Recombinant Rat ATP synthase subunit a (Mt-atp6)
Description
Definition and Basic Properties
Recombinant Rat ATP synthase subunit a (Mt-atp6) is a laboratory-produced version of the naturally occurring mitochondrial protein encoded by the Mt-atp6 gene. This protein constitutes a critical component of the mitochondrial ATP synthase complex (Complex V), which produces ATP from ADP in the presence of a proton gradient across the inner mitochondrial membrane . The recombinant version is typically produced in expression systems such as Escherichia coli with an affinity tag (commonly His-tag) to facilitate purification and subsequent experimental applications .
The full-length rat Mt-atp6 protein consists of 226 amino acids and functions as a membrane-embedded subunit of the F0 portion of ATP synthase . The protein contains multiple transmembrane domains that anchor it within the mitochondrial inner membrane, where it plays a crucial role in proton translocation coupled to ATP synthesis .
Expression and Purification
Recombinant Rat Mt-atp6 is typically expressed in bacterial systems, with E. coli being the most common expression host . The protein is engineered with an N-terminal histidine tag (His-tag) that enables efficient purification using immobilized metal affinity chromatography (IMAC) . Following expression and purification, the protein is commonly supplied as a lyophilized powder, requiring reconstitution before use in experimental applications .
The purification process typically yields a protein with greater than 90% purity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . This high level of purity ensures reliable experimental results when the protein is used in biochemical and structural studies.
Role in ATP Synthesis
Mt-atp6 functions as a critical component of the ATP synthase complex, which represents the primary cellular machinery for ATP production via oxidative phosphorylation. This protein is specifically located in the F0 portion of the ATP synthase, which is embedded within the inner mitochondrial membrane . Mt-atp6 plays a direct role in the translocation of protons across the membrane, which is coupled to ATP synthesis in the catalytic F1 domain of the complex .
The protein contains a proton channel that facilitates the movement of hydrogen ions down their electrochemical gradient from the intermembrane space to the mitochondrial matrix . This proton movement drives the rotational mechanism of the ATP synthase, which in turn catalyzes the synthesis of ATP from ADP and inorganic phosphate .
Evolutionary Conservation
The Mt-atp6 protein shows significant evolutionary conservation across species, reflecting its essential role in cellular energy production. Comparative analysis of Mt-atp6 sequences from various organisms reveals conserved residues that are critical for protein function . These conserved regions typically correspond to the proton channel and other functionally important domains of the protein.
Research examining mutations in conserved residues has demonstrated their importance for proper ATP synthase function. Studies using yeast models have shown that alterations in these conserved regions can lead to significant deficits in ATP production and impaired cellular respiration .
Disease Modeling and Pathogenicity Assessment
Recombinant Mt-atp6 has proven invaluable for investigating the pathogenicity of mutations associated with mitochondrial diseases. Researchers have employed yeast models expressing variants of Mt-atp6 to assess the functional consequences of specific mutations identified in patients with various disorders .
Studies have demonstrated that mutations affecting conserved residues of Mt-atp6 can lead to significant deficits in ATP production and mitochondrial function. For example, investigations using yeast models have shown that certain mutations can reduce oxygen consumption (state 3 respiration) by approximately 18-25% and ATP synthesis rates by similar or greater proportions . These findings help establish the pathogenicity of specific variants and contribute to our understanding of mitochondrial disease mechanisms.
Mitochondrial Diseases Linked to Mt-atp6 Mutations
Mutations in the MT-ATP6 gene are associated with several mitochondrial disorders, including Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) and MT-ATP6-Related Mitochondrial Spastic Paraplegia . These conditions typically present with neurological symptoms reflecting the high energy demands of nervous system tissues.
Research using recombinant Mt-atp6 and model organisms has helped establish the pathogenicity of specific mutations. Studies have demonstrated that mutations affecting conserved residues can significantly compromise ATP synthase function, leading to bioenergetic deficits that underlie disease manifestations .
Broader Disease Associations
Beyond defined mitochondrial disorders, MT-ATP6 has been implicated in several other conditions, including Leber hereditary optic neuropathy, Parkinson's disease, multiple sclerosis, and systemic lupus erythematosus . These associations highlight the broader relevance of mitochondrial function to human health and disease.
The investigation of these disease associations often involves functional studies using recombinant proteins and model organisms. By assessing the impact of specific mutations on ATP synthase function, researchers can better understand the molecular mechanisms underlying disease pathogenesis and potentially identify targets for therapeutic intervention.
Mutation Studies Using Model Organisms
Research using yeast models expressing variants of Mt-atp6 has provided valuable insights into the functional consequences of specific mutations. Studies have demonstrated that mutations affecting conserved residues can significantly compromise ATP synthase function and cellular energy production .
For example, investigation of eight MT-ATP6 gene variants identified in patients with various disorders revealed that three variants (m.8950G>A, m.9025G>A, and m.9029A>G) significantly compromised ATP synthase function in yeast models . These variants led to significant defects in respiration-dependent growth and deficits in ATP production, providing evidence of their pathogenicity .
The remaining five variants showed very mild, if any, effect on mitochondrial function, suggesting that they do not have, at least alone, the potential to compromise human health . These findings highlight the utility of model organisms and recombinant proteins in assessing the pathogenicity of specific mutations.
Assembly and Stability Assessment
Research examining the impact of Mt-atp6 mutations on ATP synthase assembly and stability has provided insights into the molecular mechanisms underlying functional deficits. Studies using blue native polyacrylamide gel electrophoresis (BN-PAGE) have allowed visualization of the ATP synthase complex in its various forms (dimers, monomers, and free F1 particles) .
These investigations have revealed that some mutations can affect the assembly or stability of the ATP synthase complex, leading to reduced levels of functional enzyme complexes. Other mutations may primarily affect the proton translocation function of Mt-atp6 without significantly impacting complex assembly .
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please indicate them in your order notes, and we will fulfill your request to the best of our ability.
Note: All proteins are shipped with standard blue ice packs unless otherwise specified. If you require dry ice shipping, please communicate this in advance as 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
KEGG: rno:26197
STRING: 10116.ENSRNOP00000049769
Q&A
What is the functional role of ATP synthase subunit a in mitochondrial energy production?
ATP synthase subunit a (Mt-atp6) forms part of the F₀ region of ATP synthase and, together with the oligomeric c-subunit ring, creates the proton pathway responsible for transporting protons through the mitochondrial inner membrane. This proton movement is coupled to the rotation of the c-ring, ultimately driving ATP synthesis. The subunit is essential for converting the proton motive force generated by the electron transport chain into the mechanical energy required for ATP production .
The F₀ component, where subunit a resides, is embedded within the inner mitochondrial membrane and comprises multiple subunits (a, b, c, d, e, f, g, F6, and 8). Of these, only subunits a and 8 are encoded by the mitochondrial genome, while the others are nuclear-encoded .
How is recombinant rat Mt-atp6 typically detected in laboratory settings?
Recombinant rat Mt-atp6 can be detected using various techniques, with Western blotting being the most common approach. Specific monoclonal antibodies targeting ATP6, such as antibody 68442-1-Ig, have been developed for this purpose. These antibodies typically show reactivity with both human and rat samples .
For Western blot applications, a dilution range of 1:5000-1:50000 is recommended, though optimal dilutions should be determined for each specific testing system. The protein typically appears at 25-30 kDa on Western blots, close to its calculated molecular weight of 25 kDa .
What conservation patterns exist for ATP synthase subunit a across species?
ATP synthase is extraordinarily prevalent across all living organisms, and its subunits show remarkable evolutionary conservation. The ATP6 subunit contains numerous conserved residues that have withstood evolutionary pressure, reflecting their critical functional importance .
Amino acid sequence alignments between human and yeast ATP6 reveal this conservation. For example, human mutations at positions p.I106T, p.V142I, p.I164V, p.G167S, p.H168R, p.T178A, p.A205T, and p.Y212H correspond to positions in yeast at a-I123T, a-V159I, a-I181V, a-G184S, a-H185R, a-T195A, a-A225T, and a-Y232H respectively .
| Human Position | Human Mutation | Corresponding Yeast Position |
|---|---|---|
| p.I106T | m.8843T>C | a-I123T |
| p.V142I | m.8950G>A | a-V159I |
| p.I164V | m.9016A>G | a-I181V |
| p.G167S | m.9025G>A | a-G184S |
| p.H168R | m.9029A>G | a-H185R |
| p.T178A | m.9058A>G | a-T195A |
| p.A205T | m.9139G>A | a-A225T |
| p.Y212H | m.9160T>C | a-Y232H |
How can yeast models be effectively utilized to study MT-ATP6 mutations found in human patients?
Yeast models are valuable tools for investigating MT-ATP6 mutations due to several key advantages:
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Genetic tractability: Yeast allows for mitochondrial genetic transformation and doesn't stably maintain heteroplasmy (mixed wild-type and mutant mtDNA populations), making it easier to study the effects of specific mutations .
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Evolutionary conservation: The strong conservation between human and yeast ATP6 enables meaningful cross-species comparisons. Mutations in human MT-ATP6 can be introduced at equivalent positions in yeast ATP6 .
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Functional validation: Studies have demonstrated that MT-ATP6 mutations causing severe clinical phenotypes in humans dramatically affect yeast ATP synthase function, while mutations associated with milder diseases compromise oxidative phosphorylation less severely in yeast mitochondria .
For effective experimentation, researchers can:
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Introduce mutations into yeast ATP6 using site-directed mutagenesis
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Create diploid strains carrying the mutations
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Assess respiratory growth on non-fermentable substrates
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Analyze ATP synthase assembly using blue native polyacrylamide gel electrophoresis (BN-PAGE)
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Measure mitochondrial ATP production rates
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Identify suppressor mutations that restore function in compromised mutants
What techniques are most effective for assessing the impact of Mt-atp6 mutations on ATP synthase assembly and function?
Multiple complementary techniques provide comprehensive insights into how Mt-atp6 mutations affect ATP synthase:
How do specific missense mutations in Mt-atp6 differentially affect ATP synthase function and disease phenotypes?
Research has revealed that different MT-ATP6 mutations produce varying impacts on ATP synthase function, correlating with the severity of disease phenotypes:
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Severe functional impact mutations: Three mutations (m.8950G>A, m.9025G>A, and m.9029A>G) significantly compromise ATP synthase function. These mutations affect conserved amino acid residues and lead to substantial impairment of ATP production .
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Mild impact mutations: Five mutations (m.8843T>C, m.9016A>G, m.9058A>G, m.9139G>A, and m.9160T>C) have minimal effects on ATP synthase function, suggesting they may not independently compromise human health .
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The m.8993T>G mutation: This common pathogenic mutation causes replacement of a strictly conserved leucine residue with arginine (aL156R in humans, aL173R in yeast). In yeast models, this mutation dramatically impairs respiratory growth and causes a 90% reduction in mitochondrial ATP synthesis. The severity of this effect explains its association with Leigh syndrome and Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) .
First-site reversions at the mutation site (aR173M, aR173K, and aR173S) and second-site suppressor mutations at nearby positions can partially restore function, providing insights into structure-function relationships within the ATP synthase complex .
What are the optimal conditions for expressing and purifying recombinant rat Mt-atp6?
Expressing and purifying mitochondrially-encoded proteins like Mt-atp6 presents unique challenges due to their hydrophobic nature and integration into multiprotein complexes. Consider these approaches:
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Expression Systems:
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Bacterial expression systems can be used, but require careful optimization due to the hydrophobic nature of Mt-atp6
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Cell-free expression systems are advantageous for membrane proteins
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Baculovirus-insect cell systems may provide higher yields with proper post-translational modifications
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Solubilization and Purification:
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Use mild detergents (digitonin, n-dodecyl β-D-maltoside) to solubilize membrane proteins while maintaining native structure
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Employ affinity tags (His-tag) for initial purification
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Consider Size Exclusion Chromatography (SEC) for further purification
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Store in buffer containing glycerol (up to 50%) and appropriate detergent
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Quality Control:
What strategies can be employed to study the structural dynamics of Mt-atp6 during proton transport?
Understanding the structural dynamics of Mt-atp6 during proton transport involves sophisticated methodological approaches:
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Site-Directed Mutagenesis: Systematically alter conserved residues in the proton pathway to evaluate their roles in proton translocation and coupling to ATP synthesis .
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Cross-linking Studies: Chemical cross-linking combined with mass spectrometry can capture transient interactions between subunit a and the c-ring during rotation.
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Molecular Dynamics Simulations: Computational modeling based on known structural information can predict conformational changes during proton transport.
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Single-Molecule Techniques: Fluorescence resonance energy transfer (FRET) or high-speed atomic force microscopy (HS-AFM) can monitor real-time structural changes in functioning ATP synthase.
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Suppressor Mutation Analysis: Identify second-site mutations that restore function in compromised mutants, revealing functional relationships between different regions of the protein .
How can heteroplasmy of Mt-atp6 mutations be modeled in experimental systems?
Modeling heteroplasmy (the coexistence of wild-type and mutant mtDNA) presents unique challenges:
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Cybrid Cell Lines: Human cybrid (cytoplasmic hybrid) cells containing various proportions of mutant and wild-type mtDNA can be generated by fusing enucleated patient-derived cells with mtDNA-depleted (ρ⁰) recipient cells .
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Mouse Models: Heteroplasmic mice can be created using mitochondrial targeted nucleases or base editors to introduce specific mutations at varying levels.
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Quantification Methods:
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Threshold Effect Analysis: Systematic studies with varying heteroplasmy levels can determine the threshold at which biochemical and phenotypic defects manifest, typically between 60-90% mutant load for many MT-ATP6 mutations .
Note that yeast naturally segregates to homoplasmy and cannot stably maintain heteroplasmy, which makes it unsuitable for heteroplasmy modeling but advantageous for studying homoplasmic effects of mutations .
How can MT-ATP6 research contribute to understanding and treating mitochondrial diseases?
Research on MT-ATP6 offers several pathways toward understanding and potentially treating mitochondrial diseases:
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Mutation-Specific Pathogenicity Assessment: Evaluating the functional impact of patient-derived MT-ATP6 variants helps distinguish pathogenic mutations from benign polymorphisms, improving diagnostic accuracy. Studies in yeast models have successfully validated the pathogenicity of numerous MT-ATP6 mutations .
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Structure-Function Relationships: Investigations of how specific mutations affect ATP synthase assembly, stability, and function provide insights into disease mechanisms. For example, studies of m.8993T>G (causing aL156R in humans) have revealed how this mutation dramatically impairs proton transport, explaining its severe clinical manifestations .
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Therapeutic Target Identification: Understanding how suppressor mutations can rescue ATP synthase function may identify targets for therapeutic intervention. Studies in yeast have identified several compensatory mutations that partially restore function in MT-ATP6 mutants .
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Pharmacological Approaches: Research on ATP synthase inhibitors provides insights that could be reversed to develop compounds that enhance ATP synthase function in deficient states .
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Gene Therapy Approaches: The development of mitochondrial-targeted nucleic acid delivery systems could eventually enable correction of MT-ATP6 mutations directly in affected tissues.
What is the relationship between Mt-atp6 mutations and mitochondrial reactive oxygen species (ROS) production?
The relationship between Mt-atp6 mutations and ROS production represents an important area of investigation:
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Mechanisms of Increased ROS: Mt-atp6 mutations can lead to increased ROS production through:
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Experimental Evidence: Studies have shown that inhibiting ATP synthase in rat cardiac myocytes leads to elevated oxidative stress and calcium levels, ultimately causing cell death .
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Vicious Cycle Hypothesis: Increased ROS resulting from Mt-atp6 mutations can damage additional mtDNA molecules, potentially creating a vicious cycle of mitochondrial dysfunction.
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Tissue-Specific Effects: The relationship between Mt-atp6 mutations and ROS production may vary across tissues, explaining why certain organs (brain, heart, kidneys) are particularly affected in ATP synthase disorders .
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Therapeutic Implications: Antioxidant therapies may provide benefit in cases where Mt-atp6 mutations lead to increased ROS production, suggesting a potential treatment avenue that would complement approaches aimed at improving ATP synthase function directly.
