Recombinant Artibeus jamaicensis ATP synthase protein 8 (MT-ATP8)
Description
Protein Identity and Basic Features
MT-ATP8 from Artibeus jamaicensis is a small mitochondrial protein encoded by the MT-ATP8 gene, which is located in the mitochondrial genome. The protein is also known by several alternative names including A6L and F-ATPase subunit 8 . In the UniProt database, this protein is identified with the accession number O99598, confirming its cataloging in protein databases and its recognition as a distinct biological entity . The full-length protein consists of 67 amino acids, making it relatively small compared to many other proteins involved in energy metabolism .
Genetic Information
The gene encoding this protein, MT-ATP8, has several synonyms including ATP8, ATPASE8, and MTATP8 . As a mitochondrially-encoded gene, MT-ATP8 is part of the mitochondrial genome rather than the nuclear genome. Interestingly, phylogenetic studies have noted that the ATP6 and ATP8 genes have overlapping regions in mitochondrial genomes, which can complicate certain types of genetic analyses . This overlapping gene arrangement is a common feature in mitochondrial genomes across many species and represents an evolutionary adaptation for compact genome organization.
Role in ATP Synthesis
MT-ATP8 functions as a subunit of mitochondrial ATP synthase (Complex V), the inner mitochondrial enzyme that catalyzes the final step of oxidative phosphorylation in the electron transport chain . This complex is responsible for harnessing the energy of the proton gradient across the inner mitochondrial membrane to synthesize ATP, the primary energy currency of cells. Although small in size, MT-ATP8 plays a crucial structural and functional role in the assembly and operation of the ATP synthase complex.
The protein is specifically located in the Fo portion of ATP synthase, which is embedded in the inner mitochondrial membrane and forms the proton channel through which hydrogen ions flow back into the mitochondrial matrix. This proton flow drives the rotary mechanism of ATP synthase, enabling the synthesis of ATP from ADP and inorganic phosphate.
Involvement in Cellular Signaling
Beyond its direct role in ATP synthesis, MT-ATP8 may also be connected to broader cellular signaling networks. Research indicates a potential connection to the AMP-activated protein kinase (AMPK) signaling pathway in Artibeus jamaicensis . The AMPK pathway acts as a crucial sensor of cellular energy status, responding to changes in the AMP:ATP ratio caused by various metabolic stresses. Once activated, AMPK regulates numerous metabolic processes, inhibiting energy-consuming pathways while activating energy-producing ones.
Recombinant Expression Systems
The recombinant form of Artibeus jamaicensis MT-ATP8 is typically produced using bacterial expression systems, particularly Escherichia coli . When expressed in E. coli, the protein can be fused with various tags to facilitate its purification and detection. Although specific information for A. jamaicensis MT-ATP8 is limited, comparable recombinant MT-ATP8 proteins from other species (such as Balaenoptera musculus) are often expressed with N-terminal His-tags to enable efficient purification through affinity chromatography .
The recombinant production process typically involves:
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Cloning the MT-ATP8 gene into an appropriate expression vector
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Transforming the recombinant plasmid into E. coli host cells
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Inducing protein expression under controlled conditions
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Cell lysis and extraction of the recombinant protein
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Purification using affinity chromatography based on the fusion tag
Evolutionary and Phylogenetic Studies
Mitochondrial proteins, including MT-ATP8, have been instrumental in phylogenetic studies of bat species and their relationships to other mammals. The complete mitochondrial genome of Artibeus jamaicensis has been used to develop hypotheses about the evolutionary relationships of bats to other mammalian orders . In these analyses, MT-ATP8 sequences, along with other mitochondrial proteins, provide valuable data for constructing evolutionary trees and understanding the molecular evolution of different species.
Phylogenetic analyses often employ sophisticated statistical methods such as the ProtML program in MOLPHY package and the CodeML program in PAML package with the mtREV-F model to analyze mitochondrial protein sequences, including MT-ATP8 . These analyses help elucidate the evolutionary history and taxonomic relationships among different bat species and between bats and other mammals.
Viral Infection Research
Recent research has utilized Jamaican fruit bat models, including cellular components with mitochondrial proteins like MT-ATP8, to study viral infections. Particularly noteworthy is the establishment of a Jamaican fruit bat intestinal organoid model for investigating SARS-CoV-2 infection . While these studies do not focus specifically on MT-ATP8, they demonstrate the broader research context in which Artibeus jamaicensis-derived biological materials, including recombinant proteins, are being employed.
In the SARS-CoV-2 research, Jamaican fruit bat intestinal organoids were found to support limited viral replication but not viral reproduction, with infection triggering significant increases in type I interferons and inflammatory cytokines . These findings suggest that primary Jamaican fruit bat intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection induces protective regenerative pathways in these cells.
Antibody Development and Immunological Research
Antibodies against MT-ATP8 have been developed for research applications, enabling the detection and study of this protein in various experimental contexts . While the available information specifically refers to human MT-ATP8 antibodies, similar approaches are likely applicable to the study of Artibeus jamaicensis MT-ATP8. These antibodies can be used in techniques such as Western blotting and immunoprecipitation to investigate the expression, localization, and interactions of MT-ATP8 in cellular systems.
Functional Implications of Sequence Variations
Sequence variations in MT-ATP8 between different species may have functional implications for ATP synthase efficiency, proton conductance, or assembly of the enzyme complex. Research on human MT-ATP8 has shown that mutations in this gene can have deleterious effects on ATP synthase function, leading to various neurological and neuromuscular diseases . By extension, species-specific variations in the Artibeus jamaicensis MT-ATP8 may reflect adaptations to the bat's unique metabolic requirements, possibly including adaptations related to flight, echolocation, or other bat-specific physiological traits.
Technical Challenges in Protein Production
The production and purification of functional recombinant MT-ATP8 presents several technical challenges. As a small, hydrophobic membrane protein, MT-ATP8 can be difficult to express in soluble form and may require specialized conditions for maintaining its native conformation. Future research may focus on optimizing expression systems and purification protocols to improve yield and functionality of recombinant Artibeus jamaicensis MT-ATP8.
Emerging Research Opportunities
The study of Recombinant Artibeus jamaicensis ATP synthase protein 8 offers several promising research directions:
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Investigation of its role in the exceptional longevity observed in some bat species, potentially related to mitochondrial efficiency and energy metabolism
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Exploration of its involvement in viral resistance mechanisms, particularly in the context of bats serving as natural reservoirs for numerous viruses
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Comparative analyses with MT-ATP8 from other bat species to understand the evolution of energy metabolism in flying mammals
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Structure-function studies to elucidate its precise role in ATP synthase assembly and operation
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you require a specific format, please indicate your preference 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 notify us in advance, as additional fees will apply.
Generally, liquid formulations have a shelf life of 6 months at -20°C/-80°C. Lyophilized formulations have a shelf life of 12 months at -20°C/-80°C.
Target Background
Q&A
What is the structural positioning of MT-ATP8 within the ATP synthase complex?
MT-ATP8 is located in the membrane portion of the ATP synthase stator. It forms an α-helix that spans the mitochondrial membrane and protrudes into the matrix. The protein is tightly associated with subunits a and i/j but is positioned remotely from the c-ring, suggesting it is not directly involved in catalytic proton transfer . This structural arrangement is conserved across species despite significant sequence variations.
What strategies can optimize allotopic expression of MT-ATP8?
For allotopic expression (expressing mitochondrial genes from the nucleus), several critical modifications are necessary:
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Codon optimization based on nuclear genetic code
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Addition of an efficient N-terminal mitochondrial targeting sequence (MTS) from nuclear-encoded mitochondrial proteins (e.g., ATP5G1)
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Inclusion of epitope tags (such as FLAG or MYC) for immunodetection
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Selection of appropriate promoters (e.g., CAG promoter) for consistent expression
In transgenic model studies, these modifications have enabled successful localization of recombinant MT-ATP8 to mitochondria and functional incorporation into ATP synthase complexes .
What are the optimal storage conditions for recombinant MT-ATP8 preparations?
Recombinant MT-ATP8 should be stored at -20°C or -80°C for extended periods. For working solutions, store aliquots at 4°C for no more than one week. Repeated freeze-thaw cycles should be strictly avoided as they lead to protein degradation and loss of activity .
What buffer compositions optimize MT-ATP8 stability?
Tris-based buffers containing 50% glycerol have proven effective for stabilizing recombinant MT-ATP8 . Alternative formulations include Tris/PBS-based buffers with 6% trehalose at pH 8.0 . For reconstitution of lyophilized protein, deionized sterile water should be used to achieve a concentration of 0.1-1.0 mg/mL, followed by addition of glycerol (5-50% final concentration) for long-term storage .
How can researchers verify successful incorporation of recombinant MT-ATP8 into functional ATP synthase complexes?
Verification requires multiple complementary approaches:
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Immunodetection with antibodies against epitope tags (FLAG, MYC) in purified mitochondrial fractions
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Blue Native PAGE to assess integration into assembled ATP synthase complexes
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Ratiometric analysis comparing endogenous and exogenous MT-ATP8 levels through western blotting
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Enzymatic activity assays comparing ATP synthase function between transgenic and non-transgenic controls
| Method | Purpose | Technical Considerations |
|---|---|---|
| Western blot | Quantify protein expression | Normalize to mitochondrial markers (e.g., aconitase) |
| Blue Native PAGE | Assess complex assembly | Gentle detergent solubilization preserves complexes |
| Enzymatic assays | Measure functional activity | Compare with non-transgenic controls |
| RT-qPCR | Monitor transcript levels | Assess impact on endogenous gene expression |
What experimental controls are essential when studying recombinant MT-ATP8 function?
Critical controls include:
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Non-transgenic littermates for baseline comparisons
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Transgenic animals with the same genetic background but different mitochondrial haplotypes
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Multiple tissue types to account for tissue-specific expression differences
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Age-matched samples to control for developmental variations in mitochondrial function
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Purified mitochondrial fractions to eliminate variations in tissue lysis efficiencies
How does Artibeus jamaicensis MT-ATP8 compare structurally to other mammalian homologs?
While specific structural comparisons for A. jamaicensis MT-ATP8 with other mammals aren't fully characterized, we can make inferences based on related species. The N-terminal sequence (MPQLDTSTW) shows strong conservation with blue whale MT-ATP8 (MPQLDTSTWL) , suggesting functional constraints in this region. Despite primary sequence divergence between mammalian species, the membrane domain structure tends to be preserved, allowing for structural modeling based on related species .
What methodological approaches can identify functionally critical residues in MT-ATP8?
Advanced approaches include:
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Structural analysis using "humanized" models where sequences from one species are mapped onto well-characterized structures from another species
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Yeast complementation studies, introducing mutations equivalent to those found in human populations
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In silico prediction of substitution effects on protein stability and interactions
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Comparative analysis across diverse species to identify evolutionary constraints
These methods have successfully identified that while MT-ATP8 is not involved in catalytic proton transfer, specific residues are critical for proper assembly and structural integrity of the ATP synthase complex.
How can MT-ATP8 variants be studied for their potential role in mitochondrial diseases?
Researchers can employ several approaches:
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Yeast-based modeling systems to introduce equivalent mutations and assess biochemical consequences
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Transgenic mice expressing epitope-tagged recombinant MT-ATP8 with specific variants
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Analysis of ATP synthase assembly and function in models expressing variant proteins
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Complementation studies to determine if exogenous wild-type MT-ATP8 can rescue defects caused by variants
These approaches have already provided insights into how specific amino acid substitutions might affect ATP synthase function at the molecular level.
What methodological considerations apply when investigating positive selection in MT-ATP8 across species?
Investigation of evolutionary selection requires:
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Comprehensive phylogenetic reconstruction using complete mitochondrial genomes
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Calculation of nonsynonymous/synonymous substitution ratios (dN/dS)
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Branch-site models to detect lineage-specific selection
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Correlation of molecular evolution with ecological adaptations
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Structural mapping of positively selected sites to predict functional impacts
Such analyses have revealed that mitochondrial genes, including MT-ATP8, can experience relaxed evolutionary constraints or positive selection in species adapting to novel environments .
How can transcriptome sequencing inform MT-ATP8 research in Artibeus jamaicensis?
Transcriptome sequencing of A. jamaicensis tissues has been successfully performed using both 454 and Illumina platforms, yielding comprehensive gene annotations . This approach can:
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Identify tissue-specific expression patterns of MT-ATP8
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Reveal potential splice variants or RNA editing events
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Provide insights into coordinated expression with other mitochondrial and nuclear genes
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Establish baseline expression levels for experimental comparisons
What techniques can assess the impact of recombinant MT-ATP8 expression on endogenous gene regulation?
RT-qPCR analysis can determine whether nuclear expression of recombinant MT-ATP8 affects endogenous mitochondrial gene expression. Previous studies have shown that nuclear oATP8 expression does not significantly alter the levels of endogenous ATP8 or ATP6 mRNA, suggesting limited feedback regulation between nuclear and mitochondrial gene expression systems .
How can researchers address the challenge of heteroplasmy when studying MT-ATP8 variants?
Heteroplasmy (the presence of multiple mitochondrial DNA variants within cells) presents significant technical challenges. Methodological solutions include:
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Single-cell analysis to quantify heteroplasmy at the cellular level
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Cybrid cell lines with controlled levels of variant mtDNA
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Allotopic expression systems that bypass heteroplasmy by expressing the gene from the nucleus
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Quantitative assessment of variant-to-wild-type ratios using next-generation sequencing
What approaches can verify proper subcellular localization of recombinant MT-ATP8?
Verification of mitochondrial localization requires:
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Confocal microscopy with fluorescently tagged MT-ATP8 constructs
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Subcellular fractionation followed by western blotting
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Protease protection assays to confirm membrane integration
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Co-localization with established mitochondrial markers
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Electron microscopy with immunogold labeling for highest resolution confirmation
