Recombinant Pichia canadensis ATP synthase subunit 9, mitochondrial (ATP9)

What is ATP synthase subunit 9 and what is its role in mitochondrial function?

ATP synthase subunit 9 (ATP9) is a critical component of the mitochondrial ATP synthase complex (F₀F₁-ATPase), which plays a central role in cellular energy production through oxidative phosphorylation. This protein forms part of the F₀ domain, specifically contributing to the c-ring that facilitates proton translocation across the inner mitochondrial membrane. In Pichia canadensis, ATP9 is a 76 amino acid protein situated in the mitochondrial membrane .

The functional importance of ATP9 is underscored by its high conservation across species, with studies showing that the ATP9 gene from various organisms may share between 40-60% sequence identity . This conservation reflects the protein's essential role in ATP synthesis, whereby the proton gradient established across the inner mitochondrial membrane drives the rotation of the c-ring, ultimately coupling this mechanical energy to ATP production by the F₁ component of the complex.

What is the amino acid sequence of full-length Pichia canadensis ATP9?

The full-length Pichia canadensis ATP synthase subunit 9, mitochondrial (ATP9) protein consists of 76 amino acids with the following sequence:

MQLVLAAKYIGAAIATIGLLGAGIGIAIVFAALINGTSRNPSLRNTLFPFAILGFALSEATGLFCLMISFLLLYGV

This sequence reveals several important structural features characteristic of mitochondrial ATP9 proteins. The sequence includes numerous hydrophobic amino acids arranged in patterns consistent with membrane-spanning domains, which allows the protein to embed properly in the mitochondrial membrane. The N-terminal region contains a mitochondrial targeting sequence that directs the protein to its correct subcellular location, similar to what has been observed in other organisms like Trypanosoma brucei .

Analysis of this sequence demonstrates that the protein likely forms alpha-helical structures that span the mitochondrial membrane, contributing to the formation of the c-ring structure within the F₀ domain of ATP synthase. The specific arrangement of hydrophobic and hydrophilic residues allows ATP9 to participate in proton translocation, which is essential for the chemiosmotic coupling that drives ATP synthesis.

How does ATP9 from Pichia canadensis compare to ATP9 from other organisms?

ATP9 demonstrates significant evolutionary conservation across species while maintaining organism-specific adaptations. Comparative analysis reveals that ATP9 from Pichia canadensis shares structural and functional similarities with homologous proteins from other organisms, though with distinct variations that reflect evolutionary divergence and adaptation to specific cellular environments.

Research on the T. brucei ATPase subunit 9 demonstrated that it shares between 40-60% identity with subunit 9 from various organisms . While specific comparison data between P. canadensis and other organisms is not directly provided in the search results, we can infer from studies of similar proteins that the core functional domains are likely well-conserved.

Key differences between ATP9 proteins from different species are often found in:

The genomic organization of ATP9 also varies across species. In some organisms, like Petunia species, ATP9 genes can undergo intergenomic recombination, as demonstrated in hybrid lines . In T. brucei, the ATP9 gene is found in the nuclear genome rather than the mitochondrial genome (kinetoplast DNA) . This genomic location variation has important implications for evolutionary studies and genetic manipulation approaches.

How is recombinant ATP9 from Pichia canadensis typically expressed in laboratory settings?

Recombinant Pichia canadensis ATP9 protein is typically expressed using heterologous expression systems, with E. coli being a common choice for laboratory-scale production . This approach offers advantages including rapid growth, high protein yields, and well-established protocols for genetic manipulation and protein purification.

For expression in E. coli, the following methodological steps are typically employed:

• Vector design and construction: The ATP9 gene (encoding amino acids 1-76) is cloned into an expression vector with an N-terminal His-tag for purification purposes . The His-tag facilitates downstream purification using metal affinity chromatography while minimizing interference with protein function.

• Transformation and expression: The recombinant vector is transformed into an appropriate E. coli strain optimized for membrane protein expression. Expression conditions (temperature, induction time, inducer concentration) are carefully optimized to maximize yield while maintaining proper protein folding.

• Cell lysis and protein extraction: Given ATP9's hydrophobic nature, specialized detergent-based extraction methods are typically employed to solubilize the protein from bacterial membranes.

• Purification: The His-tagged protein is purified using immobilized metal affinity chromatography (IMAC), potentially followed by additional purification steps such as size exclusion chromatography to achieve >90% purity .

• Storage: The purified protein is often lyophilized to enhance stability during storage at -20°C or -80°C, with aliquoting recommended to avoid repeated freeze-thaw cycles .

While E. coli is commonly used, alternative expression systems may be employed depending on research needs. For instance, yeast-based expression systems like Pichia pastoris can be advantageous for proteins requiring specific post-translational modifications or proper membrane insertion.

What methodologies are available for studying ATP9's role in ATP synthase assembly?

Investigating ATP9's role in ATP synthase assembly requires sophisticated experimental approaches. Several methodological strategies can be employed:

• Site-directed mutagenesis and functional complementation: Specific residues in ATP9 can be mutated to assess their importance in assembly and function. Mutant proteins can be expressed in cells depleted of native ATP9 to determine whether they rescue ATP synthase assembly and function. This approach allows mapping of critical interaction sites and functional domains.

• Protein-protein interaction studies: Techniques such as co-immunoprecipitation, yeast two-hybrid assays, or proximity labeling approaches (BioID, APEX) can identify direct interaction partners of ATP9 during assembly. Cross-linking mass spectrometry is particularly valuable for capturing transient interactions during the assembly process.

• In vitro reconstitution assays: Purified recombinant ATP9 can be combined with other purified ATP synthase components to reconstitute assembly in vitro. This controlled approach allows detailed analysis of assembly intermediates and kinetics.

• Cryo-electron microscopy: High-resolution structural analysis of assembly intermediates can reveal the progressive incorporation of ATP9 into the c-ring and subsequent integration into the larger ATP synthase complex.

• Time-resolved pulse-chase experiments: By pulse-labeling newly synthesized proteins and tracking their incorporation into complexes over time, researchers can determine the sequence and kinetics of ATP9 assembly into functional ATP synthase.

• Genetic approaches using hybrid genes: The study of intergenomic recombination between ATP9 genes, as demonstrated in Petunia hybrid lines , provides insights into the functional domains of ATP9 critical for assembly and function. Similar approaches could be applied to study P. canadensis ATP9.

• Native gel electrophoresis: Blue native PAGE combined with western blotting or mass spectrometry can identify assembly intermediates containing ATP9 and track their progression to fully assembled complexes.

These methodologies can be complementary, providing a comprehensive understanding of ATP9's role in the complex process of ATP synthase assembly. Researchers often employ multiple approaches to validate findings and address different aspects of the assembly process.

How can I use flow cytometry to screen for high-expressing ATP9 recombinant strains?

Flow cytometry offers a powerful approach for rapidly identifying high-expressing recombinant strains. Based on recent methodological advances in Pichia pastoris systems, a similar approach could be adapted for screening ATP9 expression:

• Reporter system construction: Create a fusion construct linking ATP9 expression to a fluorescent reporter. Following the methodology developed for P. pastoris, you could replace the endogenous endoplasmic reticulum transmembrane protein with a functional Sec63-EGFP fusion protein . This allows indirect monitoring of recombinant protein expression through fluorescence intensity.

• Validation of correlation: Establish the relationship between EGFP fluorescence intensity and ATP9 expression levels. Studies with P. pastoris have demonstrated excellent linear correlation (0.8<|R|<1) between EGFP fluorescence and recombinant protein expression levels . This correlation must be validated specifically for ATP9 expression.

• Flow cytometry sorting protocol: Prepare a mixed population of transformants with different ATP9 expression levels and sort them based on fluorescence intensity using a flow cytometer sorter. The cells can be separated into distinct sub-populations (high, medium, and low fluorescence) .

• Post-sorting validation: Verify ATP9 expression levels in the sorted populations using biochemical methods (Western blotting, activity assays). In P. pastoris, this approach identified high-fluorescence strains with expression levels 4.09 times higher than low-fluorescence strains after 120 hours of methanol induction .

• Scale-up and stability assessment: Evaluate the stability of ATP9 expression in high-producing clones over multiple generations to ensure consistent performance.

This flow cytometry-based approach offers significant advantages over traditional screening methods, including:

• Increased throughput: Thousands of cells can be screened per second

• Enhanced sensitivity: Detection of subtle differences in expression levels

• Time efficiency: Reduction of screening time from weeks to days

• Versatility: Applicable to different expression systems and target proteins

For optimal results, the screening protocol should be integrated with other high-throughput technologies such as droplet microfluidics, which can further increase screening efficiency and precision.

What are the implications of ATP9 mutations on mitochondrial function?

Mutations in ATP9 can have profound implications for mitochondrial function due to its central role in energy production. The consequences of such mutations extend beyond ATP synthesis to affect multiple cellular processes:

• Compromised ATP synthesis: Mutations affecting proton translocation or c-ring assembly can directly impair ATP production capacity. This energy deficit has cascading effects on all ATP-dependent cellular processes, particularly in high-energy-demanding tissues.

• Altered mitochondrial membrane potential: The proton-translocating function of ATP9 contributes to maintaining the electrochemical gradient across the inner mitochondrial membrane. Mutations can disrupt this gradient, affecting not only ATP synthesis but also other membrane potential-dependent processes like protein import.

• Respiratory chain dysfunction: ATP9 mutations can lead to compensatory changes in respiratory chain components, potentially increasing reactive oxygen species (ROS) production. Studies in T. brucei have shown developmental regulation of ATP9 expression , suggesting a coordinated relationship between ATP synthase and respiratory chain components.

• Mitochondrial morphology changes: Severe ATP9 mutations can alter mitochondrial ultrastructure, particularly the organization of cristae, which are enriched in ATP synthase complexes. These structural changes further compromise mitochondrial function.

• Calcium homeostasis disturbances: ATP synthase participates in calcium handling within mitochondria, and ATP9 mutations can disrupt this process, with potential implications for signaling pathways and cell death mechanisms.

• Metabolic reprogramming: Cells with ATP9 mutations often exhibit metabolic adaptation to compensate for reduced oxidative phosphorylation. This may include increased reliance on glycolysis, similar to the metabolic shifts observed during the life cycle of T. brucei, where ATP9 expression varies significantly between life stages .

• Development and differentiation effects: The developmental regulation of ATP9 observed in organisms like T. brucei suggests that proper ATP9 function may be particularly important during specific developmental stages or cellular differentiation processes.

Experimental approaches to study these effects include creating site-specific mutations in ATP9, expressing the mutant proteins in appropriate model systems, and assessing functional parameters such as oxygen consumption, ATP production, membrane potential, and ROS generation. Advanced techniques such as live-cell imaging, metabolic flux analysis, and proteomics can provide comprehensive insights into the cellular consequences of ATP9 mutations.

How can I design experiments to study the interaction of ATP9 with other ATP synthase subunits?

Designing experiments to investigate ATP9 interactions with other ATP synthase subunits requires strategic approaches that capture both stable and transient interactions within this complex multiprotein assembly. Here are methodological strategies for such studies:

• Chemical cross-linking combined with mass spectrometry (XL-MS):

  • Apply membrane-permeable cross-linkers to intact mitochondria expressing tagged ATP9

  • Isolate cross-linked complexes through affinity purification

  • Perform proteolytic digestion and analyze by mass spectrometry

  • Identify cross-linked peptides to map interaction interfaces

  • This approach can capture direct interactions between ATP9 and neighboring subunits within native membrane environments

• Förster Resonance Energy Transfer (FRET):

  • Generate fusion constructs of ATP9 and potential interaction partners with appropriate fluorophore pairs

  • Express in relevant cell systems and monitor FRET signals in live cells

  • Quantify interaction strengths through FRET efficiency measurements

  • This approach provides spatial and temporal resolution of interactions in living cells

• Bioluminescence Resonance Energy Transfer (BRET):

  • Similar to FRET but using luciferase-fluorophore pairs

  • Offers advantages of lower background and no requirement for external illumination

  • Particularly useful for detecting ATP9 interactions in native membrane environments

• Proximity-based labeling approaches:

  • Fuse ATP9 with enzymes like BioID (biotin ligase) or APEX (ascorbate peroxidase)

  • These enzymes biotinylate or modify proteins in close proximity

  • Identify labeled proteins by streptavidin pulldown and mass spectrometry

  • This method can capture both stable and transient interactions within the native cellular context

• Recombinant protein approaches:

  • Express and purify recombinant ATP9 along with potential interaction partners

  • Conduct in vitro binding assays using techniques such as surface plasmon resonance

  • Perform co-reconstitution experiments in liposomes to assess functional interactions

  • These approaches allow precise control of experimental conditions for quantitative analysis

• Genetic approaches:

  • Generate hybrid constructs similar to the intergenomic recombination observed in Petunia ATP9 genes

  • Create chimeric ATP9 proteins with domains from different species to map interaction regions

  • Test functionality through complementation assays in cells depleted of native ATP9

  • This approach helps identify functionally important interaction domains

• Structural biology approaches:

  • Apply cryo-electron microscopy to purified ATP synthase complexes

  • Focus on the interface between ATP9 (c-ring) and other subunits

  • Complement with computational modeling to predict interaction dynamics

  • These methods provide atomic-level details of interaction interfaces

When implementing these approaches, it's important to include appropriate controls to distinguish specific interactions from non-specific associations. Validation across multiple complementary techniques strengthens confidence in the identified interactions. Additionally, studying interactions under different physiological conditions may reveal dynamic changes in the ATP synthase complex assembly and function.

How can amino acid metabolism be customized to enhance recombinant ATP9 production?

Optimizing amino acid metabolism represents a sophisticated strategy for enhancing recombinant ATP9 production. Research on Pichia pastoris provides valuable insights that can be adapted for ATP9 expression:

• Transcriptomic and metabolomic profiling: First, conduct comprehensive systems-level analyses of the metabolome and transcriptome of your production strain during ATP9 expression . This reveals metabolic bottlenecks specific to ATP9 production, particularly in amino acid metabolism pathways.

• Targeted overexpression of amino acid biosynthesis genes: Based on metabolomic data, identify and overexpress key genes involved in amino acid biosynthesis. Research in P. pastoris has shown that constitutive overexpression of genes related to amino acid metabolism can significantly impact recombinant protein production . For ATP9, which contains 76 amino acids , focus on pathways producing the most abundant amino acids in its sequence.

• Supplementation with energetically expensive amino acids: The ATP9 sequence contains several energetically expensive amino acids (such as leucine, isoleucine, phenylalanine, and tyrosine) . Supplementing the cultivation media with these specific amino acids can unburden cellular metabolism. Studies have shown that addition of energetically expensive amino acids or glutamate increases recombinant protein production in P. pastoris .

• Manipulation of transcription factors: Overexpression of transcription factors regulating amino acid metabolism can lead to global improvements in amino acid availability. This approach affects multiple pathways simultaneously and may overcome limitations of single-gene overexpression strategies.

• Engineering of amino acid transporters: Enhance the uptake of supplemented amino acids by overexpressing specific amino acid transporters. This ensures that externally provided amino acids effectively reach the intracellular synthesis machinery.

• Metabolic flux redirection: Implement strategies to redirect carbon flux toward amino acid biosynthesis. For example, studies in P. pastoris have revealed redistribution of intracellular carbon fluxes toward the TCA-cycle and ATP production during recombinant protein production , which could be further optimized for ATP9 expression.

• Media optimization based on compositional analysis: Analyze the amino acid composition of ATP9 (MQLVLAAKYIGAAIATIGLLGAGIGIAIVFAALINGTSRNPSLRNTLFPFAILGFALSEATGLFCLMISFLLLYGV) and design a custom supplementation strategy that matches the stoichiometric requirements of this specific protein.

These approaches should be implemented with careful monitoring of cellular stress responses, as overexpression of metabolic genes or accumulation of certain amino acids may trigger unfavorable cellular responses. An iterative optimization approach, combining multiple strategies and fine-tuning based on experimental results, typically yields the best outcomes for enhancing recombinant protein production.

What are the optimal conditions for expressing recombinant Pichia canadensis ATP9 in E. coli systems?

Expressing hydrophobic membrane proteins like ATP9 in E. coli presents specific challenges that require careful optimization. Based on successful expression strategies for recombinant Pichia canadensis ATP9 and related proteins, the following methodological approach is recommended:

• Expression vector selection:

  • Choose vectors with tunable promoters (T7-based systems with lac operator control are often effective)

  • Incorporate an N-terminal His-tag for purification while preserving protein function

  • Consider fusion partners (e.g., MBP, SUMO) that enhance solubility if expression yields are low

• E. coli strain optimization:

  • Use C41(DE3) or C43(DE3) strains designed for membrane protein expression

  • Consider BL21(DE3)pLysS to reduce leaky expression prior to induction

  • For proteins with rare codons, use Rosetta strains with additional tRNAs

• Culture conditions optimization:

  • Temperature: Lower temperatures (16-25°C) often improve proper folding

  • Media composition: Enriched media (TB, 2xYT) generally yield higher biomass

  • Inducer concentration: Use lower IPTG concentrations (0.1-0.5 mM) for more controlled expression

  • Induction timing: Induce at mid-log phase (OD₆₀₀ ≈ 0.6-0.8) for optimal balance between growth and expression

• Membrane extraction protocol:

  • Use mild detergents (DDM, LDAO) for efficient solubilization without denaturation

  • Include glycerol (10-15%) in extraction buffers to stabilize the protein

  • Optimize detergent:protein ratios to maximize extraction while maintaining native structure

• Purification strategy:

  • Employ IMAC with imidazole gradient elution for initial capture via the His-tag

  • Follow with size exclusion chromatography to achieve >90% purity

  • Consider ion exchange chromatography as an additional polishing step if needed

• Stabilization approaches:

  • Add phospholipids during purification to maintain native-like environment

  • Include appropriate protease inhibitors to prevent degradation

  • Optimize buffer composition (pH, salt concentration, additives) based on protein stability assessments

• Storage considerations:

  • Lyophilize purified protein for long-term stability

  • Store at -20°C/-80°C and avoid repeated freeze-thaw cycles

  • Prepare single-use aliquots to maintain protein integrity

Systematic optimization using Design of Experiments (DoE) approaches is recommended to efficiently identify optimal conditions with minimal experimental iterations. Expression levels should be monitored using Western blotting, while protein quality can be assessed using circular dichroism or functional assays appropriate for ATP9.

How can I troubleshoot low expression yields of recombinant ATP9?

When encountering low expression yields of recombinant ATP9, a systematic troubleshooting approach addressing multiple aspects of the expression system is crucial. Here's a methodological framework for addressing common issues:

• Genetic construct optimization:

  Issue	Diagnostic Approach	Solution Strategy
  Codon bias	Analyze CAI score of ATP9 sequence	Synthesize codon-optimized gene for E. coli
  mRNA stability	Predict mRNA secondary structures	Modify 5' region to eliminate strong secondary structures
  Toxicity	Observe growth curves post-induction	Use tighter promoter control or specialized strains (C41/C43)
  Translation efficiency	Examine Shine-Dalgarno sequence	Optimize ribosome binding site strength

• Expression conditions optimization:

  Parameter	Test Range	Monitoring Method
  Temperature	16°C, 25°C, 30°C, 37°C	SDS-PAGE, Western blot
  Induction timing	Early (OD₆₀₀=0.4), mid (OD₆₀₀=0.8), late (OD₆₀₀=1.2)	Growth curve, protein yield quantification
  Inducer concentration	0.1mM, 0.5mM, 1.0mM IPTG	Western blot, activity assay
  Media composition	LB, TB, 2xYT, defined media	Biomass measurement, protein yield per cell

• Protein solubility enhancement:

  • Test different fusion partners (MBP, SUMO, Trx) to improve folding

  • Co-express molecular chaperones (GroEL/ES, DnaK/J) to assist folding

  • Add membrane-mimetic compounds to culture media (specific phospholipids)

  • Consider alternative solubilization methods for improved protein extraction

• Post-translational modifications and protein stability:

  • Evaluate protein degradation using protease inhibitor cocktails

  • Analyze potential cleavage sites and design constructs to minimize proteolysis

  • Assess protein half-life in expression host

  • Monitor for formation of inclusion bodies using microscopy and fractionation

• Alternative expression systems:

  • If E. coli consistently yields poor results, consider expression in Pichia pastoris

  • For P. pastoris expression, implement flow cytometry screening to identify high-producing clones

  • Evaluate cell-free expression systems for membrane proteins

  • Consider specialized E. coli strains designed for membrane protein expression

• Scale-up considerations:

  • Optimize oxygen transfer rates in larger culture volumes

  • Monitor pH stability throughout the fermentation process

  • Implement fed-batch strategies to maintain optimal growth rates

  • Consider continuous cultivation approaches for sensitive proteins

When implementing these troubleshooting strategies, maintain careful documentation of all experiments and outcomes. Quantify protein yields using consistent methods across experiments to enable direct comparisons. Remember that membrane proteins like ATP9 often require special handling, and what works for soluble proteins may need significant modification for optimal results with membrane-embedded proteins.

What are the future research directions for Pichia canadensis ATP9 studies?

Research on Pichia canadensis ATP9 continues to evolve, with several promising future directions emerging from current findings. These research avenues span from fundamental structural biology to applied biotechnological applications:

• Structural dynamics investigations: Advanced structural studies using cryo-electron microscopy and molecular dynamics simulations could reveal the dynamic behavior of ATP9 within the c-ring structure. This would provide insights into the precise mechanisms of proton translocation and rotational catalysis.

• Evolutionary conservation analysis: Comprehensive comparative studies between P. canadensis ATP9 and homologues from diverse organisms could identify universally conserved features versus species-specific adaptations. The identity levels observed between ATP9 from various organisms (40-60%) suggest both conserved functional domains and species-specific adaptations worthy of investigation.

• Post-translational modification mapping: Identification and functional characterization of potential post-translational modifications of ATP9 could reveal regulatory mechanisms that modulate ATP synthase activity in response to cellular energetic demands.

• Interactomics approaches: Comprehensive identification of ATP9's protein interaction network beyond the core ATP synthase complex could uncover unexpected roles in mitochondrial organization or signaling pathways.

• Development of ATP9-based biotechnological tools: The recombinant expression systems established for ATP9 could be leveraged to develop novel biotechnological applications, including energy-harvesting devices or biosensors.

• Integration with high-throughput screening technologies: Building on methodologies developed for P. pastoris , sophisticated screening approaches could be developed specifically for ATP9 research, enabling rapid identification of variants with enhanced properties.

• Metabolic engineering applications: Insights from studying amino acid metabolism optimization in recombinant protein production could be specifically applied to ATP9 expression systems, potentially leading to novel bioproduction platforms.

• Synthetic biology approaches: Creation of hybrid ATP9 proteins, similar to the intergenomic recombination observed in Petunia , could generate variants with novel properties for fundamental research or biotechnological applications.