Recombinant Cricetulus griseus ATP synthase protein 8 (MT-ATP8)
Description
Introduction to MT-ATP8
ATP synthase represents one of the most fundamental protein complexes in cellular bioenergetics, serving as the primary mechanism for ATP production through oxidative phosphorylation. This molecular machine consists of two main domains: the F₁ catalytic domain and the F₀ membrane-embedded proton channel . MT-ATP8, also referred to as A6L or F-ATPase subunit 8, constitutes an essential component of the F₀ complex within the mitochondrial inner membrane . Originally identified as an unidentified reading frame (URF A6L) in early mitochondrial genome studies, MT-ATP8 has emerged as a critical element for proper ATP synthase assembly and function .
In Cricetulus griseus (Chinese hamster), MT-ATP8 shares fundamental structural and functional characteristics with its homologs across mammalian species, making it a valuable model for investigating conserved aspects of mitochondrial energy production . The gene encoding this protein is located within the mitochondrial genome, highlighting its evolutionary significance as one of the few proteins still encoded by mitochondrial DNA rather than nuclear DNA . This conservation underscores the protein's essential role in cellular energetics across diverse mammalian species.
ATP Synthase Complex Overview
ATP synthase functions as a rotary molecular motor consisting of multiple subunits organized into two primary domains. The F₁ domain contains the catalytic core responsible for ATP synthesis, while the F₀ domain forms the transmembrane proton channel that harnesses the proton gradient established across the inner mitochondrial membrane . This proton gradient, generated through the electron transport chain, drives the rotation of the F₀ complex, which mechanically couples to the F₁ domain to catalyze ATP production . The entire complex represents a marvel of molecular engineering, efficiently converting electrochemical energy into chemical energy in the form of ATP.
Evolutionary Significance of MT-ATP8
The MT-ATP8 gene demonstrates variable sequence conservation across different taxonomic groups, with significant differences observed between metazoa, plants, and fungi . Despite this sequence divergence, the structural role of subunit 8 appears largely preserved, suggesting functional constraints on its evolution . In mammalian systems, MT-ATP8 has been retained in the mitochondrial genome alongside MT-ATP6, with which it shares a 46-nucleotide overlap - an unusual genomic arrangement highlighting their interrelated functions .
Structure and Functional Characteristics
The MT-ATP8 protein from Cricetulus griseus consists of 67 amino acids with the sequence MPQLDTSTWFTTVLASSITLFILMQLKISFHDLHKKPSNKYLKLFKPTNPWEQKWTKIYSPLSLPQP . This relatively small protein (approximately 8 kDa) plays a disproportionately important role in the structural organization of the ATP synthase complex . Structurally, MT-ATP8 forms an α-helical domain that spans the mitochondrial inner membrane, with its C-terminal portion extending into the mitochondrial matrix .
Functional Role
The precise functional contribution of MT-ATP8 to ATP synthase activity remains somewhat enigmatic, though structural evidence suggests it serves as an integral component of the stator stalk in mitochondrial F-ATPases . This stator structure anchors into the mitochondrial membrane and prevents futile rotation of ATPase subunits relative to the rotor during coupled ATP synthesis and hydrolysis . By helping maintain the spatial organization of the complex, MT-ATP8 contributes to the efficiency of proton flow and energy conversion within the ATP synthase machinery.
Interaction with Other Subunits
MT-ATP8 functions within a complex network of protein-protein interactions that collectively establish the architecture of the ATP synthase complex. Its positioning adjacent to subunit a appears particularly significant, as this subunit forms part of the critical proton channel with the c-ring . Additionally, MT-ATP8 interacts with other membrane-embedded subunits to form a stable stator structure that resists the torque generated during rotary catalysis .
Recombinant Expression and Characteristics
Recombinant Cricetulus griseus MT-ATP8 protein provides researchers with a valuable tool for investigating this important mitochondrial component outside its native environment. The recombinant protein retains the complete 67-amino acid sequence of the native protein, potentially with additional tag sequences depending on the expression system employed .
Expression Systems and Purification
While specific expression systems for Cricetulus griseus MT-ATP8 are not detailed in the provided sources, related mitochondrial proteins such as Balaenoptera musculus MT-ATP8 have been successfully expressed in E. coli systems with N-terminal His-tags to facilitate purification . These expression strategies likely involve optimization of codon usage for prokaryotic expression and careful consideration of the hydrophobic nature of membrane proteins . Purification typically involves affinity chromatography leveraging tag systems, with final products achieving greater than 90% purity as determined by analytical methods such as SDS-PAGE .
Comparative Analysis with Other Species
The amino acid sequence of Cricetulus griseus MT-ATP8 (MPQLDTSTWFTTVLASSITLFILMQLKISFHDLHKKPSNKYLKLFKPTNPWEQKWTKIYSPLSLPQP) demonstrates both similarities and differences when compared with other mammalian homologs . For instance, the Blue whale (Balaenoptera musculus) MT-ATP8 sequence (MPQLDTSTWLLTILSMLLTLFVLFQLKISKHSYSPNPKLVPTKTQKQQTPWNITWTKIYLPLL) shows conservation in the N-terminal region but divergence in other portions of the protein . These sequence variations provide valuable insights into the evolutionary constraints on different regions of the protein and may correlate with functional or structural requirements.
Research Applications
Recombinant Cricetulus griseus MT-ATP8 provides researchers with numerous applications across biochemical, structural, and functional studies of mitochondrial energy production.
Functional Assays and Enzyme Kinetics
Recombinant MT-ATP8 can be incorporated into reconstituted systems to investigate its contribution to ATP synthase function and stability . Though not directly involved in catalytic proton transfer, its structural role may significantly impact the efficiency of ATP synthesis under various conditions . Comparative functional studies using wild-type and mutant forms of the protein can reveal the importance of specific residues for proper positioning and function within the complex.
Immunological Applications
As a purified antigen, recombinant MT-ATP8 has applications in antibody production and immunoassay development . Enzyme-linked immunosorbent assays (ELISA) utilizing this recombinant protein enable detection and quantification of antibodies against MT-ATP8, which may be relevant in certain autoimmune conditions or as biomarkers for mitochondrial dysfunction .
Disease Relevance and Mutation Studies
The MT-ATP8 gene has attracted increasing attention in the context of mitochondrial disorders, with several pathogenic variants identified in human patients.
MT-ATP8 Variants and Disease Association
Research has identified multiple variants in the human MT-ATP8 gene associated with various mitochondrial diseases and syndromes . For example, the m.8381A>G (T6A) variant has been linked to maternally inherited diabetes and deafness (MIDD) and left ventricular non-compaction cardiomyopathy, while m.8403T>C (I13T) has been associated with episodic weakness and progressive neuropathy . The table below summarizes some of the reported MT-ATP8 variants and their clinical associations:
| mtDNA Variant | Amino Acid Change | Associated Disease/Syndrome | Pathogenic Score |
|---|---|---|---|
| m.8381A>G | T6A | MIDD/LVNC cardiomyopathy | 0.47 |
| m.8382C>T | T6I | Episodic paralysis | 0.58 |
| m.8403T>C | I13T | Episodic weakness and progressive neuropathy | 0.77 |
| m.8411A>G | M16V | Severe mitochondrial disorder | 0.63 |
These disease associations highlight the clinical significance of MT-ATP8 and the potential consequences of disrupting its structure or function .
Model Systems for Mutation Studies
Yeast (Saccharomyces cerevisiae) has proven valuable as a model system for investigating the effects of mutations in MT-ATP8 and related genes . Despite differences in primary sequence between yeast and mammalian MT-ATP8, the structural role appears sufficiently conserved to provide insights into the functional consequences of specific mutations . Such model systems enable researchers to characterize the biochemical and bioenergetic impacts of mutations identified in human patients, potentially revealing mechanisms of pathogenesis and therapeutic targets .
Future Research Directions
The availability of recombinant Cricetulus griseus MT-ATP8 opens numerous avenues for future research that may enhance our understanding of mitochondrial function and dysfunction.
Comparative Mitochondrial Biology
The availability of recombinant MT-ATP8 from Cricetulus griseus facilitates comparative studies with homologs from other species, potentially revealing evolutionary adaptations in mitochondrial function across different taxonomic groups . Such comparative approaches may identify conserved functional elements that have remained under selective pressure throughout evolutionary history, distinguishing them from more variable regions that may reflect species-specific adaptations .
Therapeutic Developments
As our understanding of MT-ATP8's role in health and disease advances, this knowledge may inform the development of therapeutic strategies targeting mitochondrial dysfunction . For instance, compounds that stabilize ATP synthase structure in the presence of destabilizing mutations could potentially mitigate the consequences of certain pathogenic variants . Additionally, gene therapy approaches targeting mitochondrial DNA may eventually provide more direct interventions for MT-ATP8-related disorders .
Product Specs
Please note: We will prioritize shipping the format that is currently in stock. However, if you have specific requirements for the format, please indicate your needs in the order notes, and we will prepare the product accordingly.
Note: All protein orders are shipped with standard blue ice packs unless otherwise requested. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, the shelf life for the liquid form is 6 months at -20°C/-80°C. The lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: cge:3979187
Q&A
What is the molecular structure and function of MT-ATP8 in Cricetulus griseus?
MT-ATP8 (ATP synthase protein 8) is a small, hydrophobic subunit of the mitochondrial ATP synthase complex comprising 67 amino acids with the sequence MPQLDTSTWFTTVLASSITLFILMQLKISFHDLHKKPSNKYLKLFKPTNPWEQKWTKIYSPLSLPQP . It functions as an essential component of the F0 sector of ATP synthase, contributing to proton translocation across the inner mitochondrial membrane. Structurally, MT-ATP8 contains a transmembrane domain that anchors it within the membrane and interacts with other subunits to maintain the integrity of the ATP synthase complex. Its relatively small size belies its critical importance in energy production within mitochondria.
How is the MT-ATP8 gene organized in mitochondrial DNA and how does this affect its expression?
In Cricetulus griseus, as in other mammals, MT-ATP8 is encoded by the mitochondrial genome and typically exists in a bicistronic arrangement with ATP6, forming a shared transcript . This genetic organization has significant implications for expression regulation. Research methodologies for studying this organization include:
-
Northern blot analysis to detect the bicistronic ATP8/ATP6 mRNA
-
RT-PCR with primers spanning the junction region
-
RNA-seq to quantify expression levels and identify potential processing intermediates
-
Mitochondrial DNA sequencing to characterize strain-specific variations
The bicistronic nature of this transcript creates a complex regulatory landscape where translation of both genes must be coordinated, often through sophisticated post-transcriptional mechanisms .
What experimental approaches can distinguish between MT-ATP8 and nuclear-encoded ATP synthase components?
Differentiating between mitochondrial-encoded MT-ATP8 and nuclear-encoded ATP synthase components requires specialized experimental approaches:
| Approach | Methodology | Application |
|---|---|---|
| Differential inhibition | Chloramphenicol (mitochondrial) vs. cycloheximide (cytosolic) translation inhibitors | Pulse-labeling experiments |
| Genome editing | CRISPR-Cas9 for nuclear genes vs. mitochondrial targeting nucleases | Creating knockout models |
| Expression kinetics | Short-term vs. long-term inhibition recovery | Measuring synthesis rates |
| Subcellular fractionation | Mitochondrial isolation followed by proteomic analysis | Protein localization |
| Import assays | In vitro transcription/translation with mitochondrial import | Testing protein targeting |
These approaches allow researchers to study the specific contribution of MT-ATP8 to ATP synthase function without confounding effects from nuclear-encoded components.
What expression systems are most effective for producing functional recombinant MT-ATP8?
The optimal expression system depends on research objectives, with several options available:
| Expression System | Advantages | Limitations | Yield | Post-translational Modifications |
|---|---|---|---|---|
| E. coli | Cost-effective, rapid, scalable | Limited PTMs, inclusion bodies | High | Minimal |
| CHO cells | Native-like processing, proper folding | Higher cost, slower growth | Moderate | Extensive, native-like |
| Insect cells | Higher yield than mammalian, some PTMs | Less authentic PTMs than CHO | High-Moderate | Intermediate |
| Cell-free systems | Rapid, membrane protein-friendly | Expensive, limited scale | Variable | Customizable |
For functional studies of MT-ATP8, mammalian expression systems, particularly CHO cells, often provide the most physiologically relevant results despite lower yields. Methodological considerations include codon optimization for the target organism, use of appropriate signal sequences, and selection of tags that minimally interfere with function .
What strategies can overcome the challenges in purifying hydrophobic MT-ATP8?
Purification of hydrophobic membrane proteins like MT-ATP8 presents significant challenges. Effective methodological approaches include:
-
Detergent screening protocol:
-
Test multiple detergent classes (mild, moderate, strong) at varying concentrations
-
Assess protein stability using size-exclusion chromatography
-
Optimize detergent-to-protein ratios for extraction efficiency
-
-
Solubilization strategy:
-
Use fusion partners (MBP, SUMO) to enhance solubility
-
Employ amphipols or nanodiscs for maintaining native-like membrane environment
-
Implement gentle extraction conditions with appropriate buffer components
-
-
Purification workflow:
-
Initial capture using affinity chromatography (His-tag, GST-tag)
-
Intermediate purification using ion exchange chromatography
-
Polishing step with size exclusion chromatography to ensure homogeneity
-
Researchers should monitor protein quality throughout purification using techniques such as circular dichroism and thermal shift assays to ensure the protein maintains its native conformation .
How can researchers validate the functional integrity of purified recombinant MT-ATP8?
Validating functional integrity requires multiple complementary approaches:
-
ATP synthesis assays using reconstituted proteoliposomes
-
Proton translocation measurements with pH-sensitive fluorescent dyes
-
Binding assays with known interaction partners using surface plasmon resonance
-
Structural integrity assessment through limited proteolysis
-
Oligomeric state determination using analytical ultracentrifugation
These methodologies collectively provide a comprehensive assessment of whether purified MT-ATP8 retains its native structural and functional properties after recombinant expression and purification.
How does MT-ATP8 contribute to ATP synthase assembly and what methods best study this process?
MT-ATP8 plays a critical role in the assembly and stability of the F0 sector of ATP synthase. Research methodologies to investigate its contribution include:
-
Assembly intermediate characterization:
-
Blue native PAGE to resolve assembly complexes
-
Immunoprecipitation followed by mass spectrometry
-
Pulse-chase labeling to track assembly kinetics
-
FRET-based approaches for studying subunit interactions
-
-
Mutagenesis approaches:
-
Site-directed mutagenesis of key residues
-
Deletion analysis to identify essential domains
-
Chimeric constructs to map functional regions
-
Temperature-sensitive mutants to study assembly dynamics
-
Cross-linking studies combined with mass spectrometry have been particularly informative, revealing proximity relationships between MT-ATP8 and other subunits within the assembled complex .
What experimental designs best elucidate the regulatory relationship between F1 ATPase and MT-ATP8 expression?
Research has established that F1 ATPase components regulate MT-ATP8 translation in mitochondria . To study this relationship:
-
Translation analysis methods:
-
In organello translation assays with radiolabeled amino acids
-
Ribosome profiling of mitochondrial mRNAs
-
Polysome analysis to assess translation efficiency
-
Reporter gene constructs (e.g., ARG8m substitution approaches)
-
-
Genetic manipulation strategies:
-
F1 subunit knockout/knockdown studies
-
Overexpression of F1 components to test sufficiency
-
Chaperone manipulation (e.g., Atp11p, Atp12p)
-
Use of translation inhibitors with time-course analysis
-
-
Interaction studies:
-
RNA-protein interaction assays (RIP, CLIP)
-
Two-hybrid screens for translation factor interactions
-
Structural studies of regulatory complexes
-
Computational modeling of regulatory networks
-
Such approaches have revealed that mutants lacking α or β subunits of F1, or the Atp11p and Atp12p chaperones that promote F1 assembly, have normal levels of the bicistronic ATP8/ATP6 mRNAs but fail to synthesize Atp6p and Atp8p .
How can CRISPR-Cas9 technology be applied to study MT-ATP8 function despite its mitochondrial encoding?
Studying mitochondrially-encoded genes presents unique challenges for genome editing. Advanced methodological approaches include:
-
Allotopic expression strategies:
-
Nuclear expression with mitochondrial targeting sequences
-
Codon optimization for cytosolic translation
-
Careful validation of proper targeting and processing
-
-
Indirect manipulation approaches:
-
Targeting nuclear genes involved in MT-ATP8 translation
-
Modifying mitochondrial import machinery
-
Engineering mitochondria-targeted nucleases
-
-
Heteroplasmy manipulation:
-
Selective inhibition of mutant or wild-type mtDNA replication
-
Mitochondrial targeted restriction endonucleases
-
Leverage of natural mtDNA segregation mechanisms
-
-
Surrogate systems:
-
Using the ARG8m reporter system as demonstrated in yeast studies
-
Developing equivalent reporter systems for mammalian cells
-
Creating cellular models with trackable MT-ATP8 variants
-
These approaches circumvent the limitations of direct CRISPR-Cas9 editing of mitochondrial genes while still providing valuable insights into MT-ATP8 function .
What techniques best characterize the interaction between MT-ATP8 and host cell protein (HCP) production in recombinant systems?
Understanding how MT-ATP8 affects and is affected by the HCP landscape is critical for optimizing recombinant protein production. Advanced methodological approaches include:
-
Secretome engineering:
-
Multiple gene knockout strategies targeting HCPs
-
Systems biology modeling of energy allocation
-
Proteomics-guided identification of interacting proteins
-
Multiplex CRISPR-Cas9 for simultaneous modification of multiple targets
-
-
Bioenergetic analysis:
-
Seahorse XF analysis of mitochondrial function
-
In situ measurements of ATP production rates
-
Metabolic flux analysis using stable isotope labeling
-
Real-time monitoring of mitochondrial membrane potential
-
Research has demonstrated that targeted genome editing guided by omics analyses can substantially reduce HCP content, with reductions of 40-70% observed in engineered cell lines, potentially freeing energy for recombinant protein production .
How can researchers investigate the potential role of MT-ATP8 in mitochondrial disease models?
MT-ATP8 mutations have been implicated in various mitochondrial disorders. Methodological approaches include:
-
Disease-relevant mutation modeling:
-
Introduction of patient-derived mutations in cellular models
-
Phenotypic characterization of energy metabolism defects
-
Rescue experiments with wild-type protein expression
-
Compensatory pathway identification
-
-
Multi-omics characterization:
-
Transcriptomic profiling to identify affected pathways
-
Metabolomic analysis of energy intermediates
-
Proteomic assessment of mitochondrial complex assembly
-
Lipidomics to detect membrane composition alterations
-
-
Functional assays:
-
High-resolution respirometry
-
ATP synthesis rate measurements
-
Reactive oxygen species quantification
-
Mitochondrial network morphology analysis
-
These approaches provide comprehensive insights into how MT-ATP8 variants contribute to disease pathophysiology and identify potential therapeutic targets.
What methods can determine the stoichiometry of MT-ATP8 within the ATP synthase complex under different conditions?
Accurate stoichiometry determination is critical for understanding ATP synthase assembly and function:
| Method | Description | Resolution | Sample Requirements | Limitations |
|---|---|---|---|---|
| Quantitative Mass Spectrometry | Absolute quantification using labeled standards | High | Purified complex or enriched fractions | Requires specialized equipment |
| Cryo-EM | Direct visualization of complex architecture | Near-atomic | Highly purified complex | Sample heterogeneity challenges |
| FRET/BRET | Energy transfer between labeled subunits | Medium | Genetically modified cells | Potential interference from tags |
| Single-molecule counting | Direct observation of labeled subunits | High | Surface-immobilized complexes | Technical complexity |
| Native MS | Mass analysis of intact complexes | High | Purified complex in MS-compatible detergent | Limited by complex size |
Combining multiple orthogonal techniques provides the most reliable assessment of stoichiometry, particularly when investigating dynamic changes under different physiological conditions.
What strategies address low expression yields of recombinant MT-ATP8?
Low yields of small, hydrophobic proteins like MT-ATP8 are common. Methodological solutions include:
-
Expression optimization protocol:
-
Systematic testing of promoter strength
-
Evaluation of signal sequence variations
-
Codon optimization for expression host
-
Induction condition screening (temperature, inducer concentration, time)
-
-
Stabilization approaches:
-
Co-expression with interaction partners
-
Addition of chemical chaperones
-
Use of specialized strains with enhanced membrane protein expression
-
Implementation of fusion partners with demonstrated solubility benefits
-
-
Harvest timing optimization:
-
Time-course analysis of expression
-
Viability monitoring to prevent degradation
-
Controlled induction systems for toxic proteins
-
Selective permeabilization techniques for enhanced recovery
-
Researchers have reported significant variations in protein yield based on harvest timing, with optimal results when cultures maintain >90% viability .
How can researchers distinguish between genuine MT-ATP8 function and artifacts in experimental systems?
Differentiating true biological effects from experimental artifacts requires rigorous controls:
-
Essential control experiments:
-
Tag-only controls to assess tag interference
-
Inactive mutant versions as negative controls
-
Complementation assays to confirm functionality
-
Dose-response relationships to establish specificity
-
-
Validation across multiple systems:
-
Comparison between different expression hosts
-
Correlation between in vitro and cellular assays
-
Cross-species conservation of observed phenomena
-
Integration of computational predictions with experimental results
-
-
Artifact identification techniques:
-
Analysis of protein aggregation state
-
Assessment of post-translational modification status
-
Membrane integration verification
-
Complex assembly validation
-
These methodological approaches collectively strengthen the validity and reproducibility of MT-ATP8 functional studies.
What analytical methods effectively detect and quantify MT-ATP8 incorporation into ATP synthase complexes?
Detection and quantification of MT-ATP8 within assembled complexes present technical challenges due to its small size and hydrophobicity:
-
Enhanced detection workflow:
-
Specialized extraction protocols for membrane complexes
-
Optimized gel systems for small hydrophobic proteins
-
Custom antibody development targeting accessible epitopes
-
Cross-linking prior to analysis to stabilize interactions
-
-
Quantitative approaches:
-
Targeted proteomics with heavy-labeled peptide standards
-
Fluorescent labeling with quantum yield calibration
-
Western blot with recombinant protein standard curves
-
Complex stoichiometry modeling based on multiple measurements
-
-
Localization confirmation:
-
Super-resolution microscopy with specific antibodies
-
Proximity labeling techniques (BioID, APEX)
-
Subcellular fractionation with marker validation
-
Protease protection assays for topology determination
-
These analytical methods provide complementary information about MT-ATP8's presence, abundance, and interactions within the ATP synthase complex under various experimental conditions .
