Recombinant Mouse ATP synthase subunit a (Mtatp6)
Description
Definition and Origin
MT-ATP6, or Mitochondrially Encoded ATP Synthase 6, is a protein encoded by the mitochondrial genome that forms an essential subunit (subunit a) of the ATP synthase enzyme complex. This protein is crucial for normal mitochondrial function and serves as a critical component in cellular energy production . The recombinant form of mouse MT-ATP6 refers to artificially produced versions of this protein created specifically for research purposes, typically through heterologous expression systems.
Role in ATP Synthase Complex
MT-ATP6 forms one part (subunit a) of the large ATP synthase enzyme, also known as complex V in the mitochondrial respiratory chain. This subunit integrates into the F0 portion of ATP synthase, which spans the inner mitochondrial membrane . Within this complex, MT-ATP6 plays a critical role in facilitating the flow of protons (H⁺) across the inner mitochondrial membrane, creating an electrochemical gradient that drives ATP synthesis .
Energy Production Mechanism
The primary function of MT-ATP6 within the ATP synthase complex is to allow positively charged protons to flow across the specialized inner mitochondrial membrane. This proton flow generates energy that is harnessed by another segment of the enzyme to convert adenosine diphosphate (ADP) to adenosine triphosphate (ATP), which serves as the main energy source for cellular processes . This process, known as oxidative phosphorylation, is fundamental to aerobic energy metabolism across nearly all eukaryotic cells.
Expression Systems
Recombinant mouse MT-ATP6 can be produced using several different expression systems, each with distinct advantages for research applications. The table below compares the primary expression systems used for MT-ATP6 production:
Purification and Protein Characteristics
Recombinant MT-ATP6 proteins are typically purified using affinity chromatography techniques facilitated by fusion tags. For instance, mouse MT-ATP6 proteins are available commercially with Strep-Tag or His-Tag conjugations . These affinity tags enable one-step purification processes while potentially maintaining the protein's enzymatic functionality, although this must be verified experimentally .
The purified recombinant proteins are suitable for various experimental applications, including ELISA, SDS-PAGE, and Western blotting, making them valuable tools for studying mitochondrial protein function and interactions .
Investigation of Mitochondrial Diseases
Recombinant MT-ATP6 proteins serve as critical tools for investigating mitochondrial diseases associated with mutations in the MT-ATP6 gene. These proteins enable researchers to study the functional consequences of these mutations in controlled experimental systems. Significant MT-ATP6-related diseases include:
Leigh Syndrome
Mutations in the MT-ATP6 gene have been found in approximately 10 percent of individuals with Leigh syndrome, a progressive brain disorder that typically manifests in infancy or early childhood . The most common genetic change associated with this syndrome is the T8993G mutation, which replaces the nucleotide thymine with guanine at position 8993 in the MT-ATP6 gene .
NARP and Related Disorders
The syndrome of Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) represents another significant MT-ATP6-related disorder. Research has shown that approximately 20% of patients with MT-ATP6 mutations exhibit the complete NARP phenotype . A study of a large clinical cohort revealed that the majority of patients with MT-ATP6 mutations present with a combination of cerebellar ataxia (90%), peripheral neuropathy (100%), and cognitive dysfunction (80%) .
Experimental Models and Pathogenicity Assessment
Researchers have developed multiple experimental models to assess the pathogenicity of MT-ATP6 variants. For example, yeast models have been constructed to study the functional consequences of patient-derived MT-ATP6 variants . These models allow for the investigation of eight specific MT-ATP6 gene variants found in patients with various mitochondrial disorders, including mutations that lead to amino acid substitutions such as p.I106T, p.V142I, p.I164V, and others .
The following table summarizes key MT-ATP6 mutations and their associated clinical features:
Pathophysiology of MT-ATP6 Mutations
Mutations in the MT-ATP6 gene can significantly impair the function or stability of the ATP synthase complex, thereby inhibiting ATP production and disrupting oxidative phosphorylation . While the exact mechanisms remain incompletely understood, research suggests that impaired oxidative phosphorylation can lead to cell death due to decreased energy availability within affected cells .
Tissue-Specific Effects
Tissues with high energy demands, such as the brain, muscles, and heart, appear particularly sensitive to decreases in cellular energy production resulting from MT-ATP6 mutations . This tissue specificity helps explain the neurological and muscular symptoms predominantly observed in MT-ATP6-related disorders, including cerebellar ataxia, lower limb spasticity, and movement disorders .
Quality Considerations
When selecting recombinant MT-ATP6 proteins for research applications, several quality factors should be considered, including expression system compatibility, purification method, tag position and type, and functional validation status. Made-to-order proteins may be advantageous for specialized applications, as they can be designed specifically for the intended experimental purpose .
Therapeutic Potential
Ongoing research using recombinant MT-ATP6 proteins may contribute to the development of potential therapeutic approaches for MT-ATP6-related mitochondrial disorders. By facilitating the detailed characterization of mutation effects on protein function, these recombinant proteins enable researchers to identify potential targets for intervention.
Diagnostic Applications
Recombinant MT-ATP6 proteins also show promise as tools for developing improved diagnostic methods for mitochondrial disorders. They may serve as standards or controls in assays designed to detect mutations or assess ATP synthase function in patient samples.
Product Specs
Note: We will prioritize shipping the format we have in stock. However, if you have specific requirements for the format, please indicate your needs when placing the order. We will accommodate your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance. Additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
- Mice were created that allotopically express either a mutant (A6M) or wildtype (A6W) mt-Atp6 transgene. PMID: 22778551
KEGG: mmu:17705
STRING: 10090.ENSMUSP00000080996
Q&A
What is mouse ATP synthase subunit a and how does it function within ATP synthase?
Mouse ATP synthase subunit a (encoded by the mitochondrial MT-ATP6 gene) forms a crucial component of the F₀ sector of mitochondrial ATP synthase (Complex V). This protein plays an essential role in the final step of oxidative phosphorylation, which converts the energy from food into ATP, the cell's main energy source . Specifically, subunit a forms part of the proton channel through which positively charged particles (protons) flow across the inner mitochondrial membrane . This proton movement drives the rotary mechanism that enables ATP synthesis from ADP by the F₁ segment of the complex .
ATP synthase functions through a mechanism called "rotary catalysis," where the flow of protons through the F₀ portion (which includes subunit a) causes rotation of the c-ring and the attached central stalk (γ, δ, and ε subunits) . This rotation drives conformational changes in the catalytic sites located in the β subunits of the F₁ sector, facilitating ATP production . Subunit a is particularly important as it provides part of the pathway for protons to access the c-ring, thus enabling the proton-motive force to drive ATP synthesis .
How does mouse MT-ATP6 differ from human MT-ATP6?
While both mouse and human MT-ATP6 genes encode the subunit a protein of ATP synthase with similar core functions, several differences exist in their genetic and protein characteristics:
What are the common challenges in expressing recombinant mouse ATP synthase subunit a?
Expression of functional recombinant mouse ATP synthase subunit a presents several technical challenges:
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Mitochondrial encoding: MT-ATP6 is encoded by mitochondrial DNA rather than nuclear DNA, complicating standard recombinant expression approaches .
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Membrane protein solubility: As a hydrophobic membrane protein, subunit a is difficult to express in soluble form without proper membrane incorporation .
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Complex assembly requirements: Subunit a normally functions as part of the larger ATP synthase complex, requiring appropriate interactions with other subunits for proper folding and function .
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Coordinated assembly: ATP synthase assembly involves multiple pathways that must converge properly. Research indicates that in yeast, ATP synthase forms from three distinct modules (c-ring, F₁, and the Atp6p/Atp8p complex), with subunit a incorporation occurring at late stages of assembly .
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Post-translational modifications: Proper function may require specific mitochondrial-specific modifications not replicated in common expression systems.
These challenges typically necessitate specialized expression systems and purification strategies that maintain the protein's native conformation and functionality.
What are the optimal expression systems for producing functional recombinant mouse ATP synthase subunit a?
Based on current research practices, several expression systems have been explored for producing recombinant mitochondrial proteins, each with specific advantages for ATP synthase subunit a:
For structural studies, E. coli expression with solubility-enhancing tags (such as His-SUMO) has shown success for mitochondrial proteins . The recombinant mouse ATP synthase subunit beta has been successfully expressed in E. coli with His-SUMO tags, achieving >90% purity as determined by SDS-PAGE . Similar approaches may be adapted for subunit a, though its higher hydrophobicity presents additional challenges.
For functional studies, yeast or mammalian expression systems that preserve the protein's native environment are generally preferable, particularly when studying interactions with other ATP synthase components .
What role does ATP synthase subunit a play in mitochondrial diseases and how can recombinant proteins help study these conditions?
ATP synthase subunit a plays a critical role in several mitochondrial diseases, most notably Leigh syndrome . Mutations in the MT-ATP6 gene have been found in approximately 10% of people with this progressive brain disorder . The most common genetic change replaces the nucleotide thymine with guanine at position 8993 (T8993G) .
Recombinant protein studies can advance understanding of these diseases through:
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Structure-function analysis: Comparing wild-type and mutant forms of recombinant subunit a can reveal how specific mutations alter protein structure and complex assembly .
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Interaction studies: Recombinant proteins enable investigation of how subunit a interacts with other ATP synthase components, particularly how disease mutations disrupt these interactions .
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Drug screening platforms: Purified recombinant proteins can serve as targets for small molecule screening to identify compounds that might restore function in mutant forms .
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Bioenergetic assays: Incorporating recombinant subunit a into reconstituted systems allows measurement of proton translocation and ATP synthesis activities to quantify the impact of mutations .
When studying disease mechanisms, researchers should note that mutations in MT-ATP6 impair ATP synthase function or stability, inhibiting ATP production and compromising oxidative phosphorylation . This impairment is believed to lead to cell death due to energy deficiency, particularly affecting tissues with high energy demands like the brain, muscles, and heart .
How can researchers effectively validate the functionality of recombinant mouse ATP synthase subunit a?
Validating the functionality of recombinant ATP synthase subunit a requires multiple complementary approaches:
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Structural integrity assessment:
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Circular dichroism spectroscopy to confirm secondary structure
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Limited proteolysis to verify proper folding
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Size exclusion chromatography to assess oligomeric state
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Membrane incorporation:
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Liposome reconstitution experiments
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Electron microscopy to visualize membrane insertion
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Complex assembly validation:
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Functional assays:
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Proton translocation measurements using pH-sensitive fluorescent dyes
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ATP synthesis activity in reconstituted systems
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Membrane potential measurements across proteoliposomes
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Complementation studies:
Research indicates that ATP synthase assembly can be monitored using techniques like CN-PAGE, which has shown that in human mitochondria lacking mtDNA (and therefore subunits a and A6L), ATP synthase can still assemble into a complex with a mass of 550 kDa, slightly smaller than the complete 597 kDa holocomplex . This suggests that subunit a incorporation is a late step in assembly, providing a means to assess successful incorporation of recombinant subunit a.
What are the key considerations for designing experiments with recombinant mouse ATP synthase subunit a?
When designing experiments with recombinant mouse ATP synthase subunit a, researchers should consider:
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Expression strategy selection:
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Choose between full-length protein or functional domains based on experimental goals
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Consider fusion tags that enhance solubility while minimizing functional interference
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The recombinant mouse ATP synthase subunit beta has been successfully expressed with His-SUMO tags in E. coli systems, providing a potential model for subunit a expression
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Purification approach:
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Develop detergent strategies that maintain native-like membrane protein environments
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Consider amphipols or nanodiscs for stabilizing the purified protein
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Implement quality control steps at each purification stage
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Functional reconstitution:
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Select appropriate lipid compositions that mimic the mitochondrial inner membrane
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Consider co-expression or co-reconstitution with interacting subunits
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ATP synthase assembly in yeast involves separate pathways (F₁/Atp9p and Atp6p/Atp8p/stator subunits) that converge at the end stage, suggesting similar approaches may be needed for mouse proteins
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Controls and validation:
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Include well-characterized mutants as functional controls
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Utilize complementary biophysical techniques to verify structural integrity
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Confirm activity matches expectations based on native enzyme benchmarks
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Physiological relevance:
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Consider whether the experimental conditions reflect the protein's native environment
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Account for species-specific differences when comparing to human MT-ATP6
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What techniques are most effective for studying ATP synthase assembly and function using recombinant subunits?
Research on ATP synthase assembly and function with recombinant subunits benefits from multiple complementary techniques:
Studies have shown that ATP synthase assembly in mammals involves separate assembly of the F₁ sector, the c-ring, and the peripheral stalk, followed by their combination and the final addition of mitochondrially-encoded subunits a and A6L . Clear native PAGE has revealed that human ATP synthase can form a 550 kDa complex (without subunits a and A6L) compared to the complete 597 kDa holocomplex . These approaches can be adapted to study recombinant mouse ATP synthase subunit a incorporation and function.
How can researchers troubleshoot common issues in experiments with recombinant ATP synthase subunit a?
Common experimental issues and their solutions include:
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Poor expression yields:
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Protein aggregation:
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Screen various detergents systematically
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Implement on-column detergent exchange during purification
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Test amphipathic polymers (amphipols) for stabilization
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Consider nanodiscs or liposomes for membrane mimetics
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Use glycerol in storage buffers (50% glycerol has been used successfully for ATP synthase subunits)
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Inactive protein:
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Verify proper folding through limited proteolysis
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Check redox state of any critical cysteine residues
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Ensure appropriate lipid environment for function
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Consider co-expression with interacting partners
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Test activity in the presence of other ATP synthase components
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Assembly failure:
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Follow the natural assembly pathway discovered in yeast models
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Co-express with assembly factors like TMEM70, which influences complex V amount in mitochondria
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Test progressive reconstitution of subcomplex modules
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ATP synthase assembly occurs through step-wise addition of modules, with subunit a typically added in late stages
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Functional assay sensitivity:
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Optimize proton gradient formation in reconstituted systems
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Enhance detection sensitivity through fluorescent reporters
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Minimize background ATPase activity with specific inhibitors
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Implement real-time assays rather than endpoint measurements
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How does the structure-function relationship of mouse ATP synthase subunit a compare to other species?
The structure-function relationship of ATP synthase subunit a shows both conservation and species-specific adaptations:
Despite sequence divergence, the core function of subunit a in proton translocation and ATP synthesis is conserved across species . The detailed structure of bovine ATP synthase subunits (closely related to mouse) has been resolved by X-ray crystallography by John Walker's group, providing valuable structural insights into mammalian ATP synthase .
Research indicates that subunit a works with subunit A6L to stabilize the complete ATP synthase complex (holocomplex V) . These subunits also play important roles in ATP synthase dimerization and oligomerization, which influences mitochondrial cristae formation .
What role does ATP synthase subunit a play in mitochondrial permeability transition?
Although ATP synthase subunit c (not subunit a) has been primarily implicated in forming the mitochondrial permeability transition pore (mPTP) , current research suggests complex interrelationships between various ATP synthase subunits in regulating mitochondrial permeability:
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Structural contributions: While subunit a itself may not form the pore, it plays a critical role in stabilizing the ATP synthase complex, potentially influencing conformational changes that regulate pore opening .
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Bioenergetic regulation: By participating in proton translocation, subunit a helps maintain the proton gradient and membrane potential (Δψm), which are critical factors in mPT activation .
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Complex assembly and stability: Subunit a, along with A6L, provides stability to holocomplex V, and this stability may influence the susceptibility of ATP synthase to form the mPT pore under stress conditions .
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Interaction with c-ring: Subunit a interfaces with the c-ring, which has been implicated as housing the leak channel involved in mitochondrial permeability transition during excitotoxic ischemic insult .
Understanding these relationships is critical for research into ischemia-reperfusion injury, neurodegenerative diseases, and other conditions where mitochondrial permeability transition plays a pathological role.
What are the emerging applications of recombinant ATP synthase subunits in drug discovery?
Recombinant ATP synthase subunits, including subunit a, are becoming valuable tools in drug discovery pipelines:
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Target-based screening: Purified recombinant ATP synthase subunit a can serve as a target for high-throughput screening of compound libraries to identify molecules that modulate its function or stabilize disease-causing mutants .
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Structure-based drug design: The availability of recombinant protein enables structural studies that can guide rational design of therapeutics targeting specific functional domains or interaction surfaces .
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Neuroprotective strategies: Given the role of ATP synthase in mitochondrial permeability transition, which is implicated in neurodegeneration, compounds that modulate subunit a function may have neuroprotective potential .
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Leigh syndrome therapeutics: Since mutations in MT-ATP6 account for approximately 10% of Leigh syndrome cases, recombinant systems can facilitate screening for compounds that might rescue function in specific mutations like T8993G .
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Bioenergetic enhancers: Compounds that optimize ATP synthase efficiency could potentially address mitochondrial dysfunction in aging and metabolic disorders.
Researchers pursuing drug discovery applications should consider that ATP synthase is involved not only in energy production but also in energy dissipation and cell death pathways , offering multiple potential intervention points for therapeutic development.
