Recombinant Mouse ATP synthase lipid-binding protein, mitochondrial (Atp5g1)

What are the alternative names for ATP5G1 in scientific literature?

When conducting literature searches, researchers should be aware of multiple nomenclatures for ATP5G1:

• ATP synthase F(0) complex subunit C1, mitochondrial

• ATP synthase lipid-binding protein

• ATP synthase proteolipid P1

• ATP synthase proton-transporting mitochondrial F(0) complex subunit C1

• ATPase protein 9

• ATPase subunit c

Using these alternative names in literature searches will ensure comprehensive coverage of relevant research.

What are the standard methods for detecting ATP5G1 in experimental systems?

For detection of ATP5G1 in experimental systems, Western blotting is commonly employed using specific antibodies. Commercial monoclonal antibodies, such as the mouse monoclonal antibody raised against full-length recombinant ATP5G1, are available and validated for Western blot applications. The recommended dilution for Western blot applications is typically 1:500-1:1000 .

Other detection methods include:

• Immunofluorescence microscopy for localization studies

• Mass spectrometry for protein identification and characterization

• qRT-PCR for mRNA expression analysis

When using antibodies, it's important to verify specificity, as the target specificity of commercially available antibodies is often for ATP5G1 (AAH04963, 18 a.a. ~ 136 a.a) full-length recombinant protein with GST tag, with the GST tag alone having a MW of 26 KDa .

How should researchers design experiments to study ATP5G1 function in mitochondria?

When designing experiments to study ATP5G1 function in mitochondria, researchers should consider several approaches:

• Overexpression systems: Introducing wild-type or mutant forms of ATP5G1 in cell culture models to examine effects on mitochondrial function. This approach has been successfully used to demonstrate that ectopic expression of ATP5G1 variants affects cellular resilience to metabolic stress .

• CRISPR/Cas9 gene editing: For introducing specific mutations or amino acid substitutions to study their functional consequences. Base editing approaches have been particularly useful in examining the functional significance of individual amino acid residues such as the L32P substitution found in Arctic ground squirrel ATP5G1 .

• Mitochondrial functional assays: Measuring parameters such as oxygen consumption rate, ATP production, membrane potential, and spare respiratory capacity to assess the impact of ATP5G1 modifications .

• Mitochondrial morphology analysis: Examining changes in mitochondrial network structure, fusion/fission dynamics, and branch length in response to ATP5G1 variants or under stress conditions like FCCP exposure .

When designing these experiments, it's crucial to include appropriate controls, such as expression of human ATP5G1 alongside the variant of interest, to distinguish species-specific effects from general overexpression effects.

What techniques are optimal for analyzing ATP5G1 lipid-binding properties?

To analyze the lipid-binding properties of ATP5G1, researchers should consider multiple complementary approaches:

• X-ray crystallography: High-resolution structural studies have been valuable in resolving lipid binding sites in ATP synthase complexes. Crystal structures of related cytochrome oxidase complexes have revealed conserved lipid-binding sites and the residues that form them .

• Mass spectrometry: This technique can identify not only the head groups but also the fatty acid chains of bound lipids, as demonstrated in studies of bovine heart mitochondria .

• Mutagenesis of conserved residues: Targeted mutation of highly conserved residues involved in lipid interactions can reveal their functional significance. Previous studies have shown that mutations affecting lipid binding can lead to altered enzyme activity and increased tendency for suicide inactivation .

• Molecular dynamics simulations: Computational approaches to model and predict lipid-protein interactions based on known structures.

When analyzing lipid binding, it's important to note that one of the two fatty acid chains in many conserved lipid sites appears to be more tightly bound and more highly conserved than the other, suggesting a possible anchoring strategy where lipids are shared between the protein and the bilayer .

How does the naturally occurring variant of ATP5G1 in Arctic ground squirrels contribute to cytoprotection?

The Arctic ground squirrel (AGS) variant of ATP5G1 contains specific amino acid substitutions that contribute to cytoprotection against metabolic stress. Research has demonstrated that:

• The AGS-specific L32P substitution in ATP5G1 plays a causal role in mediating cytoprotection, as revealed through both ectopic expression in mouse cells and CRISPR/Cas9 base editing of endogenous AGS loci .

• Expression of AGS ATP5G1 in mouse neural progenitor cells (NPCs) confers resilience to metabolic stressors including hypoxia, hypothermia, and rotenone exposure .

• The cytoprotective mechanism involves modulation of mitochondrial morphology and metabolic functions, specifically:

  • Increasing spare respiratory capacity

  • Reducing mitochondrial fission in response to stress

  • Decreasing mitochondrial fragmentation

  • Increasing branch length of mitochondria when exposed to FCCP

Interestingly, while the L32P substitution is critical, it does not completely account for all cytoprotective effects, suggesting that variants of other genes may also contribute to metabolic stress resistance. Other identified AGS-unique amino acid substitutions (N34D, T39P) did not significantly affect survival of mouse NPCs exposed to stress conditions .

What is the relationship between ATP5G1 and neuropsychiatric disorders?

Research has identified potential links between ATP5G1 and neuropsychiatric disorders, particularly major depressive disorder (MDD):

• Co-expression network analysis has revealed that ATP5G1 is significantly down-regulated in MDD compared to control groups (t = -3.94, p-value = 0.0009) .

• Methylation analysis using the GSE88890 dataset identified highly significant differentially methylated positions (DMPs) in the ATP5G1 gene in the BA25 brain region:

  • cg25495775 (t = 2.82, p-value = 0.008)

  • cg25856120 (t = -2.23, p-value = 0.033)

  • cg23708347 (t = -2.24, p-value = 0.032)

• The data suggest that ATP5G1 may be involved in the pathogenesis of depression, potentially through influencing purine metabolism .

These findings indicate that ATP5G1 may serve as a potential biomarker or therapeutic target for neuropsychiatric disorders. Researchers investigating this relationship should consider both gene expression and epigenetic regulation of ATP5G1 in relevant brain regions.

How can researchers effectively study ATP5G1's role in mitochondrial membrane dynamics?

To effectively study ATP5G1's role in mitochondrial membrane dynamics, researchers should implement multiple methodological approaches:

• Live-cell imaging with fluorescent probes: Using mitochondria-targeted fluorescent proteins to visualize changes in mitochondrial morphology, distribution, and dynamics in real-time.

• Electron microscopy: For high-resolution analysis of mitochondrial ultrastructure and membrane organization.

• Quantitative analysis of mitochondrial network parameters: Measuring branch length, fragmentation, and connectivity using specialized software to quantify morphological changes.

• Stress-response assays: Examining how ATP5G1 variants affect mitochondrial responses to stressors such as FCCP. Studies have shown that while mouse cells demonstrate significant mitochondrial fission when exposed to FCCP, AGS cells with their variant ATP5G1 appear largely resistant to this stress-induced mitochondrial fission .

• Co-immunoprecipitation studies: To identify protein-protein interactions between ATP5G1 and other components of the mitochondrial fusion/fission machinery.

The comparative approach, studying differences between species with varying stress tolerance (e.g., mouse vs. AGS), has proven particularly valuable for understanding how ATP5G1 variants influence mitochondrial membrane dynamics under stress conditions .

What are the critical quality control parameters for recombinant ATP5G1 protein preparations?

When working with recombinant ATP5G1 protein, researchers should implement the following quality control measures:

• Purity assessment: SDS-PAGE and Western blot analysis to confirm protein size and purity, with expected molecular weight of approximately 14 kDa for the native protein (though tag additions may alter this) .

• Functional validation: Assays to confirm that the recombinant protein retains its biological activity, particularly its ability to incorporate into ATP synthase complexes.

• Subcellular localization verification: Confirmation of proper mitochondrial targeting, as improper localization may indicate issues with protein folding or modifications. Both human and AGS ATP5G1 constructs should properly target to mitochondria when expressed in cells .

• Storage stability testing: Recombinant ATP5G1 is typically stored in Tris-based buffer with 50% glycerol at -20°C. Repeated freezing and thawing should be avoided, and working aliquots should be stored at 4°C for up to one week .

• Tag interference assessment: If using tagged constructs, researchers should verify that the tag does not interfere with protein function or localization. For commercially available proteins, the tag type may vary and should be determined during the production process .

What are the most effective methods for studying ATP5G1 gene expression regulation?

To effectively study ATP5G1 gene expression regulation, researchers should consider multiple complementary approaches:

• Quantitative RT-PCR: For measuring mRNA expression levels in different tissues or experimental conditions.

• RNA-Seq: For comprehensive transcriptomic analysis to identify co-expressed genes and regulatory networks associated with ATP5G1.

• Methylation analysis: Examining DNA methylation patterns at specific positions in the ATP5G1 gene, as differential methylation has been associated with conditions like depression. Techniques like bisulfite sequencing or methylation-specific PCR can be employed .

• Chromatin immunoprecipitation (ChIP): To identify transcription factors that bind to the ATP5G1 promoter region.

• Reporter gene assays: Using luciferase or GFP reporters linked to the ATP5G1 promoter to study its regulation under different conditions.

When studying methylation patterns specifically, researchers should pay attention to specific CpG sites like cg25495775, cg25856120, and cg23708347, which have shown significant differential methylation in previous studies .

How can researchers leverage ATP5G1 variants for developing cytoprotective strategies?

Researchers looking to develop cytoprotective strategies based on ATP5G1 variants should consider the following approaches:

• Targeted gene editing: Using CRISPR/Cas9 base editing to introduce specific amino acid substitutions (such as L32P) that have been shown to confer cytoprotection in Arctic ground squirrel ATP5G1 .

• Pharmacological mimetics: Developing small molecules that can bind to ATP5G1 and induce conformational changes similar to those caused by protective mutations.

• Combinatorial approaches: Since the L32P substitution alone does not account for all protective effects, investigating combinations with other cytoprotective factors identified in hibernating species.

• Mitochondrial-targeted delivery systems: Developing methods to deliver modified ATP5G1 or mimetic compounds specifically to mitochondria in tissues vulnerable to ischemia-reperfusion injury.

• Translational models: Testing cytoprotective effects in models of human diseases characterized by metabolic stress, such as stroke, myocardial infarction, or neurodegenerative disorders.

These approaches could potentially lead to novel therapeutic strategies for conditions characterized by metabolic stress, hypoxia, or ischemia-reperfusion injury .

What are the emerging technologies for investigating ATP5G1's interactions with other mitochondrial proteins?

Emerging technologies for investigating ATP5G1's interactions with other mitochondrial proteins include:

• Proximity labeling techniques: Methods such as BioID or APEX2 that can identify proteins in close proximity to ATP5G1 within the mitochondrial membrane.

• Single-molecule imaging: Super-resolution microscopy techniques that allow visualization of individual protein complexes and their dynamics within mitochondria.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): For mapping protein-protein interaction surfaces and conformational changes in ATP5G1 and its binding partners.

• Cryo-electron microscopy: For high-resolution structural analysis of ATP5G1 within the context of the complete ATP synthase complex.

• Native mass spectrometry: For analyzing intact protein complexes and their stoichiometry in near-native conditions.

These technologies can help elucidate how ATP5G1 interacts with other components of the ATP synthase complex and how these interactions are affected by genetic variants or post-translational modifications.

What are common challenges in ATP5G1 research and how can they be addressed?

Researchers working with ATP5G1 may encounter several challenges:

• Protein solubility issues: As a membrane protein, ATP5G1 can be difficult to solubilize while maintaining its native conformation. Solution: Use mild detergents specifically optimized for mitochondrial membrane proteins and consider including lipids that are known to interact with ATP5G1.

• Specificity of antibodies: Commercial antibodies may cross-react with other ATP synthase subunits. Solution: Validate antibody specificity using knockout controls or competing peptides, and use the recommended dilutions (typically 1:500-1:1000 for Western blotting) .

• Expression system compatibility: Some expression systems may not properly process or target mitochondrial proteins. Solution: Verify mitochondrial localization using fluorescent tags or subcellular fractionation followed by Western blotting.

• Functional redundancy: Multiple ATP5G genes exist (ATP5G1, ATP5G2, ATP5G3) that encode identical mature proteins, making it challenging to study isoform-specific functions. Solution: Use 5' UTR-targeted approaches or study tissue-specific expression patterns.

• Storage stability: Recombinant ATP5G1 may lose activity during storage. Solution: Store at -20°C in 50% glycerol, avoid repeated freeze-thaw cycles, and use working aliquots stored at 4°C for up to one week .

Addressing these challenges requires careful experimental design and appropriate controls to ensure reliable and reproducible results.