Recombinant Bovine ATP synthase lipid-binding protein, mitochondrial (ATP5G2)

What is ATP5G2 and what is its role in ATP synthase complex?

ATP5G2 is one of three nuclear-encoded isoforms (along with ATP5G1 and ATP5G3) that make up the C-subunit of mitochondrial ATP synthase. These C-subunit proteins are critical components of the ATP synthase complex, primarily located in the inner mitochondrial membrane. While the three C-subunit proteins have identical sequences in their mature form after processing, they are encoded by different genes and cannot functionally substitute for one another . All three isoforms are required to constitute a fully functional C-subunit of ATP synthase, which is essential for oxidative phosphorylation and ATP production in cells .

How does bovine ATP5G2 differ from human and other mammalian orthologs?

Bovine ATP5G2 shares high sequence homology with human and other mammalian orthologs, particularly in the C-terminal membrane-spanning segment which is largely invariant across species. The primary differences between species are observed in the N-terminal region, which contains the mitochondrial targeting sequence. This region undergoes cleavage during protein maturation but can also directly modulate mitochondrial function through mechanisms that are not fully understood . Comparative sequence analysis should be performed when designing experiments that involve cross-species antibody recognition or when considering the functional implications of species-specific differences.

What are the best approaches for expressing recombinant bovine ATP5G2 in heterologous systems?

For successful expression of recombinant bovine ATP5G2, researchers should consider:

• Expression vector selection: Use vectors containing strong promoters suitable for the host system (bacterial, yeast, insect, or mammalian cells).

• Codon optimization: Optimize codons for the host organism to improve expression efficiency.

• Tag placement: For detection and purification, add tags to the N-terminus rather than C-terminus to avoid interfering with membrane insertion.

• Signal sequence consideration: Include the native mitochondrial targeting sequence if studying processing or targeting, or remove it for direct expression of the mature protein.

• Host selection: Mammalian expression systems often provide the most physiologically relevant post-translational modifications and processing.

In published studies examining ATP5G proteins, researchers have successfully used expression plasmids that include fluorescent tags such as paGFP to enable visualization of protein trafficking . When designing such constructs, verification of correct assembly into the ATP synthase complex can be confirmed using co-immunoprecipitation with antibodies against other ATP synthase subunits, as demonstrated for ATP5B-paGFP fusion proteins .

What methods are most effective for purifying recombinant bovine ATP5G2?

Purification of recombinant bovine ATP5G2 requires special consideration due to its hydrophobic nature and membrane association. The most effective purification strategy includes:

• Membrane fraction isolation: Differential centrifugation to isolate membrane fractions.

• Detergent solubilization: Use mild detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin to solubilize without denaturing.

• Affinity chromatography: Using His, FLAG, or other affinity tags for initial purification.

• Size exclusion chromatography: For further purification and to assess oligomeric state.

• Verification: Western blotting with ATP5G-specific antibodies and mass spectrometry to confirm identity and purity.

When analyzing purified ATP5G proteins, it's important to note that standard SDS-PAGE western blot techniques have been successfully used to detect these proteins in mitochondrial extracts, with citrate synthase (CS) often serving as a loading control for mitochondrial proteins .

How can researchers effectively detect and localize ATP5G2 in bovine cells and tissues?

For detection and localization of ATP5G2 in bovine cells and tissues, researchers can employ:

• Immunofluorescence microscopy: Using validated antibodies specific to ATP5G2 or to the conserved regions of ATP5G proteins. This can be combined with mitochondrial markers like MitoTracker or antibodies against TOM20 to confirm mitochondrial localization .

• Flow cytometry: For quantitative assessment of protein expression levels in cell populations. This has been used effectively to measure ectopic ATP synthase expression on cell surfaces .

• Cell fractionation: For biochemical verification of subcellular localization, separating mitochondrial, cytosolic, and plasma membrane fractions.

• Super-resolution microscopy: For detailed spatial analysis of ATP5G2 localization within mitochondria or at the plasma membrane .

• Live-cell imaging: Using fluorescently tagged ATP5G2 to track movement and dynamics in living cells .

It's important to note that some ATP synthase subunits, including ATP5G proteins, can be found both in mitochondria and ectopically on the plasma membrane in certain cell types . Therefore, proper controls and permeabilization protocols are essential when distinguishing between these populations.

How does ATP5G2 contribute to the assembly and stability of ATP synthase compared to ATP5G1 and ATP5G3?

While all three ATP5G isoforms produce identical mature proteins after processing, they cannot functionally substitute for one another . This suggests distinct roles in assembly or regulation of the ATP synthase complex:

Research has shown that the full complement of all three isoforms is required for optimal ATP synthase function . When investigating the specific contribution of ATP5G2, researchers should consider CRISPR/Cas9-based approaches for selective knockout or modification of this isoform while maintaining expression of ATP5G1 and ATP5G3.

What role does ATP5G2 play in ectopic ATP synthase localization to the plasma membrane?

Recent research has demonstrated that ATP synthase complexes can be found on the plasma membrane (eATP synthase) of various cell types, including cancer cells . The trafficking mechanism involves:

• Assembly in mitochondria: The complete ATP synthase complex is first assembled in mitochondria .

• Microtubule-dependent transport: The complex is transported to the cell surface along microtubules .

• Transport proteins: This process involves dynamin-related protein 1 (DRP1) and kinesin family member 5B (KIF5B) .

• Membrane fusion: Mitochondrial membrane fuses with the plasma membrane to anchor ATP synthase on the cell surface .

When investigating the specific role of ATP5G2 in this process, researchers should consider its potential interactions with transport machinery proteins and whether it contains specific sequences that facilitate this trafficking. Live-cell imaging using photoactivatable GFP fused to ATP5G2 (similar to the ATP5B-paGFP approach) could provide valuable insights into its dynamic localization .

How do mutations or variations in ATP5G2 affect mitochondrial function and cellular resilience to stress?

Studies of natural variants of ATP5G proteins, such as the Arctic ground squirrel (AGS) variant of ATP5G1, have shown that specific amino acid substitutions can confer cytoprotective effects against metabolic stressors like hypoxia, low temperature, and mitochondrial toxins . For ATP5G2 research:

• Evolutionary analysis: Comparing bovine ATP5G2 sequences with those from species adapted to extreme environments may reveal functional variants.

• Directed mutagenesis: CRISPR/Cas9 base editing approaches can be used to test the functional significance of specific amino acid substitutions .

• Stress response assays: Evaluating cellular responses to metabolic stressors (hypoxia, temperature variation, rotenone exposure) can reveal phenotypic effects of ATP5G2 variants .

• Mitochondrial function assessment: Measuring parameters like membrane potential, ATP production, and respiratory capacity to assess functional consequences.

Research has shown that even single amino acid substitutions in ATP5G proteins can significantly impact cellular resilience to stress, as demonstrated by the L32P substitution in AGS ATP5G1 . Similar approaches could be applied to investigate functional variations in bovine ATP5G2.

What are the most accurate methods for quantifying ATP5G2 expression at the mRNA and protein levels?

For accurate quantification of ATP5G2:

mRNA Level:

• qRT-PCR: Design isoform-specific primers targeting unique regions of ATP5G2 mRNA.

• RNA-Seq: For comprehensive transcriptomic analysis, including splice variants.

• Digital droplet PCR: For absolute quantification with higher sensitivity.

Protein Level:

• Western blotting: Using antibodies specific to ATP5G2 or common epitopes in ATP5G proteins.

• Mass spectrometry: For absolute quantification and identification of post-translational modifications.

• ELISA: For high-throughput quantification in multiple samples.

When evaluating ATP5G2 expression, researchers should be aware that in most cell types, expression of ATP5G3 and ATP5G2 is greater than ATP5G1, though the relative abundance can vary by tissue type and physiological state . Quantification should include appropriate housekeeping controls and normalization strategies.

How can researchers differentiate between the three ATP5G isoforms in functional studies?

Differentiating between ATP5G isoforms presents challenges due to their identical mature protein sequences. Strategies include:

• Gene-specific knockdown/knockout: Using siRNA or CRISPR targeting unique 5' UTR or intronic regions.

• Promoter analysis: Studying isoform-specific promoters to understand differential regulation.

• N-terminal tagging: Tagging the unique N-terminal regions before processing.

• Isoform-specific antibodies: Developing antibodies against the unique N-terminal regions.

• Pulse-chase experiments: To study different kinetics of synthesis, processing, and turnover.

Research has demonstrated that despite their identical mature forms, the three ATP5G isoforms cannot functionally substitute for one another, suggesting unique roles in ATP synthase complex assembly or regulation . When designing experiments to differentiate between isoforms, researchers should consider both their pre-processed forms and their contributions to the fully assembled complex.

What assays can accurately measure the functional activity of recombinant bovine ATP5G2 in ATP synthase complex?

To assess functional activity of recombinant bovine ATP5G2:

• ATP synthesis assay: Measuring ATP production rates in isolated mitochondria or reconstituted systems.

• Oxygen consumption rate (OCR): Using platforms like Seahorse XF Analyzer to measure mitochondrial respiration.

• Membrane potential assays: Using fluorescent dyes like TMRM or JC-1 to assess proton gradient.

• Complex V enzymatic activity: Spectrophotometric assays with or without oligomycin to assess ATP synthase function .

• Hydrogen/deuterium exchange mass spectrometry: To assess structural dynamics and conformational changes.

When assessing ATP synthase activity, it's important to normalize to mitochondrial content, which can be done using citrate synthase activity or other mitochondrial markers . Researchers should also include controls with specific inhibitors like oligomycin to confirm that measured activity is specifically due to ATP synthase function.

How is ATP5G2 involved in mitochondrial dynamics and stress responses?

Recent research indicates that ATP synthase components, including ATP5G proteins, play roles in mitochondrial dynamics:

Research on Arctic ground squirrel ATP5G1 has demonstrated that specific amino acid substitutions can enhance cytoprotection against metabolic stress by modulating mitochondrial morphological changes and metabolic functions . Similar mechanisms could be investigated for bovine ATP5G2, particularly in tissues that undergo significant metabolic fluctuations.

How does post-translational modification affect ATP5G2 function and localization?

Post-translational modifications (PTMs) of ATP5G2 can significantly impact its function:

• N-terminal processing: Cleavage of the mitochondrial targeting sequence is essential for proper integration into the ATP synthase complex.

• Phosphorylation: May regulate assembly, stability, or activity of the ATP synthase complex.

• Acetylation: Could affect protein-protein interactions or enzymatic activity.

• Oxidative modifications: May occur during oxidative stress and alter function.

• Ubiquitination: May regulate protein turnover and quality control.

The N-terminal region of ATP5G proteins undergoes cleavage but can also directly modulate mitochondrial function through mechanisms that are not fully understood . Researchers investigating PTMs should consider both the pre-processed and mature forms of ATP5G2, as modifications at different stages may have distinct functional consequences.

What are the implications of ATP5G2 in disease models and potential therapeutic applications?

ATP5G2 and other ATP synthase components have emerging roles in various disease contexts:

• Cancer biology: Ectopic ATP synthase on cancer cell surfaces has catalytic activity that generates ATP in the extracellular environment, potentially creating favorable conditions for tumor progression .

• Metabolic disorders: Dysfunction of ATP synthase components can contribute to metabolic diseases.

• Neurodegenerative diseases: Mitochondrial dysfunction is implicated in numerous neurodegenerative conditions.

• Ischemia-reperfusion injury: Variants of ATP5G proteins that confer stress resilience could provide insights into protection against ischemic damage .

• Viral interactions: ATP synthase on the plasma membrane has been implicated in viral entry, including HIV-1 transportation to immune cells .

Research has identified ectopic ATP synthase as a potential molecular target for cancer therapy . Additionally, understanding the cytoprotective mechanisms of naturally occurring variants, such as those found in hibernating mammals, could lead to novel approaches for protecting cells against metabolic stress in various disease contexts .

Table 1: Comparison of ATP5G Isoforms in Mammalian Systems

Table 2: Recommended Detection Methods for ATP5G2 Research

Table 3: ATP Synthase Complex Activities in Different Contexts