrane, driving the conformational changes in the F₁ portion that result in ATP production .

Beyond its canonical role in ATP synthesis, the c-subunit has been implicated in the formation of the mitochondrial permeability transition pore (mPT), which can trigger cell death under certain conditions . The c-ring appears to form a large multi-conductance, voltage-gated ion channel that can be inhibited by the F₁ portion of ATP synthase, particularly through interactions with central stalk subunits (gamma, delta, and epsilon) .

How do the three ATP5G isoforms differ in expression and regulation?

• In neural progenitor cells (NPCs) from Arctic ground squirrels, the relative abundance of the ATP5G1 isoform is elevated nearly twofold compared to mouse NPCs .

• In clear cell renal cell carcinoma (ccRCC), all three ATP5G isoforms show significant downregulation compared to normal renal tissue, with fold changes as follows:

These data indicate that ATP5G1 shows the most dramatic downregulation in ccRCC .

What role does ATP5G2 play in the mitochondrial permeability transition?

The c-subunit of ATP synthase, which ATP5G2 encodes, has been implicated in forming the leak channel involved in mitochondrial permeability transition (mPT), a process activated during excitotoxic ischemic insult . Studies using purified human c-ring have demonstrated that it forms a large multi-conductance, voltage-gated ion channel with the following characteristics:

• The channel can be inhibited by the addition of ATP synthase F₁ portion

• This inhibition requires the presence of central stalk subunits (gamma, delta, and epsilon)

• The addition of only the α₃β₃ complex does not inhibit channel activity

• Denatured F₁ (boiled) does not inhibit the channel activity

How can CRISPR/Cas9-based approaches be used to study ATP5G2 function?

CRISPR/Cas9 has proven valuable for studying ATP5G function through gene disruption and base editing approaches:

• Gene disruption: Researchers have successfully disrupted all three ATP5G genes (ATP5G1, ATP5G2, and ATP5G3) in human HAP1 cells using pairs of guide RNAs (gRNAs) targeting specific exons in each gene. For ATP5G2, gRNAs targeted exon IV, resulting in a 97-base deletion that changed the reading frame of the coding region in the import sequence and led to protein truncation .

• Base editing: CRISPR/Cas9 base editing has been used to introduce specific amino acid substitutions in ATP5G genes. While the search results focus more on ATP5G1 variants (L32P), similar approaches could be applied to study ATP5G2 variants .

When designing CRISPR/Cas9 experiments for ATP5G2, researchers should consider:

• Targeting unique regions in exon IV to avoid off-target effects on other ATP5G isoforms

• Confirming knockout/modification success through Western blotting and DNA sequencing

• Assessing functional consequences through mitochondrial respiration, ATP synthesis, and cell viability assays

How does ATP5G2 expression change in pathological conditions?

ATP5G2 expression appears to be dysregulated in several pathological conditions, particularly in cancer. In clear cell renal cell carcinoma (ccRCC), systematic analysis revealed that ATP5G2 is significantly downregulated compared to normal renal tissue:

• Initial screening showed a -2.69-fold change

• Validation with qPCR confirmed a -2.11-fold change

This downregulation is part of a broader pattern affecting 23 out of 29 subunits of ATP synthase in ccRCC, suggesting a general dysregulation of mitochondrial energy metabolism in this cancer type.

Changes in ATP5G2 relative to other ATP5G isoforms have also been observed in neurological conditions. In Fragile X syndrome mouse models (Fmr1−/y), although the total level of c-subunit protein is elevated, this appears to be due to dysregulation of assembly rather than direct FMRP-regulated translation of c-subunit mRNAs . RT-PCR studies identified ATP5G2 as the dominant c-subunit gene in this context, but FMRP binding was not detected for any of the three c-subunit gene products .

How to express and purify recombinant ATP5G2 protein?

Recombinant ATP5G2 can be expressed and purified using several approaches, with bacterial expression systems being common for structural and functional studies:

• Expression system: E. coli is a widely used system for ATP5G2 expression. For example, recombinant full-length mouse ATP5G2 protein (residues 72-146) can be expressed with an N-terminal His tag in E. coli .

• Purification approach:

  • Affinity chromatography using the His tag for initial purification

  • Size-exclusion chromatography to obtain homogeneous protein

  • Ion-exchange chromatography for further purification if needed

• Reconstitution: For functional studies, purified c-subunit can be reconstituted into liposomes or planar lipid bilayers to study its channel properties .

When designing expression constructs, researchers should consider:

• Using only the mature protein sequence (without the mitochondrial targeting sequence) for bacterial expression

• Including appropriate affinity tags (His, GST, etc.) to facilitate purification

• Optimizing codon usage for the expression system

What techniques can be used to study ATP5G2 channel activity?

Several biophysical techniques can be employed to study the channel activity of ATP5G2:

• Planar lipid bilayer experiments: This approach has been successfully used to characterize the channel properties of purified c-ring. The technique involves:

  • Reconstituting purified c-ring into planar lipid bilayers

  • Recording channel activity under various conditions

  • Testing the effects of potential inhibitors or activators

• ACMA assay: The 9-amino-6-chloro-2-methoxyacridine (ACMA) assay measures ATP synthase enzymatic rate by assessing proton translocation. This assay uses:

  • Submitochondrial vesicles (SMVs) relatively enriched in ATP synthase

  • A membrane-impermeant H⁺ indicator (ACMA)

  • ATP addition to initiate proton sequestration

• Patch-clamp techniques: These can be used to study channel activity in mitochondrial membranes or reconstituted systems.

How to assess the impact of ATP5G2 on mitochondrial function?

Several approaches can be used to evaluate the impact of ATP5G2 on mitochondrial function:

• Respirometry: Oxygen consumption measurements can assess:

  • Basal respiration

  • ATP-linked respiration

  • Maximal respiration

  • Spare respiratory capacity

• Mitochondrial morphology analysis: Confocal microscopy with appropriate mitochondrial staining can assess:

  • Mitochondrial fragmentation

  • Branch length

  • Network connectivity

  • Response to uncouplers like FCCP

• ATP synthesis assays: Direct measurement of ATP production rates.

• Cell viability under stress conditions: Assessing survival under:

  • Hypoxia

  • Hypothermia

  • Rotenone exposure

  • Other metabolic stressors

For example, studies of ATP5G1 variants have shown that specific amino acid substitutions can affect spare respiratory capacity and mitochondrial fragmentation in response to stressors . Similar approaches could be applied to study ATP5G2 variants or manipulations.

How to analyze ATP5G2 incorporation into ATP synthase complexes?

Several techniques can be used to assess the incorporation of ATP5G2 into ATP synthase complexes:

• Native-PAGE electrophoresis: This technique allows visualization of assembled ATP synthase complexes (monomers and dimers) as well as free c-subunit. Western blotting following Native-PAGE can quantify:

  • Levels of fully assembled ATP synthase (monomer plus dimer)

  • Levels of free c-subunit

• Immunoprecipitation: Can be used to study interactions between ATP5G2 and other ATP synthase subunits.

• Immunohistochemistry: Tissue microarrays can be used to assess the expression and localization of ATP5G2 in different tissues or cell types. The procedure involves:

  • Deparaffinizing and rehydrating sections

  • Antigen retrieval using appropriate buffers (e.g., Tris/EDTA buffer pH 9.0 for ATP5G1/G2/G3)

  • Blocking endogenous peroxidase activity

  • Primary antibody incubation (anti-ATP5G1/G2/G3, dilution 1:100)

  • Signal detection with labeled polymer and counterstaining

What are common challenges in studying ATP5G2 and how to overcome them?

Several challenges can arise when studying ATP5G2, including:

• Antibody specificity: Since the mature proteins encoded by ATP5G1, ATP5G2, and ATP5G3 are identical, antibodies typically recognize all three isoforms. To study isoform-specific effects:

  • Use gene-specific nucleic acid probes for expression analysis

  • Employ genetic approaches (e.g., CRISPR) to manipulate specific isoforms

  • Use epitope tags in recombinant expression systems

• Functional redundancy: The three ATP5G genes produce identical mature proteins, creating functional redundancy. To address this:

  • Consider simultaneous manipulation of all three genes

  • Focus on unique regulatory mechanisms or expression patterns

  • Study tissues where one isoform predominates

• Membrane protein purification: As a hydrophobic membrane protein, ATP5G2 can be challenging to purify in a functional state. Optimization strategies include:

  • Using appropriate detergents for solubilization

  • Including lipids during purification

  • Employing gentle purification conditions

How to distinguish the specific contribution of ATP5G2 from other ATP5G isoforms?

Distinguishing the specific contribution of ATP5G2 from other ATP5G isoforms requires specialized approaches:

• Isoform-specific knockdown/knockout: Using siRNA or CRISPR/Cas9 to target unique regions in ATP5G2 mRNA or gene, respectively.

• Quantitative RT-PCR: To assess the relative expression levels of each isoform in different tissues or experimental conditions. For example, RT-PCR has identified ATP5G2 as the dominant c-subunit gene in some neural tissues .

• Rescue experiments: Knocking out all ATP5G genes and then reintroducing individual isoforms to assess specific contributions.

• Promoter analysis: Studying the unique regulatory elements controlling ATP5G2 expression.

What are emerging areas of ATP5G2 research?

Several promising research directions for ATP5G2 include:

• Role in disease mechanisms: Further exploration of ATP5G2 dysregulation in cancer, neurological disorders, and other diseases. The significant downregulation observed in clear cell renal cell carcinoma suggests potential roles in cancer metabolism .

• Contribution to mitochondrial permeability transition: While studies suggest that mPT can occur in the absence of c-subunit , the regulatory role of ATP5G2 in this process remains an important area for investigation.

• Isoform-specific regulation: Understanding why three genes encode identical proteins but are regulated differently could reveal important insights into mitochondrial biology.

• Therapeutic targeting: Exploring whether ATP5G2 could be a therapeutic target for conditions involving mitochondrial dysfunction.

• Structural biology: Determining high-resolution structures of the c-ring in different functional states to better understand its dual roles in ATP synthesis and channel formation.