Recombinant Human ATP synthase lipid-binding protein, mitochondrial (ATP5G1)

What is ATP5G1 and what is its fundamental role in mitochondrial function?

ATP5G1 is one of three nuclear genes (along with ATP5G2 and ATP5G3) that encode identical copies of the c-subunit of mitochondrial ATP synthase, specifically part of the membrane domain of the enzyme's rotor . These three genes produce the same mature protein but differ in their mitochondrial targeting sequences, which are cleaved during import into the organelle . The c-subunit forms a ring structure in the inner mitochondrial membrane that is essential for the rotary mechanism of ATP synthase.

ATP5G1 plays a crucial role in oxidative phosphorylation by contributing to the proton channel of the ATP synthase complex (Complex V), facilitating the flow of protons across the inner mitochondrial membrane to generate ATP . This process is fundamental to cellular energy production and mitochondrial function.

How are ATP5G1, ATP5G2, and ATP5G3 differentially regulated despite encoding identical mature proteins?

While ATP5G1, ATP5G2, and ATP5G3 encode identical mature c-subunit proteins, they are regulated distinctly at the transcriptional and post-transcriptional levels . Their differential regulation allows for tissue-specific and condition-dependent expression patterns.

Research methodology to study this differential regulation typically involves:

• Quantitative PCR to measure isoform-specific mRNA expression levels across tissues

• Promoter analysis to identify unique transcription factor binding sites

• RNA stability assays to determine post-transcriptional regulation

• Gene knockout studies to assess functional redundancy

These genes have evolved to ensure adequate c-subunit production under various physiological conditions, with ATP5G1 being particularly regulated under stress conditions . Researchers investigating differential regulation should consider tissue-specific contexts and stress response scenarios to fully understand the unique roles of each gene.

What experimental approaches are recommended for studying ATP5G1 function in mitochondria?

When investigating ATP5G1 function, researchers should employ multiple complementary approaches:

• Genetic manipulation techniques:

  • CRISPR/Cas9 gene editing to create knockout or knock-in cell lines

  • Base editing technologies (e.g., ABEmax) for precise amino acid substitutions

  • Overexpression systems using viral vectors for gain-of-function studies

• Functional assays:

  • Seahorse XF analysis to measure mitochondrial respiration parameters, particularly spare respiratory capacity

  • ATP production assays using luciferase-based methods

  • Tetramethylrhodamine methyl ester (TMRE) staining to assess mitochondrial membrane potential

• Structural biology approaches:

  • Blue Native PAGE (BN-PAGE) to analyze ATP synthase complex assembly and dimerization

  • Cryo-electron microscopy for high-resolution structural analysis

  • Coarse-grained molecular dynamics simulations to assess lipid interactions

• Stress response protocols:

  • Hypoxia chambers to simulate ischemic conditions

  • Chemical stressors such as rotenone to induce mitochondrial dysfunction

  • Temperature manipulation to study thermotolerance mechanisms

These methodologies provide complementary data on ATP5G1's role in mitochondrial function, allowing researchers to build a comprehensive understanding of its contributions to cellular physiology.

How do naturally occurring variants of ATP5G1 contribute to cellular resilience under metabolic stress?

Naturally occurring variants of ATP5G1, particularly those found in hibernating mammals like the Arctic ground squirrel (AGS), demonstrate remarkable cytoprotective effects under conditions of metabolic stress. Research has identified a specific amino acid substitution (L32P) in AGS ATP5G1 that confers significant resilience to hypoxia, hypothermia, and mitochondrial toxins like rotenone .

Experimental evidence shows that:

• Expression of AGS ATP5G1 in mouse neural progenitor cells (NPCs) significantly improves survival under metabolic stress conditions .

• The L32P substitution in AGS ATP5G1 is causally linked to:

  • Increased spare respiratory capacity in mitochondria

  • Reduced mitochondrial fragmentation during stress

  • Increased mitochondrial branch length when exposed to FCCP (a mitochondrial uncoupler)

• CRISPR/Cas9 base editing to introduce this AGS-specific variant (L32P) in mammalian cells recapitulates key aspects of stress resilience .

Interestingly, other AGS-specific amino acid substitutions (N34D, T39P) did not affect survival under metabolic stress conditions, highlighting the unique importance of the L32P substitution .

What is the relationship between ATP5G1 and mitochondrial morphology during stress conditions?

ATP5G1 variants significantly impact mitochondrial morphology, particularly during stress conditions. Research demonstrates:

• Mitochondrial network characteristics:

  • Wild-type AGS ATP5G1 expression reduces mitochondrial fragmentation under stress

  • The L32P substitution is associated with increased mitochondrial branch length when exposed to mitochondrial uncouplers like FCCP

  • ATP5G1 variants influence the balance between mitochondrial fusion and fission processes

• Quantification methodologies:
  Researchers should employ advanced imaging techniques to quantify mitochondrial morphology:

  • Confocal microscopy with mitochondrial-specific dyes or fluorescent proteins

  • Deconvolution and skeletonization using Fiji/ImageJ for unbiased quantification

  • Parameters to measure include mitochondrial length, branching patterns, and fragmentation index

  • Automated analysis algorithms to distinguish between filamentous versus punctate structures

• Mechanistic insights:
  The effects on mitochondrial morphology are likely related to:

  • Alterations in ATP synthase dimerization, which affects cristae structure

  • Changes in interactions with membrane lipids, particularly cardiolipins

  • Modified responses to calcium-induced stress

To properly evaluate morphological changes, researchers should move beyond subjective classifications of mitochondrial shapes and employ quantitative metrics such as aspect ratio, form factor, and branching analysis as recommended in recent methodological advances .

How does ATP5G1 interact with cardiolipins and other membrane lipids in the mitochondrial membrane?

ATP5G1, as part of the ATP synthase complex, engages in critical interactions with membrane lipids, particularly cardiolipins. High-resolution cryo-EM studies of mitochondrial ATP synthase have revealed:

• Key lipid binding sites:

  • Cardiolipin binding sites at the rotor-stator interface

  • Lipid interactions at the dimer interface of ATP synthase

  • A peripheral F₀ cavity that contains multiple bound cardiolipins

• Functional implications:

  • Cardiolipins appear to modulate the rotating motor function of the enzyme

  • These lipids influence how the ATP synthase complex sits in the membrane

  • Lipid-protein interactions contribute to cristae morphology formation

• Research methodologies:
  To study these interactions, researchers should employ:

  • Coarse-grained molecular dynamics simulations to assess lipid binding dynamics

  • Analysis of lipid residence times within specific protein cavities

  • Quantification of lipid entry/exit probabilities for different phospholipid types

Experimental data from molecular dynamics simulations demonstrates that cardiolipins have approximately 2.5 times higher residence time in ATP synthase binding sites compared to other phospholipids, indicating preferential binding . These cardiolipin binding sites are induced by positively charged residues from multiple subunits extending into the membrane cavity.

What is the debated relationship between ATP5G1 and the mitochondrial permeability transition pore (PTP)?

The relationship between ATP5G1 (specifically the c-subunit it encodes) and the mitochondrial permeability transition pore (PTP) has been a subject of significant controversy in mitochondrial research:

• The hypothesis:
  Several proposals suggested that the c-subunit ring of ATP synthase provides the structural basis for the mitochondrial PTP, a nonspecific channel that opens in response to calcium, leading to mitochondrial swelling and potentially cell death .

• Contradicting evidence:
  Research using CRISPR-based genetic approaches has challenged this hypothesis:

  • A clonal cell line (HAP1-A12) was generated with disruption of all three c-subunit genes (ATP5G1, ATP5G2, ATP5G3)

  • Despite being incapable of producing any c-subunit protein, these cells preserved the characteristic properties of the PTP

  • This definitively demonstrated that the c-subunit does not provide the structural basis for the PTP

• Alternative structures:

  • HAP1-A12 cells assemble a vestigial ATP synthase with intact F₁-catalytic and peripheral stalk domains

  • This vestigial complex includes supernumerary subunits e, f, and g, but lacks membrane subunits ATP6 and ATP8

  • A similar vestigial complex was characterized in human 143B ρ⁰ cells, which maintain PTP function

• Current consensus:
  The data indicates that none of the membrane subunits of ATP synthase directly involved in transmembrane proton translocation (including those encoded by ATP5G1) form the PTP . This represents a significant revision to earlier hypotheses about PTP structure and function.

Researchers studying PTP should consider these findings when designing experiments and interpreting results related to mitochondrial permeability transition.

How can CRISPR/Cas9-based approaches be optimized for studying ATP5G1 function?

CRISPR/Cas9 technologies offer powerful tools for investigating ATP5G1 function, with several specialized approaches particularly valuable for ATP5G1 research:

• Base editing for precise amino acid substitutions:

  • Adenine base editors (ABEmax) have been successfully used to introduce single nucleotide changes in ATP5G1

  • This approach enables conversion of specific codons to study functional consequences of amino acid substitutions

  • Example: The AGS ATP5G1 L32P variant was successfully created by introducing a cytosine-to-thymine substitution in the (-) strand of the gene

• Complete gene knockout strategies:

  • For ATP5G1 research, consider the functional redundancy with ATP5G2 and ATP5G3

  • Triple knockout approaches may be necessary to completely eliminate c-subunit expression

  • Verification should include mRNA, protein, and functional assays

• Experimental design considerations:

  • Generate multiple clonal lines for both edited and control cells

  • Include base-edited controls that underwent editing but maintained wild-type sequence

  • Confirm that editing did not affect mRNA/protein expression or complex V activity

• Phenotypic validation:
  After successful editing, comprehensive phenotypic analysis should include:

  • Mitochondrial respiratory function (basal and maximal respiration, spare capacity)

  • Mitochondrial morphology analysis

  • Stress resilience assays (hypoxia, hypothermia, mitochondrial toxins)

  • ATP synthase assembly and activity measurements

When interpreting results from CRISPR-edited cells, researchers should consider potential compensatory mechanisms that may emerge during cell propagation. For instance, metabolic adaptations like increased glycolysis may develop in response to altered ATP5G1 function .

What are the implications of ATP5G1 research for understanding ischemic diseases and developing neuroprotective strategies?

ATP5G1 research, particularly studies of naturally occurring variants with cytoprotective properties, has significant implications for understanding and potentially treating ischemic diseases:

• Mechanistic insights:

  • The AGS ATP5G1 L32P variant demonstrates that single amino acid substitutions can substantially enhance cellular resilience to hypoxia and metabolic stress

  • These findings suggest that modulating ATP synthase function through ATP5G1 could be a viable approach for increasing cellular survival during ischemic events

• Therapeutic development potential:

  • Small molecules targeting ATP5G1 or mimicking the effects of protective variants could serve as neuroprotective agents

  • Gene therapy approaches might involve delivering modified ATP5G1 variants to vulnerable tissues

  • Understanding the downstream effects of ATP5G1 variants may identify additional therapeutic targets

• Neural stem cell applications:

  • Enhanced ATP5G1 variants could improve survival of neural stem cell grafts in ischemic environments

  • This approach might address a major challenge in regenerative medicine for neurological disorders

• Research models:

  • The in vitro paradigms established using ATP5G1 variants provide robust models for investigating other gene perturbations that may contribute to stress resilience

  • These models can serve as screening platforms for compounds that enhance mitochondrial resilience

Future research should focus on translating these basic science findings into therapeutic applications, particularly for conditions involving ischemia-reperfusion injury such as stroke, myocardial infarction, and organ transplantation.

How does ATP5G1 contribute to the unique stress resistance properties of hibernating mammals?

The ATP5G1 variant found in the Arctic ground squirrel (AGS) represents a fascinating example of evolutionary adaptation to extreme physiological conditions:

• Evolutionary significance:

  • The L32P substitution in AGS ATP5G1 appears to be an adaptive mechanism contributing to the remarkable stress resistance of hibernating mammals

  • This adaptation allows AGS cells to survive conditions that would be lethal to cells from non-hibernating mammals

• Functional advantages:

  • The AGS ATP5G1 variant promotes alterations in mitochondrial physiology that enhance resilience under stress conditions

  • These include improved spare respiratory capacity and reduced mitochondrial fragmentation

  • Such adaptations likely support the extreme physiological challenges faced during hibernation, including dramatic temperature fluctuations and periods of reduced oxygen availability

• Comparative research approach:

  • Studying ATP5G1 across hibernating and non-hibernating species provides insights into convergent or divergent evolutionary strategies

  • Cross-species analysis of ATP5G1 may identify additional adaptive variants

  • Functional testing across temperature ranges can reveal temperature-specific adaptations

• Broader implications:

  • ATP5G1 is likely one component of a larger adaptive network in hibernators

  • The study of such naturally evolved cytoprotective mechanisms offers a blueprint for developing human therapeutic strategies

  • The "hibernation phenotype" represents a natural model of ischemia tolerance that has evolved over millions of years

Future research should continue to explore the complex interplay between ATP5G1 variants and other adaptations in hibernating species, potentially revealing additional cytoprotective mechanisms with therapeutic relevance.

What role might ATP5G1 play as a therapeutic target for mitochondrial diseases?

ATP5G1 represents a promising therapeutic target for mitochondrial diseases, particularly those involving energy production deficits or sensitivity to metabolic stress:

• Target validation:

  • Natural variants of ATP5G1 demonstrate that modulating this protein can enhance mitochondrial function and stress resistance

  • The effects on spare respiratory capacity and mitochondrial morphology are particularly relevant to mitochondrial disease states

• Potential therapeutic approaches:

  • Gene therapy to deliver optimized ATP5G1 variants

  • Small molecule modulators that mimic beneficial ATP5G1 variant effects

  • RNA-based therapeutics to enhance ATP5G1 expression or modify its function

  • Cell-based therapies using cells with engineered ATP5G1 variants

• Disease relevance:

  • Conditions involving ischemia-reperfusion injury

  • Neurodegenerative disorders with mitochondrial dysfunction components

  • Primary mitochondrial diseases affecting ATP synthesis

  • Age-related conditions with declining bioenergetic capacity

• Research priorities:

  • Determine tissue-specific effects of ATP5G1 modulation

  • Assess long-term consequences of altered ATP5G1 function

  • Develop delivery methods for targeting specific tissues

  • Identify patient populations most likely to benefit from ATP5G1-targeted approaches

The development of ATP5G1-based therapeutics would benefit from further research on structure-function relationships and the precise mechanisms by which variants like L32P confer protective effects . Additionally, understanding how ATP5G1 interacts with lipids and other ATP synthase components may reveal additional intervention points for therapeutic development .

What are the recommended protocols for assessing mitochondrial function in models with altered ATP5G1?

When evaluating mitochondrial function in models with altered ATP5G1, researchers should implement a comprehensive assessment protocol:

For comprehensive assessment, researchers should:

• Perform both baseline and stress-induced measurements to capture dynamic responses

• Include appropriate controls (e.g., cells with wild-type ATP5G1 that underwent the same manipulation process)

• Consider compensatory mechanisms that may emerge over time in modified cells

• Assess possible metabolic reprogramming (e.g., glycolytic adaptation) in response to ATP5G1 alterations

These protocols provide a robust framework for characterizing the functional consequences of ATP5G1 modifications in various experimental models.

How can researchers effectively study the interaction between ATP5G1 variants and cardiolipin in the mitochondrial membrane?

To study ATP5G1-cardiolipin interactions effectively, researchers should employ a multi-faceted approach combining experimental and computational methods:

• Structural analysis techniques:

  • High-resolution cryo-electron microscopy of ATP synthase with bound lipids

  • Mass spectrometry-based lipidomics to identify and quantify associated lipids

  • Crosslinking approaches to capture protein-lipid interactions

• Molecular dynamics simulations:

  • Coarse-grained simulations of ATP synthase embedded in phospholipid membranes

  • Analysis of cardiolipin residence time compared to other phospholipids

  • Calculation of lipid entry/exit probabilities in specific binding sites

  • Simulation parameters should include approximately 20% cardiolipin to mimic mitochondrial inner membrane composition

• Functional assays:

  • Measurement of ATP synthase activity in liposomes with varying cardiolipin content

  • Assessment of proton translocation efficiency with modified lipid compositions

  • Evaluation of how ATP5G1 variants alter lipid binding profiles

• Visualization approaches:

  • Fluorescently labeled cardiolipin analogs to track distribution in mitochondria

  • Super-resolution microscopy to visualize cardiolipin clustering near ATP synthase complexes

  • FRET-based assays to detect proximity between ATP5G1 and cardiolipin

Research has shown that cardiolipin has approximately 2.5 times higher residence time at ATP synthase binding sites compared to other phospholipids, with positively charged residues from multiple subunits (including regions near ATP5G1) mediating this interaction . When designing experiments, researchers should consider that protein-enclosed membrane cavities in ATP synthase can form specialized lipid environments that affect enzyme function and stability.

What are the major challenges in working with recombinant ATP5G1 and how can they be addressed?

Researchers working with recombinant ATP5G1 face several technical challenges that require specific approaches:

• Expression and purification difficulties:

  • Challenge: ATP5G1 is highly hydrophobic and tends to aggregate during expression and purification

  • Solution: Use specialized expression systems such as bacteria with enhanced membrane protein production capabilities (C41/C43 strains)

  • Consider fusion tags that enhance solubility (MBP, SUMO)

  • Optimize detergent selection for extraction and purification (digitonin often preserves native conformation)

• Functional assessment:

  • Challenge: Determining if recombinant ATP5G1 is correctly folded and functional

  • Solution: Reconstitution into liposomes followed by proton translocation assays

  • Assembly assays with other ATP synthase components

  • Structural analysis by circular dichroism or NMR to confirm secondary structure

• Mitochondrial targeting:

  • Challenge: Ensuring proper mitochondrial localization when expressing in mammalian cells

  • Solution: Confirm integrity of the mitochondrial targeting sequence

  • Use microscopy to verify co-localization with mitochondrial markers

  • Perform subcellular fractionation to biochemically verify mitochondrial enrichment

• Distinguishing from endogenous protein:

  • Challenge: Differentiating recombinant ATP5G1 from endogenous protein

  • Solution: Use epitope tags that don't interfere with function or localization

  • Consider species-specific antibodies if using ATP5G1 from different organisms

  • Create CRISPR knockouts of endogenous ATP5G1/G2/G3 as background for expression studies

• Compensatory mechanisms:

  • Challenge: Cells may adjust expression of ATP5G2/G3 to compensate for ATP5G1 alterations

  • Solution: Monitor expression of all three ATP5G genes

  • Assess ATP synthase assembly and activity

  • Consider triple knockout approaches for complete functional studies

Researchers have successfully addressed these challenges through careful experimental design. For example, studies have confirmed that ATP5G1 variants maintain proper mitochondrial localization despite amino acid substitutions in the targeting sequence , and CRISPR-based approaches have successfully generated cells with modified ATP5G1 while maintaining ATP synthase complex assembly .

How can researchers resolve inconsistent results when studying ATP5G1 function across different cell types?

When facing inconsistent results in ATP5G1 studies across different cell types, researchers should consider:

• Metabolic profile variations:

  • Cell types differ significantly in their reliance on oxidative phosphorylation versus glycolysis

  • Solution: Characterize baseline metabolic profiles using Seahorse analysis

  • Normalize ATP5G1 effects to the relative importance of oxidative phosphorylation in each cell type

  • Consider how glycolytic compensation varies between cell types

• Expression level differences of ATP5G1/G2/G3:

  • Challenge: The relative expression of the three ATP5G genes varies across tissues

  • Solution: Quantify baseline expression of all three genes in each cell type

  • Assess compensatory upregulation following manipulation of ATP5G1

  • Consider the total c-subunit pool rather than just ATP5G1-derived protein

• Mitochondrial content and quality:

  • Challenge: Mitochondrial mass and functional quality vary substantially between cell types

  • Solution: Normalize data to mitochondrial content

  • Assess mitochondrial quality metrics (membrane potential, respiratory coupling)

  • Consider mitochondrial turnover rates in different cell types

• Experimental design adaptations:

  • Challenge: Standard protocols may not be optimal across all cell types

  • Solution: Adjust stress conditions based on baseline resilience of each cell type

  • Optimize transfection/transduction protocols for each cell type

  • Develop cell-type specific assay conditions

• Data interpretation framework:

  • Challenge: Distinguishing biological variation from technical artifacts

  • Solution: Use multiple methodological approaches to validate findings

  • Perform rescue experiments to confirm specificity

  • Consider relative rather than absolute changes compared to appropriate controls

Researchers have observed that the effects of ATP5G1 variants can differ between cell types based on their metabolic profiles and stress resistance. For example, the cytoprotective effects of AGS ATP5G1 may be more pronounced in cells with high oxidative metabolism or those particularly vulnerable to metabolic stress , suggesting that cell type selection should be guided by the specific research questions being addressed.

How does ATP5G1 research relate to broader studies of mitochondrial dynamics and quality control?

ATP5G1 research intersects with several aspects of mitochondrial dynamics and quality control: