Recombinant Cyprinus carpio ATP synthase protein 8 (mt-atp8), partial

What is the structural and functional role of ATP synthase protein 8 in Cyprinus carpio mitochondria?

ATP synthase protein 8 (MT-ATP8) in Cyprinus carpio is a small membrane-spanning subunit of the F₀ complex within the larger F₀F₁-ATPase (ATP synthase) enzyme. Structurally, MT-ATP8 forms an α-helix that spans the inner mitochondrial membrane and protrudes into the matrix . It is tightly associated with subunits a and i/j within the membrane portion of the ATP synthase stator . While not directly involved in catalytic proton transfer (as it is positioned away from the c-ring), MT-ATP8 plays an essential structural role in maintaining the integrity and proper functioning of the ATP synthase complex .

The functional significance of MT-ATP8 stems from its contribution to the final step of oxidative phosphorylation. The F₀F₁-ATPase complex facilitates the flow of protons across the inner mitochondrial membrane, harnessing this energy to convert ADP to ATP . In carp specifically, the activity of F₀F₁-ATPase shows significant temperature-dependent variation, with cold-acclimated fish (10°C) exhibiting approximately twofold higher enzyme activity compared to warm-acclimated (30°C) counterparts . This adaptation likely helps maintain energy production under varying environmental temperatures.

What expression systems are most effective for producing recombinant Cyprinus carpio MT-ATP8?

For recombinant Cyprinus carpio MT-ATP8 production, yeast expression systems have demonstrated particular effectiveness according to available data. Commercial recombinant carp MT-ATP8 (amino acids 1-54) is successfully expressed in yeast with His-tag purification, yielding proteins with >90% purity suitable for applications such as ELISA . Yeast systems offer advantages for membrane protein expression, including proper post-translational modifications and a eukaryotic cellular environment that facilitates correct protein folding.

The choice of expression system should consider:

• Protein authenticity: Yeast provides a eukaryotic system that can better replicate the native folding environment compared to bacterial systems

• Codon optimization: Adjusting codons for optimal expression in the chosen host

• Fusion tags: His-tagging (as seen in commercial preparations) facilitates purification while minimizing interference with protein structure

• Growth conditions: Temperature, media composition, and induction parameters must be optimized

For researchers requiring alternative expression systems, mammalian cell lines (particularly HEK-293 cells) have been successfully used for MT-ATP8 expression from other species, which could potentially be adapted for carp MT-ATP8 .

How does temperature acclimation affect MT-ATP8 expression and function in Cyprinus carpio?

Temperature acclimation induces significant changes in the expression and function of mitochondrial proteins in Cyprinus carpio, including components of the ATP synthase complex. Studies have shown that cold acclimation (10°C) results in substantial upregulation of mitochondrial gene transcripts compared to warm acclimation (30°C) .

Specifically, for mitochondrially encoded genes:

• Transcripts in cold-acclimated (10°C) carp are six to seven times more abundant than in warm-acclimated (30°C) carp when measured per unit weight of total RNA

• Nuclear-encoded mitochondrial genes show a less dramatic but still significant increase of approximately twofold in cold-acclimated fish

Functionally, F₀F₁-ATPase activity measurements at various temperatures (10°C, 25°C, and 30°C) consistently show nearly twofold higher activity in cold-acclimated fish compared to warm-acclimated counterparts . This enhanced activity likely represents a compensatory mechanism to maintain adequate ATP production at lower temperatures where enzyme kinetics would otherwise be reduced.

These expression and activity changes suggest that temperature-dependent regulation of ATP synthase components, including MT-ATP8, plays a critical role in the energy homeostasis of carp across varying environmental conditions.

What purification strategies yield the highest purity for recombinant Cyprinus carpio MT-ATP8?

Purification of recombinant Cyprinus carpio MT-ATP8 presents several challenges due to its small size, hydrophobicity, and membrane-associated nature. Commercial preparations achieve >90% purity through His-tag affinity chromatography , suggesting this approach as a primary purification strategy.

A comprehensive purification protocol should include:

• Affinity Chromatography: His-tagged MT-ATP8 can be effectively purified using nickel or cobalt affinity resins. The commercially available recombinant carp MT-ATP8 utilizes His-tagging for this purpose .

• Detergent Selection: Appropriate detergents are crucial for solubilizing membrane proteins while maintaining native structure. Mild non-ionic detergents (e.g., n-dodecyl-β-D-maltoside) are often preferred.

• Additional Purification Steps:

  • Size exclusion chromatography to separate monomeric protein from aggregates

  • Ion exchange chromatography as a polishing step

  • Consideration of on-column refolding methods for proteins expressed in inclusion bodies

• Quality Assessment: Purity should be assessed by:

  • SDS-PAGE followed by Coomassie or silver staining

  • Western blotting with specific antibodies

  • Mass spectrometry for accurate molecular weight determination

Commercial recombinant carp MT-ATP8 preparations achieve >90% purity suitable for applications such as ELISA , demonstrating that high-purity preparations are attainable with appropriate purification strategies.

What methodological approaches can resolve the apparent contradiction in Cyprinus carpio MT-ATP8 gene presence across different studies?

Interestingly, there appears to be a contradiction in the literature regarding the presence of the atp8 gene in Cyprinus carpio. Some studies suggest that "atp8 is missing" in certain analyses , while others clearly demonstrate the existence of this gene, as evidenced by the commercial availability of recombinant Cyprinus carpio MT-ATP8 protein .

To resolve this apparent contradiction, researchers should implement a multi-faceted methodological approach:

• Comprehensive Genomic Analysis:

  • Perform deep sequencing of mitochondrial DNA with multiple coverage

  • Utilize various bioinformatic algorithms specifically designed to detect short or divergent genes

  • Employ both reference-guided and de novo assembly approaches

  • Examine intergenic regions where truncated or highly diverged atp8 sequences might reside

• Transcriptomic Verification:

  • Conduct RNA-Seq across various tissues and developmental stages

  • Employ strand-specific sequencing to detect potentially overlapping transcripts

  • Use quantitative RT-PCR with multiple primer sets to validate expression

• Proteomic Confirmation:

  • Apply targeted mass spectrometry to detect MT-ATP8 peptides

  • Use enrichment techniques for mitochondrial membrane proteins

  • Compare results across different tissue types and environmental conditions

• Comparative Genomic Approach:

  • Analyze MT-ATP8 sequences across closely related cyprinid species

  • Examine synteny and gene arrangements in mitochondrial genomes

  • Consider evolutionary models that might explain gene loss or significant divergence

This contradictory situation could stem from extreme sequence divergence, strain-specific differences, annotation errors, or methodological limitations in gene detection. The fact that recombinant Cyprinus carpio MT-ATP8 protein is commercially available strongly suggests the gene exists, at least in some strains or populations of common carp.

How can recombinant MT-ATP8 be utilized to study temperature adaptation mechanisms in Cyprinus carpio?

Recombinant Cyprinus carpio MT-ATP8 provides a valuable tool for investigating the molecular basis of temperature adaptation mechanisms in this eurythermal fish species. Research has established that F₀F₁-ATPase activity in carp exhibits significant temperature-dependent variation, with cold-acclimated fish showing approximately twofold higher enzyme activity than warm-acclimated counterparts .

Advanced methodological approaches utilizing recombinant MT-ATP8 include:

• In vitro Reconstitution Studies:

  • Reconstitute ATP synthase complexes with recombinant MT-ATP8 from cold-adapted versus warm-adapted carp populations

  • Measure enzymatic activity across temperature gradients (10-30°C) to quantify functional differences

  • Assess how MT-ATP8 variants influence proton conductance and ATP synthesis efficiency

• Structure-Function Analysis:

  • Generate site-directed mutants of recombinant MT-ATP8 targeting residues hypothesized to contribute to thermal adaptation

  • Perform circular dichroism spectroscopy to assess secondary structure stability at different temperatures

  • Utilize hydrogen-deuterium exchange mass spectrometry to identify temperature-sensitive regions

• Protein-Protein Interaction Studies:

  • Investigate how temperature affects the interaction between MT-ATP8 and other ATP synthase subunits

  • Employ techniques such as microscale thermophoresis or surface plasmon resonance at varying temperatures

  • Determine binding affinities and kinetics as a function of temperature

• Cellular Bioenergetic Analysis:

  • Develop cellular models expressing recombinant wild-type or mutant MT-ATP8

  • Measure oxygen consumption rates, ATP production, and mitochondrial membrane potential across temperature ranges

  • Correlate bioenergetic parameters with structural adaptations in MT-ATP8

Such studies would provide mechanistic insights into how alterations in MT-ATP8 contribute to the remarkable sixfold to sevenfold increase in mitochondrial gene transcripts observed in cold-acclimated carp , furthering our understanding of molecular adaptation to environmental temperature fluctuations.

What are the most effective experimental designs for analyzing interactions between recombinant MT-ATP8 and other ATP synthase subunits?

Analyzing the interactions between recombinant Cyprinus carpio MT-ATP8 and other ATP synthase subunits requires sophisticated experimental approaches that address the challenges of working with membrane proteins while preserving native-like interactions. MT-ATP8 forms tight associations with subunits a and i/j within the membrane portion of the ATP synthase stator , making these interactions particularly important to characterize.

Effective experimental designs include:

• Crosslinking Mass Spectrometry (XL-MS):

  • Utilize membrane-permeable crosslinkers of varying lengths to capture transient and stable interactions

  • Apply both chemical (e.g., DSS, BS3) and photoactivatable crosslinkers for comprehensive coverage

  • Analyze crosslinked peptides using high-resolution mass spectrometry with specialized search algorithms

  • Compare interaction maps between recombinant systems and native mitochondrial complexes

• Förster Resonance Energy Transfer (FRET):

  • Label recombinant MT-ATP8 and potential interaction partners with appropriate fluorophore pairs

  • Measure FRET efficiency in reconstituted proteoliposomes or detergent micelles

  • Perform acceptor photobleaching FRET to confirm specific interactions

  • Implement temperature-controlled FRET to assess interaction stability under varying conditions

• Protein Complementation Assays:

  • Split reporter systems (e.g., split GFP, split luciferase) fused to MT-ATP8 and potential interaction partners

  • Express constructs in appropriate eukaryotic systems that support mitochondrial import

  • Quantify signal as an indicator of protein-protein interaction

  • Design controls to verify specificity and physiological relevance

• Cryo-Electron Microscopy with Focused Classification:

  • Purify ATP synthase complexes with recombinant MT-ATP8 variants

  • Apply 3D classification focused on the membrane domain

  • Generate high-resolution structures revealing MT-ATP8 interaction networks

  • Compare structures from different temperature acclimation states

These experimental approaches should be complemented by molecular dynamics simulations to predict interaction interfaces and guide experimental design. The resulting data would provide valuable insights into how MT-ATP8 contributes to ATP synthase stability and function across different physiological conditions in Cyprinus carpio.

How can functional genomics approaches enhance our understanding of MT-ATP8 evolution in Cyprinidae?

Functional genomics approaches offer powerful tools for understanding the evolution of MT-ATP8 in Cyprinidae (the carp family) and resolving questions about its presence, absence, or divergence across different lineages. The apparent discrepancy regarding ATP8 in some studies versus its confirmed presence in others highlights the need for comprehensive functional genomics investigations.

Advanced methodological approaches include:

• Comparative Mitogenomics with Functional Validation:

  • Sequence mitochondrial genomes from diverse Cyprinidae species with deep coverage

  • Apply multiple gene prediction algorithms and manual curation to identify MT-ATP8 candidates

  • Validate predictions through heterologous expression and functional complementation

  • Construct phylogenetic trees based on MT-ATP8 sequences and compare with trees from other mitochondrial genes

• Evolutionary Rate Analysis:

  • Calculate selection pressures (dN/dS ratios) acting on MT-ATP8 across Cyprinidae lineages

  • Identify sites under positive, neutral, or purifying selection

  • Correlate evolutionary rates with environmental factors (temperature ranges, habitat types)

  • Apply branch-site models to detect lineage-specific selection patterns

• Experimental Evolution Studies:

  • Subject carp cell lines or whole organisms to controlled temperature regimes over multiple generations

  • Sequence MT-ATP8 at regular intervals to track molecular evolution in real-time

  • Measure fitness parameters in relation to sequence changes

  • Utilize CRISPR-Cas9 to introduce specific MT-ATP8 variants and assess their fitness effects

• Ancestral Sequence Reconstruction:

  • Infer ancestral MT-ATP8 sequences at key nodes in the Cyprinidae phylogeny

  • Synthesize and express these reconstructed ancestral proteins

  • Compare biochemical properties and thermal stability across ancestral and extant sequences

  • Test hypotheses about evolutionary trajectories of MT-ATP8 function

This integrated approach would not only resolve questions about the presence and conservation of MT-ATP8 across Cyprinidae but also provide insights into how this gene has contributed to adaptation across diverse thermal environments. The findings could further explain the remarkable capability of carp to adjust mitochondrial gene expression in response to temperature changes .

What analytical techniques best characterize the impact of recombinant MT-ATP8 on proton conductance and ATP synthesis efficiency?

Characterizing how recombinant Cyprinus carpio MT-ATP8 influences proton conductance and ATP synthesis efficiency requires sophisticated biophysical and biochemical techniques that can measure these parameters with high precision while maintaining physiologically relevant conditions.

Recommended analytical approaches include:

• Reconstituted Proteoliposome Assays:

  • Reconstitute purified ATP synthase complexes with wild-type or variant recombinant MT-ATP8 into liposomes

  • Establish a proton gradient using acid-base transitions or bacteriorhodopsin-mediated light-driven proton pumping

  • Measure ATP synthesis rates using luciferase-based luminescence assays

  • Quantify proton conductance through pH-sensitive fluorescent dyes (e.g., ACMA, pyranine)

  MT-ATP8 Variant	ATP Synthesis Rate (nmol/min/mg)	Proton Conductance (H⁺/s)	P/O Ratio
  Wild-type	[Experimental data]	[Experimental data]	[Ratio]
  Thermally Adapted	[Experimental data]	[Experimental data]	[Ratio]
  Site-Directed Mutants	[Experimental data]	[Experimental data]	[Ratio]

• Patch-Clamp Electrophysiology:

  • Incorporate ATP synthase with recombinant MT-ATP8 into giant unilamellar vesicles or planar lipid bilayers

  • Apply patch-clamp techniques to measure proton currents directly

  • Assess the effects of membrane potential, pH, and temperature on conductance

  • Compare conductance properties between MT-ATP8 variants from cold- and warm-acclimated carp

• High-Resolution Respirometry:

  • Measure oxygen consumption rates in isolated mitochondria or submitochondrial particles containing recombinant MT-ATP8

  • Determine respiratory control ratios and P/O ratios (ATP produced per oxygen consumed)

  • Assess the impact of specific inhibitors on proton leak and ATP synthesis

  • Compare efficiency parameters across temperature ranges (10-30°C) relevant to carp physiology

• Single-Molecule FRET Approaches:

  • Label key residues in MT-ATP8 and adjacent subunits with appropriate FRET pairs

  • Track conformational changes during catalytic cycles

  • Correlate structural dynamics with functional parameters

  • Compare kinetic parameters between MT-ATP8 variants at different temperatures

These analytical techniques would provide detailed mechanistic insights into how MT-ATP8 contributes to the remarkable temperature adaptability of carp F₀F₁-ATPase, which shows nearly twofold higher activity in cold-acclimated fish compared to warm-acclimated counterparts . Such data would be particularly valuable for understanding how structural variations in MT-ATP8 translate to functional differences in energy production under varying environmental conditions.