Recombinant Pisaster ochraceus ATP synthase protein 8 (MT-ATP8), partial

What is the structural role of ATP synthase protein 8 in Pisaster ochraceus mitochondria?

ATP synthase protein 8 (MT-ATP8) in Pisaster ochraceus functions as an essential component of the mitochondrial ATP synthase complex (Complex V). This protein is encoded by the mitochondrial genome and forms part of the peripheral stalk subcomplex that helps anchor the F₁ catalytic domain to the membrane-embedded F₀ region. The protein plays a crucial role in maintaining the structural stability of the ATP synthase complex during the rotational catalysis process that drives ATP production. In echinoderms like Pisaster ochraceus, MT-ATP8 likely contributes to the unique conformational states observed in the catalytic β subunits, which differ from those seen in bacterial and mammalian ATP synthases .

How does MT-ATP8 contribute to ATP synthesis in sea star mitochondria?

MT-ATP8 contributes to ATP synthesis by helping maintain the structural integrity of the ATP synthase complex during proton translocation. ATP synthases produce ATP from ADP and inorganic phosphate using energy from the transmembrane proton motive force. Protons move through the membrane-embedded F₀ region via offset half-channels, driving rotation of the rotor subcomplex. This rotation induces conformational changes in the catalytic F₁ region, facilitating ATP production. MT-ATP8 is part of the peripheral stalk that prevents the F₁ region from rotating with the rotor during catalysis. This structural support is critical for coupling proton movement to ATP synthesis efficiently in sea star mitochondria .

How does Pisaster ochraceus MT-ATP8 differ from homologous proteins in other species?

While the search results don't provide specific sequence comparisons for Pisaster ochraceus MT-ATP8, we can infer likely differences based on evolutionary relationships. As an echinoderm, P. ochraceus likely shows significant sequence divergence from vertebrate and bacterial ATP8 proteins while maintaining functional conservation.

ATP synthase complexes show species-specific differences in inhibition mechanisms and conformational states. For example, bacterial ATP synthases from Bacillus PS3 show different catalytic β subunit conformations ('open', 'closed', and 'open') compared to E. coli F₁-ATPase ('half-closed', 'closed', and 'open') and chloroplast/mitochondrial ATP synthases ('closed', 'closed', and 'open') . These differences suggest MT-ATP8 in P. ochraceus likely has unique structural features that contribute to echinoderm-specific regulation of ATP synthesis.

What expression systems are most effective for producing recombinant P. ochraceus MT-ATP8?

Based on approaches used for other ATP synthase components, bacterial expression systems using Escherichia coli would likely be most effective for initial attempts at expressing recombinant P. ochraceus MT-ATP8. The protocol could be adapted from successful ATP synthase expression approaches, such as:

• Cloning the MT-ATP8 gene into an expression vector with a suitable tag (His-tag, FLAG-tag)

• Transforming the construct into an E. coli expression strain optimized for membrane proteins

• Inducing expression under controlled conditions (temperature, IPTG concentration)

• Harvesting cells and lysing them in a buffer containing detergents suitable for membrane proteins

• Purifying using affinity chromatography followed by size exclusion chromatography

For bacterial ATP synthase components, researchers have successfully used E. coli expression systems with glycol-diosgenin (GDN) detergent for solubilization, followed by HisTrap HP column purification and Superose 6 increase 10/300 column size exclusion chromatography . Similar approaches could be adapted for the P. ochraceus MT-ATP8 protein.

How can researchers overcome challenges in expressing this hydrophobic membrane protein?

Expressing hydrophobic mitochondrial membrane proteins like MT-ATP8 presents several challenges. Based on successful approaches with other ATP synthase components, researchers should consider:

• Fusion partners: Using solubility-enhancing fusion partners like MBP (maltose-binding protein) or SUMO to improve folding and solubility

• Specialized strains: Employing E. coli strains specifically designed for membrane protein expression (C41(DE3), C43(DE3))

• Expression conditions: Lowering induction temperature (16-20°C) and IPTG concentration to slow expression and promote proper folding

• Detergent screening: Testing multiple detergents for solubilization (glycol-diosgenin, n-dodecyl-β-D-maltopyranoside, digitonin)

• Co-expression strategies: Potentially co-expressing with other ATP synthase subunits to promote proper assembly

Purification should include buffer optimization with components that enhance stability, such as glycerol (10% w/v), sucrose (250 mM), and protease inhibitors (6-aminocaproic acid, benzamidine, PMSF) .

What methods can be used to assess the incorporation of recombinant MT-ATP8 into functional ATP synthase complexes?

To determine whether recombinant P. ochraceus MT-ATP8 properly incorporates into functional ATP synthase complexes, researchers should consider a multi-faceted approach:

• Subcellular fractionation: Isolate cytoplasmic and mitochondrial fractions to confirm mitochondrial localization, as demonstrated with FLAG-tagged ATP8 in transgenic mouse studies

• Blue native PAGE: Assess incorporation into intact ATP synthase complexes by analyzing migration patterns of the assembled complex

• Western blotting: Use antibodies against both the recombinant tag and other ATP synthase subunits to verify co-migration in native gels

• Immunoprecipitation: Pull down the recombinant protein and analyze co-precipitating subunits by mass spectrometry

• Cryo-EM analysis: For advanced structural confirmation, cryo-EM can determine if the recombinant protein adopts the expected position in the complex

Quantification of incorporation efficiency can be performed by normalizing band intensities to mitochondrial markers such as aconitase (ACO2), as demonstrated in transgenic mice studies with oATP8 .

How can researchers measure the functional impact of recombinant MT-ATP8 on ATP synthase activity?

To assess the functional impact of recombinant MT-ATP8 on ATP synthase activity, researchers should employ these complementary approaches:

• ATP synthesis assays: Measure ATP production rates in isolated mitochondria or reconstituted liposomes containing the recombinant protein

• Oxygen consumption analysis: Use respirometry to assess coupled respiration dependent on functional ATP synthase

• Membrane potential measurements: Employ fluorescent probes (TMRM, JC-1) to determine if the proton motive force is properly utilized

• ATPase activity assays: Measure ATP hydrolysis rates with colorimetric phosphate detection methods

• Proton translocation assays: Use pH-sensitive fluorescent probes to monitor proton movement across membranes

Comparing these parameters between systems with native and recombinant MT-ATP8 will reveal any functional alterations. Additionally, researchers should conduct these assays under various conditions (pH values, temperatures) to assess the impact on enzyme kinetics and stability .

How might the genetic diversity of Pisaster ochraceus populations affect MT-ATP8 structure and function?

The genetic diversity observed in Pisaster ochraceus populations, particularly following selective pressure from events like sea star wasting disease, may influence MT-ATP8 structure and function. Research indicates that P. ochraceus underwent significant allele frequency shifts following a mass mortality event in 2013, with median mortality of 81% across populations . These genetic shifts could potentially affect mitochondrial genes including MT-ATP8.

Researchers should consider:

• Sequencing MT-ATP8 from multiple geographic populations to identify polymorphisms

• Comparing pre- and post-disease outbreak sequences to identify potential adaptive mutations

• Characterizing functional differences between variant forms using enzyme kinetics and thermal stability assays

• Correlating genetic variants with environmental factors (temperature, habitat type) that may drive local adaptation

• Examining whether genetic variants affect oligomerization patterns of ATP synthase, which influence cristae membrane shaping

The distinct morphologies observed between sea stars from prey-abundant and prey-depleted habitats may correlate with metabolic adaptations, potentially involving mitochondrial genes like MT-ATP8.

What adaptations in MT-ATP8 might contribute to the environmental resilience of Pisaster ochraceus?

Pisaster ochraceus inhabits intertidal zones with fluctuating environmental conditions, suggesting potential adaptive features in its mitochondrial proteins. MT-ATP8 adaptations might contribute to:

• Temperature tolerance: Structural modifications that maintain ATP synthase function during tidal temperature fluctuations

• Desiccation resistance: Features that help preserve mitochondrial membrane integrity during low tide exposure

• Metabolic flexibility: Adaptations allowing efficient ATP production during varying oxygen availability

• Growth regulation: Potential role in the demonstrated capability of P. ochraceus to increase or decrease its size in response to prey availability

Research approaches should include comparing MT-ATP8 sequences from populations across environmental gradients, conducting functional assays under varying conditions (temperature, pH, salinity), and examining correlations between MT-ATP8 variants and measurable physiological traits like growth rate and thermal tolerance.

How does the structure of P. ochraceus MT-ATP8 contribute to ATP synthase oligomerization and cristae formation?

ATP synthase complexes form oligomers that shape the cristae membranes of mitochondria, providing physiological benefits by establishing local proton gradients and maintaining membrane potential . The specific role of MT-ATP8 in this process for P. ochraceus has not been directly studied, but based on research in other organisms, we can propose investigation approaches:

• Cryo-electron tomography: Visualize native mitochondrial membrane architecture in P. ochraceus tissues to characterize ATP synthase dimer rows

• Site-directed mutagenesis: Identify key residues in MT-ATP8 that might facilitate dimer formation

• Cross-linking studies: Use chemical cross-linkers to identify proteins interacting with MT-ATP8 at dimer interfaces

• Computational modeling: Predict structural interactions based on homology models with known ATP synthase structures

These approaches would help determine whether MT-ATP8 contributes to the unique mitochondrial adaptations that may support P. ochraceus in its variable intertidal habitat.

What structural features of MT-ATP8 are critical for its integration into the ATP synthase complex?

Based on structural studies of ATP synthases, several features of MT-ATP8 are likely critical for proper integration into the complex:

• Transmembrane domains: Hydrophobic regions that anchor the protein in the mitochondrial inner membrane

• Interface residues: Amino acids that form contacts with adjacent subunits

• Conserved motifs: Sequence elements preserved across evolutionary distances

• Post-translational modifications: Potential phosphorylation or other modification sites

To identify these features, researchers should consider:

• Performing alanine scanning mutagenesis to identify essential residues

• Using hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Applying cross-linking mass spectrometry to identify neighboring subunits

• Conducting molecular dynamics simulations to predict stable conformations

The bacterial ATP synthase structure revealed through cryo-EM imaging provides a template for understanding how peripheral stalk components interact with membrane regions , which could inform studies of P. ochraceus MT-ATP8.

How does expression of MT-ATP8 vary across different tissues and developmental stages in Pisaster ochraceus?

Understanding the expression pattern of MT-ATP8 across tissues and developmental stages requires comprehensive profiling. Based on approaches used in other systems, researchers should consider:

• Quantitative RT-PCR: Measure MT-ATP8 transcript levels across multiple tissues (tube feet, pyloric caeca, gonads, body wall) and developmental stages

• Western blotting: Quantify protein levels using specific antibodies, normalizing to appropriate loading controls

• In situ hybridization: Visualize spatial expression patterns within tissues

• Single-cell RNA sequencing: Obtain high-resolution expression data across cell types

Data could be organized in a comparative tissue expression table:

How does environmental stress affect MT-ATP8 expression and ATP synthase assembly in P. ochraceus?

Environmental stressors likely influence MT-ATP8 expression as part of adaptive responses. Researchers should design experiments examining:

• Temperature stress: Compare expression under normal conditions versus heat shock and cold stress

• Desiccation stress: Analyze changes during simulated low tide exposure

• pH stress: Assess impacts of ocean acidification conditions

• Nutritional stress: Examine effects of prey availability, connecting to the known growth plasticity of P. ochraceus

A comprehensive experimental design would include:

• Controlled exposure to stressors in laboratory settings

• Sampling at multiple time points (acute vs. chronic exposure)

• Analysis of transcript levels, protein abundance, and ATP synthase assembly/activity

• Correlation with physiological performance metrics

This approach would provide insights into how mitochondrial energy production adapts to environmental challenges in this ecologically important intertidal species.

What are the most effective methods for generating antibodies against P. ochraceus MT-ATP8?

Generating specific antibodies against P. ochraceus MT-ATP8 presents challenges due to its hydrophobic nature and potentially limited antigenic regions. Effective approaches include:

• Synthetic peptide strategy:

  • Select 2-3 hydrophilic regions (15-20 amino acids) with high predicted antigenicity

  • Synthesize these peptides with a carrier protein (KLH or BSA)

  • Immunize rabbits or mice with these conjugates

  • Validate antibody specificity against recombinant protein and native tissue extracts

• Recombinant fragment approach:

  • Express the most hydrophilic portion of MT-ATP8 as a fusion protein

  • Purify under denaturing conditions if necessary

  • Use the purified fragment for immunization

  • Screen antibodies for specificity and minimal cross-reactivity

For validation, western blotting should be performed against both recombinant MT-ATP8 and mitochondrial extracts from P. ochraceus tissues, with appropriate controls including pre-immune serum and peptide competition assays.

How can researchers overcome challenges in MT-ATP8 protein stability during purification and analysis?

MT-ATP8, as a hydrophobic membrane protein, presents significant stability challenges during purification and analysis. Based on successful approaches with ATP synthase components, consider:

• Optimized buffer composition:

  • Include stabilizing agents: glycerol (10% w/v), sucrose (250 mM)

  • Add protective compounds: 6-aminocaproic acid (5 mM), benzamidine (5 mM)

  • Include protease inhibitors: PMSF (1 mM) and complete protease inhibitor cocktail

  • Maintain physiological pH (7.2-7.4) with appropriate buffers (Tris-HCl or HEPES)

• Detergent selection:

  • Use mild detergents like glycol-diosgenin (GDN, 0.02% w/v) that maintain protein-protein interactions

  • Consider nanodiscs or amphipols for maintaining native-like membrane environment

  • Test digitonin as an alternative for blue native PAGE applications

• Thermal stability assessment:

  • Employ differential scanning fluorimetry to identify optimal buffer conditions

  • Test various ionic strengths (150-300 mM NaCl) and divalent cation concentrations (MgCl₂ 5-10 mM)

The purification approach developed for bacterial ATP synthase, involving detergent solubilization followed by metal affinity chromatography and gel filtration , provides a starting template that can be modified for the specific properties of P. ochraceus MT-ATP8.

How can recombinant P. ochraceus MT-ATP8 be used to study the evolution of mitochondrial proteins in echinoderms?

Recombinant P. ochraceus MT-ATP8 provides an excellent model for studying the evolution of mitochondrial proteins in echinoderms for several reasons:

• Comparative structural analysis:

  • Express MT-ATP8 from multiple echinoderm species

  • Compare structural features through CD spectroscopy and limited proteolysis

  • Identify conserved domains versus lineage-specific adaptations

  • Map these changes onto the echinoderm phylogenetic tree

• Functional complementation studies:

  • Test whether P. ochraceus MT-ATP8 can rescue function in ATP8-deficient systems

  • Compare complementation efficiency across evolutionary distances

  • Identify key residues determining cross-species compatibility

• Adaptive evolution analysis:

  • Calculate dN/dS ratios to identify sites under positive selection

  • Correlate selected sites with habitat-specific adaptations

  • Examine whether selection patterns differ between deep-sea and intertidal echinoderms

This research would contribute to understanding how mitochondrial energy production has evolved in response to the diverse habitats echinoderms occupy, from intertidal zones to deep-sea environments.

What insights can P. ochraceus MT-ATP8 provide about mitochondrial genome evolution and selection pressures?

P. ochraceus MT-ATP8 can serve as a model for understanding broader patterns in mitochondrial genome evolution and selection pressures:

• Selection pressure analysis:

  • Compare sequence conservation patterns between MT-ATP8 and other mitochondrial genes

  • Identify whether selection pressures differ between nuclear-encoded and mitochondrial-encoded ATP synthase components

  • Examine whether the genetic bottleneck observed after sea star wasting disease affected mitochondrial genetic diversity

• Co-evolution patterns:

  • Analyze co-evolution between MT-ATP8 and interacting proteins (both mitochondrial and nuclear-encoded)

  • Test whether mitonuclear co-adaptation is evident in regions of protein-protein interaction

  • Examine whether population-specific nuclear variants correlate with mitochondrial variants

• Adaptive signatures:

  • Compare MT-ATP8 sequences from P. ochraceus populations across environmental gradients

  • Identify whether variants correlate with temperature, tidal exposure, or other environmental factors

  • Test functional differences between variants under simulated environmental stress conditions

This research would contribute to understanding how mitochondrial genomes respond to selective pressures and maintain functional compatibility with nuclear-encoded interacting partners.