Recombinant Schizosaccharomyces pombe ATP synthase subunit 9, mitochondrial (atp9)

What is the basic structure and function of Schizosaccharomyces pombe ATP synthase subunit 9?

ATP synthase subunit 9 (atp9) is a critical component of the mitochondrial F₀F₁ ATP synthase complex in S. pombe. The mature protein consists of 74 amino acids with the sequence: MIQAAKYIGAGLATIGVSGAGVGIGLIFSNLISGTSRNPSVRPHLFSMAILGFALTEATGLFCLMLAFLIIYAA . Functionally, it forms part of the membrane-embedded F₀ portion of ATP synthase that facilitates proton translocation across the inner mitochondrial membrane, which drives ATP synthesis through the F₁ catalytic domain. The protein contains highly hydrophobic regions that anchor it within the mitochondrial membrane, consistent with its role in forming the proton channel of the ATP synthase complex .

How does S. pombe ATP synthase subunit 9 differ from its homologs in other organisms?

While the core function of ATP synthase subunit 9 is conserved across species, important structural and functional variations exist between S. pombe and other organisms:

The P/O ratio (ATP produced per oxygen atom reduced) in S. pombe has been experimentally determined to be 1.28, which affects its bioenergetic efficiency . Additionally, S. pombe employs different energetic parameters for growth, with a growth-associated ATP maintenance (GAM) value of approximately 58.3 mmol gDW⁻¹, which is comparable to but slightly higher than the 55.3 mmol gDW⁻¹ found in S. cerevisiae .

What are the optimal storage and handling conditions for recombinant S. pombe ATP synthase subunit 9?

For optimal storage and handling of recombinant S. pombe ATP synthase subunit 9:

• Storage: Store the lyophilized protein at -20°C/-80°C upon receipt. After reconstitution, aliquot and store at -20°C/-80°C to avoid repeated freeze-thaw cycles .

• Reconstitution: Prior to opening, briefly centrifuge the vial to bring contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Add glycerol to a final concentration of 5-50% (standard recommendation is 50%) before aliquoting for long-term storage .

• Buffer conditions: The protein is typically supplied in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps maintain protein stability .

• Critical consideration: Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided. Working aliquots can be maintained at 4°C for up to one week .

What methods can be used to assess the functional integrity of recombinant atp9 in experimental systems?

To evaluate the functional integrity of recombinant atp9, researchers can employ several complementary approaches:

• ATPase activity assays: Measure ATP hydrolysis rates using purified F₁ or submitochondrial particles. Parameters to assess include:

  • pH optimum (can be shifted in mutant forms)

  • Bicarbonate activation

  • Sensitivity to inhibitors (e.g., azide)

  • Cooperative kinetics

• Respiration measurements: Cellular respiration rates in reconstituted systems or yeast strains expressing the recombinant protein can indicate functional integration into respiratory complexes .

• Growth phenotype analysis: Complementation studies in S. pombe strains lacking endogenous atp9 can demonstrate functional activity. Growth on glycerol as a non-fermentable carbon source specifically requires functional ATP synthase .

• Blue Native PAGE (BN-PAGE): Useful for analyzing the incorporation of atp9 into assembled ATP synthase complexes and respiratory chain supercomplexes .

How can computational models be used to study the role of ATP synthase subunit 9 in S. pombe metabolism?

Computational modeling approaches offer powerful tools for investigating ATP synthase function within the broader metabolic network of S. pombe:

What insights have structure-function studies provided about ATP synthase subunit 9 mutations in S. pombe?

Structure-function studies of ATP synthase in S. pombe have revealed critical insights through the analysis of mutants and revertants:

• Mutational effects on catalytic properties: Phenotypic revertants selected from S. pombe mutants lacking α or β subunits show distinct alterations in catalytic parameters. For example:

  • α subunit-modified revertant (R3.51) exhibits more acidic optimal pH, absence of bicarbonate activation, and decreased sensitivity to azide inhibition

  • β subunit-modified revertant (R4.3) demonstrates more alkaline optimal pH, higher bicarbonate activation, and increased sensitivity to azide

• Cooperativity changes: Purified F₁ from these revertants shows altered cooperativity patterns:

  • Loss of bicarbonate-sensitive negative cooperativity in R3.51

  • Increased negative cooperativity in R4.3

• Nucleotide specificity: Alterations in subunit structure can affect nucleotide preference, as demonstrated by changes in ITPase activity sensitivity to azide in R4.3 submitochondrial particles compared to wild-type .

• Functional complementation: Revertants show partial restoration of growth on glycerol respiratory medium compared to the parental mutants, although growth and cellular respiration remain reduced compared to wild-type strains, indicating that the recovered subunits retain functional limitations .

How does ATP synthase subunit 9 assembly integrate with broader mitochondrial respiratory complex formation?

The assembly of ATP synthase subunit 9 is coordinated with the biogenesis of other respiratory complexes through several mechanisms:

• Assembly factor interactions: Assembly factors like Shy1 (homolog of human SURF1) interact with components of multiple respiratory complexes. While Shy1 primarily functions in complex IV assembly, evidence suggests it may also influence ATP synthase assembly through protein-protein interactions, contributing to the formation of respiratory supercomplexes .

• Mitochondrial gene expression coordination: Expression of mtDNA-encoded genes, including those encoding ATP synthase components, is coordinated through shared regulatory mechanisms. Deletion of assembly factors like Shy1 affects the expression of mtDNA-encoded genes that may include ATP synthase subunits .

• Supercomplex formation: BN-PAGE analysis has revealed that ATP synthase assembly is linked to the formation of mitochondrial respiratory chain supercomplexes, which enhance electron transport efficiency and reduce reactive oxygen species production .

• Compensatory mechanisms: Unlike some organisms, S. pombe appears to possess compensatory mechanisms that can maintain partial mitochondrial functionality even when certain assembly factors are compromised, suggesting a robust and adaptable assembly process .

What experimental approaches can be used to study ATP synthase subunit 9 interactions with other mitochondrial proteins?

Several techniques are particularly effective for investigating protein-protein interactions involving ATP synthase subunit 9:

• Co-immunoprecipitation (Co-IP): This approach has successfully identified interactions between assembly factors like Shy1 and structural subunits of respiratory complexes in S. pombe. When applying this method to atp9:

  • Use epitope-tagged versions of atp9 (ensuring tags don't disrupt function)

  • Include appropriate controls for nonspecific binding

  • Validate interactions with multiple antibodies or tag positions

• Blue Native PAGE (BN-PAGE): This technique preserves native protein complexes and can reveal the incorporation of atp9 into assembled ATP synthase and respiratory supercomplexes:

  • Sample preparation must minimize detergent concentration to preserve complexes

  • Gradient gels (3-12% or 4-16%) provide optimal resolution

  • Combined with second-dimension SDS-PAGE for subunit identification

• Proteomics approaches:

  • Crosslinking mass spectrometry (XL-MS) can capture transient interactions

  • Proximity labeling techniques (BioID, APEX) can identify neighboring proteins in the native mitochondrial environment

  • Quantitative proteomics can identify changes in the interactome under different conditions

• Genetic interaction studies:

  • Synthetic genetic array (SGA) analysis to identify genetic interactions

  • Suppressor screens to identify functional relationships

  • CRISPR-based screens for systematic interaction mapping

How do the energetic parameters of S. pombe ATP synthase compare with those of other yeast species?

Comparative analysis of ATP synthase energetics between S. pombe and other yeasts reveals important similarities and differences:

What insights from S. pombe ATP synthase studies are potentially applicable to human mitochondrial disorders?

Research on S. pombe ATP synthase provides valuable insights that may be applicable to human mitochondrial disorders:

• Leigh Syndrome models: Studies of assembly factors like Shy1 (homolog of human SURF1) in S. pombe provide insights into mitochondrial disorders such as Leigh Syndrome, which is associated with complex IV deficiency but may also involve ATP synthase dysfunction .

• Compensatory mechanisms: S. pombe exhibits compensatory mechanisms that maintain partial mitochondrial functionality when assembly factors are compromised. Understanding these mechanisms could inform therapeutic strategies for human mitochondrial disorders .

• Supercomplexes: The assembly of respiratory chain supercomplexes in S. pombe, which include ATP synthase components, parallels similar structures in human mitochondria. Perturbations in supercomplex formation may contribute to human disease pathology .

• Experimental advantages: S. pombe offers several advantages as a model system:

  • Higher sequence conservation with humans for certain mitochondrial components

  • Ability to survive with compromised respiratory function

  • Genetic tractability combined with eukaryotic mitochondrial complexity

  • Well-characterized genome and proteome

How should researchers design experiments to study the effects of post-translational modifications on S. pombe ATP synthase subunit 9?

When investigating post-translational modifications (PTMs) of ATP synthase subunit 9, consider these methodological approaches:

• Identification of modification sites:

  • Use high-resolution mass spectrometry techniques optimized for hydrophobic proteins

  • Employ complementary fragmentation methods (HCD, ETD) to improve coverage

  • Include enrichment strategies for specific modifications (e.g., phosphopeptide enrichment)

  • Compare PTM profiles across different growth conditions and metabolic states

• Functional analysis of modifications:

  • Generate site-directed mutants that either prevent modification (e.g., S→A for phosphorylation) or mimic constitutive modification (e.g., S→D/E)

  • Assess the impact on ATP synthase assembly using BN-PAGE

  • Measure ATPase/ATP synthase activity with purified enzymes or submitochondrial particles

  • Evaluate growth phenotypes on fermentable versus non-fermentable carbon sources

• Temporal dynamics:

  • Implement pulse-chase labeling combined with mass spectrometry

  • Use inducible expression systems to study the assembly of newly synthesized subunits

  • Employ time-resolved proteomics after metabolic shifts

• Integration with computational models:

  • Update metabolic models (pomGEM, pcPombe) to incorporate the effects of PTMs on ATP synthase function

  • Predict system-level consequences of altered ATP synthase regulation

What are the critical controls and validation steps when using recombinant S. pombe ATP synthase subunit 9 in reconstitution experiments?

Reconstitution experiments with recombinant atp9 require rigorous controls and validation:

• Protein quality assessment:

  • Verify purity by SDS-PAGE (>90% purity recommended)

  • Confirm protein identity by western blot and/or mass spectrometry

  • Assess the integrity of critical structural features using circular dichroism

  • Verify proper folding using limited proteolysis

• Functional validation:

  • Compare ATP hydrolysis/synthesis rates with native enzyme preparations

  • Assess sensitivity to known inhibitors (oligomycin, azide)

  • Verify pH-dependent activity profiles match expected patterns

  • Measure proton translocation in reconstituted liposomes

• Critical controls:

  • Include inactive mutant versions as negative controls

  • Perform parallel reconstitutions with native subunits for direct comparison

  • Ensure appropriate buffer conditions (pH, ionic strength) that match physiological environment

  • Include detergent-only controls when working with membrane proteins

• Integration validation:

  • Verify incorporation into the intact ATP synthase complex

  • Assess oligomerization state by native gel electrophoresis

  • Confirm proper membrane orientation in reconstituted systems

  • Evaluate long-term stability of reconstituted complexes