Recombinant Pongo abelii ATP synthase lipid-binding protein, mitochondrial (ATP5G3)

What is ATP synthase lipid-binding protein, mitochondrial (ATP5G3)?

ATP5G3 (also known as ATP5MC3) is a nuclear-encoded mitochondrial protein that functions as a component of the F0 complex in ATP synthase. It is specifically a lipid-binding protein that forms part of the membrane-embedded proton channel of the ATP synthase complex. In Pongo abelii (Sumatran orangutan), the mature form of the protein spans amino acids 68-142 and has a distinct amino acid sequence: DIDTAAKFIGAGAATVGVAGSGAGIGTVFGSLIIGYARNPSLKQQLFSYAILGFALSEAMGLFCLMVAFLILFAM . ATP5G3 is one of three genes (along with ATP5G1 and ATP5G2) that encode the c subunit of the mitochondrial ATP synthase, contributing to the formation of the c-ring structure critical for ATP production via oxidative phosphorylation.

How does ATP5G3 compare structurally and functionally to ATP5G1 and ATP5G2?

While ATP5G1, ATP5G2, and ATP5G3 encode proteins that are identical in their mature form, they differ in their mitochondrial targeting sequences. Expression studies have shown that in both mouse and Arctic ground squirrel (AGS) neural progenitor cells (NPCs), the expression of Atp5g3 or Atp5g2 is greater than that of Atp5g1 . This differential expression pattern suggests potential tissue-specific or condition-specific regulation of these isoforms. Despite these differences in expression levels, the relative abundance of the mature ATP5G protein or oligomycin sensitivity of complex V activity appears similar across different cell types . This indicates functional redundancy at the protein level while maintaining regulatory distinction at the transcript level.

What evolutionary significance does ATP5G3 hold across species?

ATP5G proteins are highly conserved across mammalian species, reflecting their essential role in cellular energy production. Comparative genomic analyses have identified specific amino acid substitutions in certain species like the Arctic ground squirrel that may contribute to metabolic resilience under stress conditions . Studies of related ATP synthase subunits across species including Pongo abelii, Homo sapiens, Equus caballus, Bos taurus, and Oryctolagus have revealed both conserved regions essential for function and variable regions that may reflect species-specific adaptations . These evolutionary patterns provide valuable insights into structure-function relationships and potential adaptive mechanisms in response to different metabolic demands.

What expression systems are available for producing recombinant Pongo abelii ATP5G3?

Recombinant Pongo abelii ATP5G3 can be produced in multiple expression systems, each offering distinct advantages for different experimental applications:

The E. coli system is particularly well-documented, with the recombinant protein consisting of the mature protein sequence (amino acids 68-142) fused to an N-terminal His tag . This diversity of expression systems provides researchers flexibility based on their specific experimental needs and downstream applications.

What protein tags and modifications are available for recombinant ATP5G3?

Several tagging options are available for recombinant Pongo abelii ATP5G3:

• His-tag: The most common tag, facilitating purification via metal affinity chromatography. His-tagged ATP5G3 is typically expressed in E. coli with the tag positioned at the N-terminus .

• Avi-tag Biotinylated: This specialized tag involves in vivo biotinylation using E. coli biotin ligase (BirA), which specifically attaches biotin to the 15 amino acid AviTag peptide. The biotin-AviTag conjugation occurs through an amide linkage between biotin and a specific lysine residue in the AviTag . This biotinylated form (product CSB-EP716241PYX1-B) is particularly useful for applications requiring highly specific protein-protein interaction studies or for immobilization onto streptavidin surfaces.

• GST-tag: While not specifically mentioned for Pongo abelii ATP5G3, related proteins in the ATP5G family are available with GST tags, which can improve solubility and provide an alternative purification method .

The choice of tag depends on the research application, with considerations for protein function, purification strategy, and detection methods.

What are the optimal storage and reconstitution conditions for recombinant ATP5G3?

For optimal stability and activity, recombinant Pongo abelii ATP5G3 should be handled according to the following guidelines:

Storage conditions:

• Upon receipt, store at -20°C/-80°C

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• For working aliquots, store at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Aliquot for long-term storage at -20°C/-80°C

Buffer composition:

• The protein is typically provided in a Tris/PBS-based buffer containing 6% Trehalose, pH 8.0

Adherence to these storage and handling recommendations is critical for maintaining protein integrity and biological activity for experimental applications.

How can functional activity of recombinant ATP5G3 be assessed in vitro?

The functional assessment of recombinant ATP5G3 requires evaluating its incorporation into the ATP synthase complex and subsequent effects on ATP synthesis activity. Several methodological approaches can be employed:

• Oligomycin sensitivity assay: ATP5G proteins form part of the oligomycin-binding site in ATP synthase. Researchers can measure complex V activity and its sensitivity to oligomycin inhibition in reconstituted systems or in cells expressing recombinant ATP5G3 . Differences in sensitivity profiles can indicate successful incorporation and functional impact of the recombinant protein.

• Clear-native gel electrophoresis: This technique allows visualization of intact ATP synthase complexes and assessment of ATP5G3 incorporation. It can reveal alterations in ATP synthase dimer formation, which has been observed in studies of related ATP5G proteins .

• Mitochondrial respiration analysis: Oxygen consumption rate measurements using platforms like Seahorse XF Analyzer can assess the impact of ATP5G3 variants on mitochondrial respiration parameters including basal respiration, ATP production, maximal respiration, and spare respiratory capacity .

• ATP synthesis rate measurement: Direct quantification of ATP production rates in isolated mitochondria or reconstituted systems containing recombinant ATP5G3 provides the most direct assessment of functional activity.

These complementary approaches provide a comprehensive evaluation of both incorporation and functional consequences of recombinant ATP5G3.

What experimental models are suitable for studying ATP5G3 function?

Several experimental models have proven valuable for investigating ATP5G3 function:

• Reconstituted liposome systems: Purified recombinant ATP5G3 can be incorporated into artificial membrane systems along with other ATP synthase subunits to study its specific role in complex assembly and proton translocation.

• Cell lines with genetic manipulation:

  • HEK293T cells have been successfully used for lentiviral transduction of ATP synthase components

  • Neural progenitor cells (NPCs) provide a model for studying metabolic resilience

  • CRISPR/Cas9 gene editing systems allow precise manipulation of endogenous ATP5G genes to study their function

• Mitochondrial isolation and subfractionation: Isolated mitochondria from cells expressing recombinant ATP5G3 can be subfractioned to study protein localization and complex assembly.

• Stress challenge models: Exposing cells expressing recombinant ATP5G3 to stressors such as hypoxia, hypothermia, or complex I inhibitors (e.g., rotenone) provides insights into its role in stress resilience .

Selection of the appropriate model should be guided by the specific research question, considering factors such as technical feasibility, physiological relevance, and available resources.

How can CRISPR gene editing be used to study ATP5G3 function?

CRISPR gene editing represents a powerful approach for studying ATP5G3 function through precise genetic manipulation. Based on successful applications with related ATP synthase components:

• Base editing technology: Adenine base editors like ABEmax can introduce specific nucleotide substitutions without double-strand breaks. This approach has been successfully used to create point mutations in the related ATP5G1 gene (e.g., L32P substitution) .

• Knockout generation: CRISPR/Cas9 can generate ATP5G3 knockout cell lines to study loss-of-function effects, though compensatory expression of other ATP5G isoforms may complicate interpretation.

• Knock-in strategies: Specific tags or reporter genes can be knocked into the endogenous ATP5G3 locus to track protein expression, localization, and dynamics without disrupting native regulatory elements.

• Isogenic cell line creation: Creating isogenic cell lines differing only in specific ATP5G3 variants enables controlled comparison of functional effects. Researchers have successfully isolated clonal cell lines harboring desired knock-in mutations and control lines that underwent editing but remained homozygous for wild-type alleles .

The selection of specific CRISPR approach should consider factors such as editing efficiency, potential off-target effects, and the specific experimental question being addressed.

How does ATP5G3 contribute to mitochondrial structure and dynamics?

ATP5G3, as a component of ATP synthase, plays significant roles in mitochondrial structure beyond its direct function in ATP production:

• ATP synthase dimerization: ATP5G proteins are integral to the formation of ATP synthase dimers, which create the characteristic curvature of the inner mitochondrial membrane at cristae junctions. Alterations in ATP5G proteins can affect the ratio of ATP synthase dimers to monomers, as observed in studies using native gel electrophoresis .

• Cristae morphology: The arrangement of ATP synthase dimers influences cristae structure, which in turn affects respiratory efficiency and mitochondrial function under stress conditions.

• Mitochondrial permeability transition pore (MPTP): The suprastructural organization of ATP synthase is critical to mitochondrial morphology and MPTP formation. While the exact relationship between ATP5G proteins and MPTP is controversial, studies demonstrate improved bioenergetic responses and cell survival with ATP synthase dimerization .

• Mitochondrial dynamics responses: Variants of ATP5G proteins can alter mitochondrial dynamics in response to mitochondrial uncouplers like FCCP, suggesting a role in adaptive responses to bioenergetic stress .

These structural contributions highlight the multifaceted role of ATP5G3 beyond its catalytic function in ATP synthesis.

What is known about the relationship between ATP5G3 and cellular stress resilience?

Studies of ATP synthase components, particularly in species adapted to metabolic stress, provide insights into potential roles of ATP5G3 in stress resilience:

• Hypoxia tolerance: Cells with certain ATP5G variants show differential survival under hypoxic conditions. While specific data for ATP5G3 is limited, the related ATP5G1 with an L32P variant in Arctic ground squirrels demonstrates enhanced survival under hypoxia .

• Temperature stress resistance: ATP synthase components play crucial roles in adaptations to temperature extremes. Arctic ground squirrel cells with the natural ATP5G1 variant show improved survival under hypothermic conditions compared to those with the L32P substitution .

• Metabolic toxin resistance: The expression of certain ATP5G variants confers protection against mitochondrial toxins such as rotenone (a complex I inhibitor) .

• Spare respiratory capacity: ATP5G variants can significantly affect 'spare respiratory capacity,' a measure of the cell's ability to respond to increased energy demands under stress. This parameter is markedly reduced in cells with the ATP5G1L32P variant compared to those with the native Arctic ground squirrel variant .

These findings suggest that ATP5G3, as part of the same protein family, may similarly contribute to cellular resilience against metabolic stressors, though specific studies on ATP5G3 variants are needed to confirm this hypothesis.

How do post-translational modifications affect ATP5G3 function?

ATP5G3, like other mitochondrial proteins, undergoes several post-translational modifications that influence its localization, stability, and function:

• Proteolytic processing: ATP5G3 is synthesized as a precursor with an N-terminal mitochondrial targeting sequence that is cleaved during import by mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). This processing is essential for proper incorporation into the ATP synthase complex .

• TMEM70-mediated stabilization: Processing of ATP5G proteins and their incorporation into oligomeric c-rings and Complex V-Fo involve cleavage by MPP and stabilization by TMEM70, a protein critical for ATP synthase biogenesis .

• N-terminal peptide functions: Intriguingly, some studies suggest that the cleaved ATP5G N-terminal mitochondrial targeting sequence may have functions distinct from the mature C-terminal protein, potentially modulating mitochondrial function downstream of Complex IV .

• Regulatory modifications: While not specifically documented for Pongo abelii ATP5G3, ATP synthase components can undergo regulatory modifications such as phosphorylation, acetylation, or oxidative modifications that affect complex assembly or activity.

Understanding these modifications is crucial for interpreting experimental results with recombinant proteins that may lack native processing or modification patterns.

What controls should be included in experiments using recombinant ATP5G3?

To ensure reliable and interpretable results when working with recombinant Pongo abelii ATP5G3, researchers should incorporate several critical controls:

• Expression vector-only control: Cells expressing the empty vector used for ATP5G3 expression help distinguish effects of the recombinant protein from those of the expression system itself.

• Inactive ATP5G3 variant: A non-functional ATP5G3 variant (e.g., with mutations in conserved residues) can help distinguish specific functional effects from non-specific effects of protein overexpression.

• Wild-type comparisons: When studying ATP5G3 variants, include the wild-type protein expressed under identical conditions. In gene editing studies, include cells that underwent the editing process but retained the wild-type sequence .

• Related isoform controls: Given the functional overlap between ATP5G isoforms, including ATP5G1 or ATP5G2 controls can help identify isoform-specific effects.

• Oligomycin treatment: As a specific inhibitor of ATP synthase, oligomycin provides a positive control for experiments assessing ATP synthase function .

• Mitochondrial function controls: Include established mitochondrial function modifiers (e.g., FCCP for uncoupling, rotenone for complex I inhibition) as reference points for interpreting ATP5G3 effects .

What are the common technical challenges in studying recombinant ATP5G3?

Researchers working with recombinant Pongo abelii ATP5G3 should be aware of several technical challenges:

• Protein solubility and stability: As a lipid-binding membrane protein, ATP5G3 may present solubility challenges. Maintaining stability requires careful buffer optimization and potentially the inclusion of lipids or detergents.

• Functional incorporation: Ensuring proper incorporation of recombinant ATP5G3 into endogenous ATP synthase complexes requires verification through techniques such as blue native PAGE or immunoprecipitation.

• Redundancy with other isoforms: The functional redundancy between ATP5G1, ATP5G2, and ATP5G3 can mask phenotypes in overexpression or knockdown studies. Researchers should consider compensatory expression changes in related isoforms .

• Species-specific differences: When studying Pongo abelii ATP5G3 in heterologous systems, consider potential incompatibilities with the host cell's ATP synthase components.

• Mitochondrial import efficiency: Recombinant ATP5G3 requires efficient mitochondrial import and processing for function. Verification of proper localization is essential for interpreting functional studies.

• Storage and handling stability: Recombinant ATP5G3 requires careful handling to maintain activity, including avoiding repeated freeze-thaw cycles and maintaining appropriate buffer conditions .

Addressing these challenges requires careful experimental design and appropriate validation steps to ensure meaningful results.

How can researchers quantify ATP5G3 expression and incorporation?

Accurate quantification of ATP5G3 expression and its incorporation into functional complexes is critical for experimental interpretation. Several complementary approaches are recommended:

• Transcript quantification:

  • Quantitative RT-PCR with isoform-specific primers allows precise measurement of ATP5G3 mRNA levels relative to other ATP5G isoforms

  • Care must be taken to design primers that distinguish between highly similar ATP5G transcripts

• Protein detection:

  • Western blotting with antibodies against the mature ATP5G protein or epitope tags

  • Clear-native or blue-native gel electrophoresis to assess incorporation into ATP synthase complexes

  • Immunocytochemistry to confirm mitochondrial localization

• Functional incorporation:

  • Oligomycin sensitivity assays to confirm functional integration into ATP synthase

  • ATP synthesis rate measurements in isolated mitochondria

  • Respiratory capacity measurements using platforms like Seahorse XF Analyzer

• Complex assembly analysis:

  • Separation of ATP synthase dimers versus monomers by native gel electrophoresis provides insights into structural incorporation and supramolecular organization

These multi-level analyses provide a comprehensive picture of ATP5G3 expression, localization, and functional incorporation, essential for accurate experimental interpretation.

What are the most promising future research applications for recombinant ATP5G3?

Several promising research directions for recombinant Pongo abelii ATP5G3 include:

• Comparative studies across species: Investigating species-specific variations in ATP5G3 structure and function, particularly in species adapted to metabolic stress, may reveal novel mechanisms of mitochondrial adaptation .

• Therapeutic applications: Understanding the cytoprotective mechanisms of ATP5G variants could lead to novel strategies for treating human ischemic disorders, including stroke and heart attack .

• Structural biology: High-resolution structural studies of ATP5G3 within the ATP synthase complex could provide insights into its precise role in proton translocation and energy coupling.

• Mitochondrial disease models: Recombinant ATP5G3 could be used to model and potentially correct defects in mitochondrial diseases associated with ATP synthase dysfunction.

• CRISPR-based therapeutic development: The successful application of base editing to modify related ATP5G genes suggests potential for precision genetic therapies targeting ATP synthase components .

These research directions highlight the broad potential impact of ATP5G3 research beyond basic mitochondrial biology to applications in human health and disease treatment.

What key knowledge gaps remain in ATP5G3 research?

Despite advances in understanding ATP synthase components, several critical knowledge gaps remain regarding ATP5G3:

• Isoform-specific functions: The precise functional distinctions between ATP5G1, ATP5G2, and ATP5G3 remain poorly understood, particularly regarding tissue-specific expression patterns and stress responses.

• Post-translational regulation: The regulatory mechanisms controlling ATP5G3 processing, stability, and incorporation into ATP synthase complexes require further elucidation.

• Evolutionary adaptations: The evolutionary pressures driving variations in ATP5G3 across species and their functional significance remain to be fully characterized.

• Interaction with other mitochondrial processes: The relationship between ATP5G3 and other mitochondrial functions such as calcium handling, reactive oxygen species production, and mitochondrial dynamics needs further investigation.

• Therapeutic potential: The potential for targeting ATP5G3 or leveraging protective variants for treating ischemic or mitochondrial disorders remains largely unexplored.