Recombinant Solanum lycopersicum ATP synthase subunit 9, mitochondrial (ATP9)

What is the basic structure and function of ATP synthase subunit 9 in tomato mitochondria?

ATP synthase subunit 9 (ATP9) in tomato mitochondria is a critical component of the F₀ domain of the mitochondrial ATP synthase complex. Within the membrane domain (F₀), subunit 9 forms an oligomeric ring structure (typically composed of 10 identical subunits) that works in conjunction with subunit 6 to transport protons across the mitochondrial inner membrane. This proton translocation is coupled to ATP synthesis in the extra-membrane F₁ domain of the ATP synthase complex .

The proton channel consists of one subunit 6 protein and the oligomeric ring of subunits 9. During ATP synthesis, the subunit 9 ring rotates as protons are translocated, which induces conformational changes in the F₁ domain that promote ATP synthesis . The protein is encoded by the mitochondrial gene ATP9 and represents one of the few ATP synthase components with mitochondrial (rather than nuclear) genetic origin.

How does tomato ATP9 compare structurally to ATP9/subunit c in other species?

While the search results don't provide explicit comparisons of tomato ATP9 to other species, research on ATP synthases across different organisms shows conservation of the basic structure with species-specific variations. In yeast and many other eukaryotes, subunit 9 (also called subunit c) forms a ring of 10 identical proteins .

Interestingly, in some anaerobic archaea, the motor subunit c has unusual characteristics that are otherwise only found in eukaryotic V₁V₀ ATPases . This evolutionary conservation suggests fundamental importance for the function of ATP synthases across diverse species.

When working with tomato ATP9, researchers should note that while the basic function is conserved, species-specific differences in sequence, post-translational modifications, and regulatory mechanisms may exist. Comparative sequence analysis is recommended before designing experiments with recombinant tomato ATP9.

What are the recommended methods for expressing recombinant tomato ATP9 in heterologous systems?

For heterologous expression of recombinant tomato ATP9, researchers should consider several systems depending on experimental goals:

• Bacterial Expression Systems:

  • E. coli expression systems with specialized vectors designed for membrane proteins

  • Codon optimization for the host system may be necessary

  • Consider fusion tags that aid solubility while not disrupting function

• Yeast Expression Systems:

  • S. cerevisiae can be advantageous as it has native ATP synthase and supports proper folding

  • Consider using strains with deletions in native ATP9 to prevent interference

• Plant-Based Expression:

  • Nicotiana tabacum transient expression systems, similar to those used for other plant membrane proteins like Cf-9

  • Agrobacterium tumefaciens-mediated transformation can be effective, as demonstrated with other recombinant proteins

When designing expression constructs, researchers should consider that ATP9 is normally encoded by the mitochondrial genome, and may require specific modifications for expression from nuclear DNA in heterologous systems, including appropriate targeting sequences for mitochondrial import.

How can researchers accurately measure ATP synthesis activity of recombinant ATP9 incorporated into proteoliposomes?

ATP synthesis activity can be measured using reconstituted proteoliposomes with the following methodology:

• Proteoliposome Preparation:

  • Reconstitute purified recombinant ATP9 along with other ATP synthase components into liposomes

  • Control the orientation of incorporation to ensure proper directionality

• Establishing Ion Gradients:

  • Create a potassium diffusion potential by preparing proteoliposomes with low internal K⁺ (approximately 0.5 mM) and exposing them to high external K⁺ (approximately 200 mM)

  • Add valinomycin to allow K⁺ entry, generating an electrical field (positive inside, around 160 mV)

  • Establish appropriate Na⁺ or H⁺ gradients depending on the ion specificity of the ATP synthase

• Measuring ATP Synthesis:

  • Add ADP to the reaction medium

  • Monitor ATP production over time using luciferase-based assays or other ATP detection methods

  • Synthesis rates can be expressed as nmol·min⁻¹·mg protein⁻¹

• Verification with Controls:

  • Include ionophore controls (such as TCS or ETH2120) that dissipate the electrochemical gradient

  • Perform reactions without ADP to confirm ATP is synthesized rather than released from binding sites

Using this approach, researchers can determine if the recombinant ATP9 integrates functionally into the ATP synthase complex and contributes to ATP synthesis activity.

What strategies can be used to create site-directed mutations in tomato ATP9 for functional studies?

Site-directed mutagenesis of tomato ATP9 can provide valuable insights into structure-function relationships. Consider these approaches:

• PCR-Based Mutagenesis:

  • Site-directed mutagenesis using overlap extension PCR

  • QuikChange or similar commercial kits optimized for high efficiency

• CRISPR-Cas9 Editing:

  • For directly modifying the mitochondrial gene in planta

  • Requires specialized techniques for targeting the mitochondrial genome

• Recombination-Based Approaches:

  • Create chimeric genes between ATP9 and related sequences to analyze domain functions

  • This approach parallels the recombination events observed in the Cf-9 studies, where chimeric genes yielded important functional insights

When designing mutations, researchers should focus on:

• Conserved residues involved in proton transport

• Interface regions important for ring formation

• Residues potentially involved in interactions with other ATP synthase subunits

Table 1: Priority Residues for Mutation Analysis in ATP9

How can researchers investigate the assembly-dependent translation of ATP9 in tomato mitochondria?

To investigate assembly-dependent translation of ATP9, researchers can adapt approaches used in yeast systems to tomato mitochondria:

• Generate Assembly-Deficient Mutants:

  • Create mutations in genes encoding other ATP synthase subunits

  • Target assembly factors specific to ATP synthase

• Measure Translation Rates:

  • Use pulse-labeling with radioactive amino acids to measure de novo synthesis

  • Employ ribosome profiling to analyze translation efficiency

• Analyze Feedback Mechanisms:

  • Investigate cis-regulatory sequences in the ATP9 gene that might respond to assembly status

  • Identify potential regulatory proteins similar to those found in yeast (like Atp25)

Research in yeast has shown that the rate of translation of ATP9 is enhanced in strains with mutations leading to specific defects in the assembly of this protein, suggesting feedback regulation . Similar mechanisms likely exist in tomato mitochondria, though with potential differences in the specific regulatory factors involved.

How should researchers address contradictory findings regarding ATP9 assembly into the ATP synthase complex?

When confronting contradictory findings about ATP9 assembly, consider these systematic approaches:

• Identify the Source of Contradiction:

  • Determine if contradictions arise from methodological differences

  • Consider species-specific differences if comparing across organisms

  • Evaluate if the contradictions represent conditional truths dependent on physiological conditions

• Systematic Comparative Analysis:

  • Categorize contradictions as self-contradictory (within single studies), pairwise contradictions (between two studies), or conditional contradictions (involving three or more interdependent findings)

  • Assess the impact of statement importance on contradiction evaluation - more important statements tend to be more reliably evaluated for contradictions

• Resolution Strategies:

  • Design experiments that directly test contradictory hypotheses under identical conditions

  • Consider temporal dynamics - some contradictions may reflect different stages of a process

For example, the traditional view that the ATP9 ring forms separately from other ATP synthase components has been contradicted by evidence suggesting assembly-dependent formation . Researchers should design experiments that specifically track the formation of the ATP9 ring under different assembly conditions to resolve this contradiction.

What statistical approaches are most appropriate for analyzing ATP9 functional data from proteoliposome experiments?

For rigorous analysis of ATP9 functional data from proteoliposome experiments:

• Appropriate Statistical Tests:

  • ANOVA for comparing multiple experimental conditions

  • Paired t-tests for comparing specific treatments

  • Non-parametric alternatives when data do not meet normality assumptions

• Controls and Normalization:

  • Include negative controls (ionophores that dissipate gradients)

  • Include positive controls (known functional ATP synthase)

  • Normalize ATP synthesis rates to protein amount or liposome internal volume

• Time-Series Analysis:

  • Establish linearity of ATP synthesis over time (typically linear for about 2 minutes)

  • Calculate initial rates from linear portion of the curve

  • Model kinetics to extract mechanistic parameters

• Presentation of Data:

  • Include both raw data and calculated rates

  • Present confidence intervals rather than just standard deviations

  • Consider visualization techniques that display both individual data points and statistical summaries

When reporting ATP synthesis rates, express them in nmol·min⁻¹·mg protein⁻¹ and include details about the electrochemical gradient (e.g., ΔμNa⁺/F of 230 mV) to allow proper comparison with other studies .

How can researchers investigate the role of ATP9 in tomato stress responses and disease resistance pathways?

To explore ATP9's role in stress responses and disease resistance:

• Integration with Defense Pathways:

  • Investigate potential connections between mitochondrial function and plant defense pathways

  • Study if ATP9 expression changes during pathogen attack, similar to defense genes upregulated in the M205 tomato mutant

• Metabolic Reprogramming:

  • Analyze how alterations in ATP9 function affect energy metabolism during stress

  • Measure ATP/ADP ratios in wild-type versus ATP9-modified plants under stress conditions

• ROS Signaling:

  • Investigate if ATP9 dysfunction leads to altered reactive oxygen species (ROS) production

  • Determine if such ROS changes activate defense signaling cascades

• Experimental Approaches:

  • Generate plants with modified ATP9 expression levels or mutated ATP9

  • Challenge these plants with pathogens and abiotic stressors

  • Monitor both energy metabolism parameters and defense response markers

The study of the M205 tomato mutant, which showed stunted growth, wilting, progressive leaf chlorosis and necrosis, and constitutive expression of defense genes , provides a conceptual framework for understanding how alterations in essential cellular components can trigger defense responses.

What are the current challenges in studying ATP9 post-translational modifications in tomato mitochondria?

Studying post-translational modifications (PTMs) of ATP9 presents several challenges:

• Technical Limitations:

  • Low abundance of ATP9 in mitochondrial samples

  • Hydrophobic nature complicating standard proteomic approaches

  • Potential lability of some modifications during purification

• Mitochondrial-Specific Challenges:

  • Limited accessibility of mitochondrial proteins to standard cellular machinery

  • Unique redox environment influencing PTM stability

  • Potential for plant-specific modifications not found in model systems

• Current Methodological Approaches:

  • Specialized extraction techniques for membrane proteins

  • Targeted mass spectrometry with multiple reaction monitoring

  • Analysis of ATP9 in partially purified ATP synthase complexes

• Biological Significance Assessment:

  • Creating site-specific mutants that mimic or prevent specific PTMs

  • Assessing if PTMs change in response to physiological or stress conditions

  • Determining if PTMs affect assembly, stability, or function of ATP9

Researchers should consider parallels with other membrane proteins, such as how phosphorylation/dephosphorylation of threonine residues (e.g., T835 in Cf-9) can act as molecular switches determining protein functional states .