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

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

ATP5G2 is one of three nuclear genes (along with ATP5G1 and ATP5G3) that encode the c-subunit (subunit 9) of the mitochondrial ATP synthase Fo complex . All three genes encode identical mature proteins but with different mitochondrial-targeting sequences that are removed during import into the organelle . The c-subunit forms a ring structure in the mitochondrial inner membrane that is essential for proton translocation, driving ATP synthesis by the F1 catalytic portion. The mature protein is highly hydrophobic and contains approximately 75 amino acids that form two transmembrane helices.

How is recombinant Rat ATP5G2 protein typically expressed and purified?

Recombinant Rat ATP5G2 can be expressed in mammalian cell systems to ensure proper folding and post-translational modifications . The protein is typically tagged (e.g., with a His-tag) to facilitate purification . According to standard protocols, the purified protein should be maintained at >80% purity with endotoxin levels below 1.0 EU per μg as determined by the LAL method . For storage, the protein can be maintained in PBS buffer at +4°C for short-term use or at -20°C to -80°C for long-term storage . When designing experiments, researchers should consider that custom production typically requires 5-9 weeks lead time .

How do expression patterns differ between ATP5G1, ATP5G2, and ATP5G3?

While ATP5G1, ATP5G2, and ATP5G3 encode identical mature c-subunits, their expression patterns show tissue-specific and condition-dependent regulation. In clear cell renal cell carcinoma (ccRCC), all three genes are significantly downregulated compared to normal renal tissue, with ATP5G1 showing the most dramatic reduction (fold changes of -8.09/-5.88 in screening/validation cohorts), followed by ATP5G3 (-3.10/-2.37) and ATP5G2 (-2.69/-2.11) . This differential regulation suggests distinct transcriptional control mechanisms for each gene, potentially allowing for context-specific modulation of ATP synthase activity under various physiological and pathological conditions.

What methodologies can be used to study ATP5G2-specific functions versus ATP5G1 and ATP5G3?

To differentiate the specific contributions of ATP5G2 from its paralogs, researchers can employ several methodological approaches:

• Gene-specific knockdown using siRNA or shRNA targeting the unique 5'-UTR or mitochondrial targeting sequence regions of ATP5G2 mRNA

• CRISPR-Cas9 gene editing targeting exon IV of ATP5G2, as demonstrated in research creating the HAP1-A12 cell line

• Quantitative RT-PCR with primers specific to unique regions of each gene to assess their relative expression levels across tissues or experimental conditions

• Protein import studies using constructs containing the ATP5G2 mitochondrial targeting sequence fused to reporter proteins

When designing these experiments, it's crucial to include appropriate controls and validation steps, as the functional redundancy between these three genes may complicate the interpretation of results when only one gene is manipulated.

How can ATP synthase activity be assessed following ATP5G2 manipulation?

Several assays can be employed to evaluate ATP synthase function after modifying ATP5G2 expression:

• The 9-amino-6-chloro-2-methoxyacridine (ACMA) assay measures ATP synthase enzymatic rate by monitoring proton translocation in submitochondrial vesicles (SMVs)

• Mitochondrial membrane potential can be assessed using fluorescent dyes like tetramethyl rhodamine methyl ester (TMRM)

• Oxygen consumption rates can be measured using substrates like glutamate/malate and succinate to confirm respiratory complex activity

• Native gel electrophoresis can analyze the oligomeric state of ATP synthase complexes, providing insights into assembly and stability

• Direct ATP production measurement using luminescent assays to quantify functional output

Each approach provides different insights into ATP synthase function, and combining multiple methods offers a more comprehensive understanding of how ATP5G2 manipulation affects mitochondrial bioenergetics.

What approaches are most effective for studying ATP5G2 in the context of the mitochondrial permeability transition?

Recent research has challenged the hypothesis that the c-subunit forms the mitochondrial permeability transition pore (PTP). To investigate this relationship, researchers can:

• Generate cell lines with disrupted ATP5G genes (individually or in combination) as demonstrated with the HAP1-A12 cell line, which lacks all three c-subunit genes yet preserves PTP properties

• Measure calcium retention capacity of mitochondria before PTP opening

• Test cyclosporin A sensitivity to assess classical PTP involvement

• Perform mitochondrial swelling assays in response to calcium or other PTP inducers

• Use co-immunoprecipitation or proximity labeling techniques to identify proteins that interact with ATP5G2

Research has shown that mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase with intact F1-catalytic and peripheral stalk domains plus supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8 . This suggests that none of the membrane subunits directly involved in proton translocation (including the c-subunit) forms the PTP .

How is ATP5G2 expression altered in cancer, and what are the methodological considerations for studying this relationship?

ATP5G2 expression is significantly downregulated in clear cell renal cell carcinoma (ccRCC), as shown in the following comparative data:

When studying ATP5G2 in cancer contexts, researchers should:

• Compare expression between matched tumor and normal tissue samples

• Validate findings in independent cohorts

• Correlate expression levels with clinical parameters including tumor stage, grade, and patient outcomes

• Investigate the functional consequences of ATP5G2 downregulation on mitochondrial metabolism in cancer cells

What is the relationship between ATP5G2 and neurological disorders?

Research has implicated ATP synthase c-subunit function in Fragile X syndrome (FXS), a neurodevelopmental disorder caused by loss of Fragile X mental retardation protein (FMRP) . Key findings include:

• FXS neurons exhibit an ATP synthase "leak" that affects cellular metabolism

• Closure of this leak channel by manipulation of c-subunit levels normalizes stimulus-induced and constitutive mRNA translation rates

• This normalization decreases lactate and key glycolytic/TCA cycle enzyme levels and triggers synapse maturation

• In wild-type neurons, FMRP regulates leak closure through stimulus-dependent ATP synthase β subunit translation, which increases the ratio of ATP synthase enzyme to its c-subunit

• In FXS, the inability to close this developmental c-subunit leak prevents stimulus-dependent synaptic maturation

When investigating ATP5G2 in neurological disorders, researchers should consider the distinct roles of mitochondrial bioenergetics in different neural cell types and brain regions, as well as the potential for compensatory mechanisms involving ATP5G1 and ATP5G3.

How can ATP5G2 function be effectively studied in disease models?

When investigating ATP5G2 in disease models, researchers should consider these methodological approaches:

• Conditional knockout or knockdown systems that allow tissue-specific and temporally controlled manipulation of ATP5G2 expression

• CRISPR-Cas9 gene editing to introduce disease-associated mutations or disrupt the gene entirely

• Disease-specific phenotyping protocols:

  • For cancer models: tumor growth, metabolism, and survival assessment

  • For neurological disorders: electrophysiology, synaptic analysis, and behavioral testing

  • For metabolic diseases: glucose tolerance, insulin sensitivity, and energy expenditure measurements

• Multi-omics approaches combining proteomics, metabolomics, and transcriptomics to comprehensively characterize molecular changes

• Pharmacological interventions targeting ATP synthase function to assess potential therapeutic applications

When designing these studies, researchers should account for the functional redundancy between ATP5G1, ATP5G2, and ATP5G3, which may require simultaneous manipulation of multiple genes to observe clear phenotypic effects.

How does the ATP5G2-encoded c-subunit interact with other components of the ATP synthase complex?

The c-subunit encoded by ATP5G2 forms a critical ring structure in the Fo portion of ATP synthase that interacts with other membrane components. Research using cells with disrupted c-subunit genes has provided valuable insights:

• The c-ring is necessary for stable incorporation of membrane subunits ATP6 and ATP8 into the complex

• Without c-subunits, a vestigial ATP synthase forms containing F1-catalytic and peripheral stalk domains plus supernumerary subunits e, f, and g, but lacking ATP6 and ATP8

• The oligomeric state of ATP synthase depends on the presence of the c-ring, as demonstrated by native gel electrophoresis comparing wild-type versus c-subunit-deficient cells

• The dimerization interface likely relies on subunits e and g and may also involve supernumerary subunits f, DAPIT, and 6.8PL

What mechanisms regulate ATP5G2 expression at transcriptional and translational levels?

Regulation of ATP5G2 expression can be studied through multiple methodological approaches:

• Promoter analysis using reporter constructs to identify regulatory elements controlling transcription

• Chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the ATP5G2 promoter

• Epigenetic profiling through bisulfite sequencing and ChIP-seq to characterize DNA methylation and histone modifications

• miRNA binding site analysis and validation using miRNA mimics or inhibitors

From existing research, we know that:

• ATP5G2 expression is significantly downregulated in clear cell renal cell carcinoma

• In Fragile X syndrome, ATP5G2 c-subunit mRNA is elevated, but its translation may not be regulated by FMRP (unlike ATP synthase β subunit)

These findings suggest complex regulatory mechanisms that may be context-dependent and involve both transcriptional and post-transcriptional controls. Studies should incorporate multiple cell types and disease states to fully characterize these regulatory pathways.

How can researchers investigate post-translational modifications of ATP5G2-encoded protein?

Studying post-translational modifications (PTMs) of the c-subunit requires specialized approaches:

• Mass spectrometry-based methods:

  • Enrichment strategies for specific PTMs (phosphorylation, acetylation, etc.)

  • Targeted proteomics to identify and quantify modified peptides

  • Comparison of PTM profiles under different physiological conditions

• Site-directed mutagenesis:

  • Generate mutants where potential modification sites are altered

  • Express these mutants in cells lacking endogenous c-subunits

  • Assess functional consequences on ATP synthase assembly and activity

• Functional correlation studies:

  • Link PTM changes to alterations in ATP synthase activity

  • Compare PTM patterns across different tissues and disease states

  • Investigate enzymes responsible for specific modifications

Researchers should be aware that studying PTMs of the mature c-subunit presents technical challenges due to its small size and high hydrophobicity. Additionally, since ATP5G1, ATP5G2, and ATP5G3 encode identical mature proteins, distinguishing modifications specific to the ATP5G2-encoded protein requires careful experimental design or the use of cells where ATP5G1 and ATP5G3 have been knocked out.

What structural biology approaches are most informative for studying ATP5G2-encoded c-subunit?

Several complementary structural biology techniques can provide insights into c-subunit structure-function relationships:

• Cryo-electron microscopy of intact ATP synthase complexes to visualize the c-ring in its native context

• X-ray crystallography of purified c-rings to determine high-resolution structural details

• NMR spectroscopy for studying dynamic aspects of c-subunit structure in membrane environments

• Molecular dynamics simulations to predict conformational changes during proton translocation

• Site-directed mutagenesis of key residues combined with functional assays to correlate structure with function

Recent advances in structural biology have improved our understanding of how the c-subunit contributes to proton translocation and ATP synthesis. Researchers should consider that extraction methods can affect the oligomeric state of ATP synthase complexes, as demonstrated by the variable results obtained with different digitonin concentrations used for extraction from 143B ρ0 cells .

How can researchers differentiate between direct effects of ATP5G2 disruption and compensatory changes involving ATP5G1 and ATP5G3?

Distinguishing direct effects from compensatory mechanisms requires carefully designed experimental approaches:

• Gene-specific knockdown with phenotype rescue:

  • Deplete ATP5G2 using targeted siRNA

  • Attempt rescue with an siRNA-resistant ATP5G2 construct

  • Compare with rescue using ATP5G1 or ATP5G3 overexpression

• Time-course experiments:

  • Use inducible knockout/knockdown systems

  • Monitor immediate versus long-term effects

  • Distinguish primary effects from secondary compensatory responses

• Combinatorial genetic manipulation:

  • Compare phenotypes of single, double, and triple knockouts

  • Identify unique versus redundant functions

  • Example: Complete disruption of all three c-subunit genes was necessary to eliminate the c-subunit from HAP1-A12 cells

• Tissue-specific analysis to identify context-dependent roles and compensation patterns

These approaches help delineate the specific contribution of ATP5G2 to mitochondrial function while accounting for the functional redundancy between the three c-subunit genes.

What high-throughput methods can be applied to comprehensively analyze ATP5G2 function?

Several high-throughput approaches can accelerate research on ATP5G2 function:

• CRISPR-based functional genomics:

  • Genome-wide CRISPR screens in ATP5G2-modified backgrounds

  • Identification of synthetic lethal interactions

  • Discovery of compensatory pathways

• Multi-omics integration:

  • RNA-seq to detect transcriptional responses to ATP5G2 manipulation

  • Proteomics to identify changes in protein abundance and modifications

  • Metabolomics focused on energy metabolism intermediates

  • Network analysis to identify coordinated responses

• High-content imaging:

  • Automated microscopy with multiple fluorescent markers

  • Simultaneous tracking of mitochondrial morphology, membrane potential, and ROS production

  • Correlation of subcellular phenotypes with ATP5G2 expression levels

• Drug screening:

  • Identification of compounds that modulate ATP synthase function

  • Testing of combinations with ATP5G2 manipulation

  • Discovery of context-specific vulnerabilities

These approaches enable systematic characterization of ATP5G2 function across different cellular contexts, potentially revealing unexpected roles beyond its canonical function in ATP synthesis.

What recent findings have clarified the relationship between ATP5G2 and the mitochondrial permeability transition pore?

Recent research has significantly revised our understanding of the relationship between c-subunits and the mitochondrial permeability transition pore (PTP):

Researchers generated a clonal cell line (HAP1-A12) with disrupted ATP5G1, ATP5G2, and ATP5G3 genes, eliminating all c-subunit production . Key findings include:

This research significantly advances our understanding of mitochondrial biology by excluding c-subunits as direct PTP components, redirecting attention to other potential structural elements of this enigmatic channel.

How is ATP5G2 being investigated in relation to neurological disorders?

Recent research has revealed interesting connections between ATP synthase c-subunits and neurological conditions, particularly Fragile X syndrome:

• In neurons lacking FMRP (Fragile X mental retardation protein), there appears to be a persistent leak in ATP synthase that affects cellular metabolism

• Closure of this leak channel through c-subunit manipulation normalizes multiple cellular phenotypes:

  • Normalizes stimulus-induced and constitutive mRNA translation rates

  • Decreases lactate and key glycolytic/TCA cycle enzyme levels

  • Triggers synapse maturation

• In wild-type neurons, FMRP regulates leak closure through stimulus-dependent ATP synthase β subunit translation, increasing the ratio of ATP synthase enzyme to its c-subunit

• This mechanism enhances ATP production efficiency and promotes synaptic growth in normal conditions but fails in Fragile X syndrome

These findings suggest potential therapeutic approaches targeting ATP synthase function in neurological disorders, including pharmacological agents that modulate ATP synthase leak or interventions targeting downstream metabolic pathways affected by altered mitochondrial function.

What computational models are advancing our understanding of ATP5G2 function?

Computational approaches are increasingly valuable for studying ATP5G2 structure, function, and interactions:

• Molecular dynamics simulations:

  • Model the behavior of the c-ring in lipid bilayers

  • Predict conformational changes during proton translocation

  • Assess the impact of mutations on structure and function

• Systems biology approaches:

  • Integrate ATP5G2 into genome-scale metabolic models

  • Predict systemic effects of ATP5G2 alterations

  • Model impact on mitochondrial energy production pathways

• Machine learning applications:

  • Predict functional consequences of ATP5G2 variants

  • Identify patterns in gene expression data

  • Discover potential regulatory mechanisms

• Network analysis:

  • Map protein-protein interaction networks involving ATP5G2

  • Identify hub proteins that coordinate with ATP5G2 function

  • Predict synthetic lethal interactions

These computational approaches complement experimental studies by generating testable hypotheses, guiding experimental design, and providing mechanistic insights that may be difficult to obtain through experimental methods alone.