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

What is ATP5G1 and what role does it play in mitochondrial function?

ATP5G1 (ATP Synthase, H+ Transporting, Mitochondrial F0 Complex, Subunit C1, Subunit 9) is a critical component of the mitochondrial ATP synthase complex. This protein functions as a lipid-binding protein within the F0 portion of the ATP synthase, which is embedded in the inner mitochondrial membrane. ATP5G1 contributes to the c-ring rotor that is essential for the rotational mechanism of ATP synthesis. The protein plays a fundamental role in coupling proton transport across the inner mitochondrial membrane to the synthesis of ATP, thereby serving as a crucial component of cellular energy production machinery .

The amino acid sequence of human ATP5G1 includes key functional regions: "TRGLIRPVSASFLSSPVNSSKQPSYSNFPLQVARREFQTSVVSRDIDTAAKFIGAGAATVGVAGSGAGIGTVFGSLIIGYARNPSLKQQLFSYAILGFALSEAMGLFCLMVAFLILFAM" . This sequence reveals the predominantly hydrophobic nature of ATP5G1, consistent with its membrane-embedded location.

How does ATP5G1 interact with lipids in the mitochondrial membrane?

In the E. gracilis ATP synthase structure, two bound cardiolipins (CDL1, CDL2) flank the horizontal helices of subunit a. These cardiolipins are coordinated by specific arginine residues from multiple subunits, suggesting they help seal the F0 complex against proton leakage and separate lipid and aqueous environments around the proton half-channels . Molecular dynamics simulations have demonstrated that the lipid-binding cavity within the ATP synthase preferentially binds cardiolipin, with cardiolipin showing approximately 2.5 times higher residence time compared to other phospholipids .

What structural features distinguish ATP5G1 from other ATP synthase subunits?

ATP5G1 is distinguished by its predominantly hydrophobic nature and its association with the c-ring of the F0 portion of ATP synthase. The protein contains multiple membrane-spanning regions that contribute to its integration into the lipid bilayer. Unlike the catalytic subunits of the F1 portion, ATP5G1 does not participate directly in ATP synthesis but instead contributes to proton translocation across the membrane, which drives the rotational mechanism of the enzyme .

A notable feature of ATP5G1 is its ability to bind specific lipids, particularly cardiolipins, which are essential for optimal enzyme function. These lipid interactions occur at defined sites and contribute to the structural integrity and functional efficiency of the ATP synthase complex. Additionally, specific amino acid residues in ATP5G1 can vary between species, leading to functional differences that may confer adaptive advantages in certain environments .

What methods are recommended for purifying recombinant ATP5G1?

Purification of recombinant ATP5G1 requires careful consideration of its hydrophobic nature and membrane association. The following methodology has proven effective:

• Expression Systems: E. coli, yeast, baculovirus, or mammalian cell expression systems can be used depending on the research requirements, with E. coli often preferred for higher yields . For functional studies requiring post-translational modifications, mammalian or insect cell systems may be more appropriate.

• Affinity Tags: Incorporating a hexahistidine tag, particularly at the N-terminus, facilitates purification using immobilized metal affinity chromatography (IMAC) . This approach has been successfully employed for yeast ATP synthase and can be adapted for pig ATP5G1.

• Detergent Solubilization: Due to its hydrophobic nature, membrane-bound ATP5G1 requires careful solubilization using mild detergents such as n-dodecyl-β-D-maltoside (DDM) or digitonin to maintain protein integrity and function.

• Quality Control: SDS-PAGE analysis should confirm purity ≥85% for most research applications . Western blotting using specific antibodies can verify protein identity, with monoclonal antibodies providing consistent results across purification batches .

• Storage Conditions: Aliquot the purified protein and store at -20°C or lower to prevent repeated freeze-thaw cycles that can degrade protein quality and function .

How can recombinant ATP5G1 be reconstituted into lipid bilayers for structural and functional studies?

Reconstitution of ATP5G1 or the entire ATP synthase complex into lipid bilayers is essential for many structural and functional studies. Two established methods have proven effective:

Method 1: Proteoliposome Formation

• Prepare a ternary mixture containing purified ATP synthase (containing ATP5G1), lipids (preferably including 20% cardiolipin to mimic the mitochondrial membrane), and an appropriate detergent.

• Remove the detergent gradually using adsorbent beads or dialysis to form proteoliposomes densely packed with the reconstituted protein.

• Verify successful reconstitution through electron microscopy or functional assays measuring ATP synthesis or hydrolysis activity .

Method 2: Supported Monolayer Technique

• Form a lipid monolayer at an air-water interface.

• Add hexahistidine-tagged ATP synthase beneath the monolayer.

• Allow the protein to associate with the lipid monolayer through the hexahistidine tag.

• This method creates a unidirectional orientation with the F1 domain attached to the lipid monolayer and the F0 domain (containing ATP5G1) exposed to the bulk solution .

The supported monolayer technique is particularly valuable for structural studies as it enables determination of c-ring stoichiometry and organization of membrane-intrinsic subunits within F0 using electron microscopy and atomic force microscopy (AFM) .

What experimental approaches can reveal ATP5G1's role in mitochondrial membrane organization?

ATP5G1's role in mitochondrial membrane organization can be investigated through several complementary approaches:

• Cryo-Electron Microscopy: This technique has successfully revealed ATP synthase dimers and their role in shaping mitochondrial cristae. For ATP5G1 specifically, cryo-EM at resolutions of 2.8 Å or better can visualize lipid-protein interactions at the subunit level .

• Molecular Dynamics Simulations: Coarse-grained molecular dynamics simulations have proven effective for studying lipid interactions with ATP5G1 and other ATP synthase subunits. These simulations can reveal lipid diffusion patterns, residence times, and preferential binding of specific lipids like cardiolipin .

• CRISPR/Cas9 Base Editing: This approach allows precise modification of specific amino acids in endogenous ATP5G1. For example, this technique has been used to study the L32P substitution in Arctic ground squirrel ATP5G1, demonstrating its causal role in cytoprotection against metabolic stress .

• Functional Mitochondrial Assays: Measurements of spare respiratory capacity, coupled with visualization of mitochondrial morphology following expression of wild-type or variant ATP5G1, can reveal its impact on mitochondrial function and structure .

• Fluorescence Microscopy with Mitochondrial Dyes: This technique can assess how ATP5G1 variants affect mitochondrial network morphology, particularly in response to stressors like FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) .

How do naturally occurring ATP5G1 variants impact mitochondrial function and cellular resilience?

Naturally occurring ATP5G1 variants can significantly impact mitochondrial function and cellular resilience to stress. The most well-documented example comes from studies of the Arctic ground squirrel (AGS), a hibernation-capable mammal with remarkable tolerance to metabolic stress and hypoxia.

The AGS-specific L32P variant of ATP5G1 has been causally linked to cytoprotection against metabolic stress. When this variant is expressed in mouse cells, it confers increased resilience to conditions of hypoxia, hypothermia, and exposure to the mitochondrial toxin rotenone . The cellular mechanisms underlying this protection involve:

• Enhanced Spare Respiratory Capacity: The L32P variant increases the mitochondrial spare respiratory capacity, providing cells with greater metabolic flexibility during stress conditions .

• Altered Mitochondrial Morphology: Expression of the L32P variant reduces mitochondrial fragmentation in response to stressors like FCCP, maintaining longer mitochondrial branch lengths and more connected networks .

• Species-Specific Effects: The cytoprotective effect appears to be specific to the L32P substitution, as other AGS-unique amino acid substitutions (N34D, T39P) did not confer similar protection when tested in mouse neural progenitor cells .

• Bidirectional Effects: Introducing the reverse mutation (P32L) into AGS ATP5G1 reduced spare respiratory capacity and increased mitochondrial fragmentation compared to wild-type AGS ATP5G1, further confirming the functional significance of this single amino acid position .

What experimental design considerations are important when studying ATP5G1 variants?

When designing experiments to study ATP5G1 variants, researchers should consider the following key factors:

• Expression System Selection: The choice between heterologous expression (e.g., expressing AGS ATP5G1 in mouse cells) versus modifying endogenous ATP5G1 (using CRISPR/Cas9 base editing) depends on the research question. Heterologous expression is faster but may introduce artifacts due to overexpression, while editing endogenous loci maintains native expression levels but requires more complex techniques .

• Control Constructs: Include both wild-type ATP5G1 from the species of interest and reciprocal mutations (e.g., if studying the AGS L32P variant, also test the reverse P32L mutation in AGS ATP5G1) to establish causality .

• Subcellular Localization Verification: Confirm that variant ATP5G1 properly localizes to mitochondria using fluorescence microscopy or subcellular fractionation followed by Western blotting. This is essential as mutations could potentially disrupt targeting sequences .

• Multi-Parameter Functional Assessment: Measure multiple aspects of mitochondrial function, including:

  • Spare respiratory capacity using Seahorse or similar technology

  • Mitochondrial morphology using appropriate fluorescent dyes

  • ATP synthesis rates under both basal and stress conditions

  • Membrane potential using voltage-sensitive dyes

  • Cell survival under various stressors (hypoxia, hypothermia, toxins)

• Dose-Response Relationships: Evaluate effects across a range of stressor intensities or durations to fully characterize the protective phenotype .

How might ATP5G1-lipid interactions be therapeutically targeted in mitochondrial disorders?

ATP5G1-lipid interactions, particularly with cardiolipins, represent a promising therapeutic target for mitochondrial disorders. Based on structural and functional studies, several potential intervention strategies emerge:

• Cardiolipin Stabilization: Given the preferential binding of cardiolipin to ATP5G1 and other ATP synthase subunits, therapies that stabilize cardiolipin content or prevent its oxidation could enhance ATP synthase function in disorders characterized by mitochondrial dysfunction .

• Biomimetic Peptides: Peptides designed to mimic the cardiolipin-binding regions of ATP5G1 could potentially modulate ATP synthase activity or stability. This approach would require detailed knowledge of the specific amino acid residues involved in lipid coordination, such as the arginine residues identified in structural studies .

• Targeted Lipid Delivery: Nanoparticle-based delivery of cardiolipin or synthetic analogs to mitochondria could potentially restore optimal ATP synthase function in conditions where mitochondrial membrane composition is altered.

• ATP5G1 Variant Expression: For conditions involving metabolic stress or ischemia-reperfusion injury, expression of cytoprotective variants like the AGS L32P ATP5G1 could potentially confer resilience to affected tissues . This might be achieved through gene therapy approaches targeted to affected tissues.

• Small Molecule Modulators: Compounds that mimic the structural effects of protective ATP5G1 variants could be developed, potentially offering pharmacological means to enhance mitochondrial resilience to stress.

What methods can be used to study ATP5G1's role in mitochondrial ATP synthase rotation?

Although direct observation of mitochondrial ATP synthase rotation has been challenging compared to bacterial ATP synthase, several methodological approaches can be employed:

• Single-Molecule FRET (Fluorescence Resonance Energy Transfer):

  • Attach fluorescent donor and acceptor molecules to different subunits of the ATP synthase complex

  • Monitor energy transfer changes during ATP synthesis or hydrolysis

  • This approach can provide information about conformational changes and rotational movement without directly visualizing rotation

• High-Speed Atomic Force Microscopy (HS-AFM):

  • Reconstitute ATP synthase into supported lipid bilayers using established methods

  • Use HS-AFM to directly visualize conformational changes in real-time

  • This technique has been successful for bacterial ATP synthase and could be adapted for mitochondrial ATP synthase

• Gold Nanoparticle Labeling:

  • Attach gold nanoparticles to the rotating portions of ATP synthase

  • Visualize rotation using dark-field microscopy

  • This approach requires careful design to ensure nanoparticles don't interfere with function

• Reconstitution Systems for Functional Studies:

  • Two methods have been described for reconstituting mitochondrial ATP synthase:
    a) Proteoliposome formation through detergent removal from ternary mixtures
    b) Supported monolayer technique with hexahistidine-tagged ATP synthase

  • These systems provide platforms for functional studies of ATP synthase rotation

• Cryo-EM of Different Conformational States:

  • Capture ATP synthase in different rotational states using cryo-EM

  • Reconstruct the rotational cycle from these static images

  • This approach has provided insights into the mechanics of ATP synthase operation

What are the emerging techniques for studying ATP5G1 interactions with other mitochondrial proteins?

Several cutting-edge techniques are advancing our understanding of ATP5G1's interactions with other mitochondrial proteins:

• Proximity Labeling Proteomics:

  • Fusion of ATP5G1 with enzymes like BioID or APEX2 that biotinylate nearby proteins

  • Identification of labeled proteins by mass spectrometry

  • This approach can reveal both stable and transient interaction partners in the native cellular environment

• Cross-Linking Mass Spectrometry (XL-MS):

  • Treatment of isolated mitochondria or purified ATP synthase with chemical cross-linkers

  • Digestion and mass spectrometric analysis to identify cross-linked peptides

  • This method can map specific interaction interfaces at amino acid resolution

• Cryo-Electron Tomography:

  • Visualization of ATP synthase in situ within mitochondria

  • Reveals native organization and interactions with other complexes

  • Particularly valuable for studying ATP synthase dimers and their role in cristae formation

• Genetic Interaction Mapping:

  • CRISPR-based screens to identify synthetic lethal or suppressor interactions with ATP5G1 variants

  • These genetic relationships can reveal functional connections even in the absence of direct physical interactions

• Super-Resolution Microscopy:

  • Techniques like STORM or PALM can visualize ATP5G1's distribution relative to other mitochondrial proteins

  • Multi-color imaging can reveal colocalization patterns and potential interaction zones

  • Live-cell super-resolution can capture dynamic associations

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

Working with recombinant ATP5G1 presents several challenges due to its hydrophobic nature and membrane association. Here are common issues and their solutions:

• Low Expression Yields:

  • Problem: ATP5G1's hydrophobic nature can lead to protein aggregation and toxicity in expression hosts.

  • Solution: Use specialized expression strains designed for membrane proteins, reduce induction temperature (e.g., to 18°C), and optimize induction time and concentration. Consider fusion partners that enhance solubility, such as MBP or SUMO .

• Protein Misfolding:

  • Problem: Improper folding affecting structure and function.

  • Solution: Express ATP5G1 in eukaryotic systems like yeast or insect cells that have more sophisticated folding machinery. Including specific lipids, particularly cardiolipins, in the expression medium can also improve folding .

• Detergent Selection Challenges:

  • Problem: Harsh detergents can denature ATP5G1, while mild detergents may not efficiently extract it from membranes.

  • Solution: Screen a panel of detergents starting with milder options like DDM, LMNG, or digitonin. Consider detergent mixtures or addition of lipids to stabilize the protein structure.

• Functional Assessment:

  • Problem: Confirming that recombinant ATP5G1 retains native function.

  • Solution: Reconstitute ATP5G1 into proteoliposomes and measure proton translocation or, for the complete ATP synthase, ATP synthesis activity. Compare activity to ATP5G1 within native ATP synthase complexes .

• Storage Stability:

  • Problem: Protein degradation during storage.

  • Solution: Store in small aliquots at -20°C or lower to avoid repeated freeze-thaw cycles. Include glycerol (10-15%) in storage buffers and consider adding specific lipids that enhance stability .

How can researchers validate that their recombinant ATP5G1 is properly folded and functional?

Validating the proper folding and functionality of recombinant ATP5G1 requires multiple complementary approaches:

• Structural Validation:

  • Circular Dichroism (CD) Spectroscopy: Assess secondary structure content, particularly alpha-helical content expected for ATP5G1.

  • Limited Proteolysis: Properly folded proteins show resistance to proteolytic digestion compared to misfolded variants.

  • Size-Exclusion Chromatography: Monomeric, well-folded protein should elute as a single, symmetrical peak.

• Lipid Binding Assays:

  • Microscale Thermophoresis (MST) or Isothermal Titration Calorimetry (ITC): Measure binding affinities for cardiolipin and other lipids.

  • Fluorescence-based assays using labeled lipids can also assess lipid binding capacity .

• Functional Reconstitution:

  • Proteoliposome Formation: Reconstitute ATP5G1 into liposomes containing other necessary ATP synthase subunits.

  • Proton Translocation: Measure pH-dependent fluorescence changes using pH-sensitive dyes.

  • ATP Synthesis: For complete ATP synthase complexes, measure ATP synthesis driven by artificial proton gradients .

• Integration into ATP Synthase Complex:

  • Co-immunoprecipitation: Verify that recombinant ATP5G1 can associate with other ATP synthase subunits.

  • Blue Native PAGE: Confirm incorporation into the complete ATP synthase complex.

• Comparative Studies with Known Variants:

  • Express well-characterized variants like the AGS L32P mutant alongside wild-type ATP5G1.

  • Compare functional outcomes such as spare respiratory capacity or response to stressors .

  • Expected differences based on literature can serve as positive controls for proper folding and function.