Recombinant Arabidopsis thaliana ATP synthase subunit 9, mitochondrial (ATP9)

What is the basic function of ATP9 in Arabidopsis thaliana mitochondria?

ATP9 (also known as subunit 9) is a component of the F0 module of mitochondrial ATP synthase in Arabidopsis thaliana. It forms part of the membrane-embedded proton channel that facilitates proton movement across the inner mitochondrial membrane. This movement drives the rotation of the enzyme's central stalk, which is coupled to ATP synthesis in the F1 catalytic domain. ATP9 exists in multiple copies (approximately 10) in the assembled complex, forming a ring structure that is essential for proton translocation .

What is the protein structure and sequence of Arabidopsis ATP9?

Arabidopsis thaliana ATP9 is an 85-amino acid protein with the sequence: MTKREYNSQPEMLEGAKLIGAGAATIALAGAAIGIGNVFSSLIHSVARNPSLAKQLFGYAILGFALTEAIALFALMMAFLILFVF . The protein is hydrophobic and forms two transmembrane α-helices connected by a loop region. In the assembled F0 complex, these helices form part of the proton channel structure .

What are the most effective methods for isolating recombinant ATP9 protein from Arabidopsis?

For effective isolation of recombinant ATP9 from Arabidopsis, researchers typically employ:

• Heterologous expression systems: Due to ATP9's hydrophobic nature, E. coli expression systems with specialized vectors containing His-tags are commonly used .

• Purification protocol:

  • Lyse cells under denaturing conditions with detergents suitable for membrane proteins

  • Use immobilized metal affinity chromatography (IMAC) with Ni-NTA resin to capture His-tagged ATP9

  • Perform size exclusion chromatography to improve purity

  • Store purified protein in buffer containing 6% trehalose at pH 8.0 to maintain stability

• Quality control: Verify purity using SDS-PAGE (>90% purity) and confirm identity via western blotting or mass spectrometry .

How can I assess ATP9 integration into the ATP synthase complex in vivo?

Several complementary approaches can be used to assess ATP9 integration into the ATP synthase complex:

• Blue Native PAGE (BN-PAGE): This technique allows visualization of intact ATP synthase complexes and can detect assembly defects when ATP9 function is compromised .

• In organello protein synthesis: Isolate mitochondria from Arabidopsis seedlings and monitor [35S]Met incorporation into newly synthesized proteins, including ATP9. This approach reveals translation rates and can identify defects in protein synthesis .

• Complex activity assays: Measure ATP synthase activity in isolated mitochondria to assess functional integration of ATP9 .

• Immunoprecipitation: Use antibodies against other ATP synthase subunits to pull down the complex and detect associated ATP9 .

• Fluorescence microscopy: Use GFP-tagged assembly factors or other subunits to visualize complex formation in living cells .

What are the consequences of ATP9 knockdown or mutation in Arabidopsis?

Research on ATP9 knockdown or mutation in Arabidopsis has revealed:

• Growth phenotypes: Plants with reduced ATP9 levels show delayed vegetative growth and reduced fertility .

• Biochemical consequences:

  • Five-fold depletion of ATP synthase abundance

  • Lowered ATP synthesis rate in isolated mitochondria

  • No change to mitochondrial electron transport chain complexes

  • Altered adenylate levels and energy charge in planta

• Cellular adaptations:

  • Differential expression of transcripts for amino acid transport

  • Upregulation of various stress response processes

  • Higher respiratory rates

  • Elevated steady-state levels of numerous amino acids, particularly those of the serine family

What approaches can be used to study ATP9 function when direct knockout is lethal?

When direct knockout of ATP9 is lethal, several alternative approaches can be employed:

How does ATP9 assembly into ATP synthase occur and what factors facilitate this process?

ATP9 assembly into the ATP synthase complex involves several steps and factors:

How do defects in ATP9 affect the stoichiometry and assembly of other ATP synthase subunits?

Defects in ATP9 have significant impacts on ATP synthase assembly and subunit stoichiometry:

• Subunit imbalances:

  • Reduced ATP9 levels lead to decreased abundance of other ATP synthase subunits

  • The stoichiometric ratio between α and β subunits in the F1 module can be altered

  • Western blot analysis and BN-PAGE have shown that the steady-state level of ATP synthase decreases when ATP9 synthesis is compromised

• Assembly intermediate accumulation:

  • Defects in ATP9 can lead to accumulation of unassembled subcomplexes

  • Both F0 and F1 modules may be affected, with incomplete assembly of both components

• Compensatory mechanisms:

  • Some evidence suggests that while Complex I, II, III, and IV abundance may not be reduced despite decreased translation rates, ATP synthase levels cannot be maintained

  • This suggests differential regulation of complex stability and turnover

How conserved is ATP9 structure and function across different plant species?

ATP9 structure and function show significant conservation across plant species, though with some important variations:

• Sequence conservation:

  • The core functional domains of ATP9 are highly conserved across plants

  • Transmembrane regions show the highest conservation, while loop regions may vary

  • The protein maintains its fundamental role in proton translocation across species

• Genomic organization:

  • ATP9 is typically encoded in the mitochondrial genome in plants

  • In Arabidopsis and other plants, it is part of a conserved operon structure

  • Some plant species have nuclear-encoded copies resulting from gene transfer events

• Expression regulation:

  • Regulatory mechanisms for ATP9 expression show both conservation and species-specific adaptations

  • Translation efficiency of ATP9 appears consistently high across species, reflecting its critical role

How does chloroplast ATP synthase subunit 9 compare to its mitochondrial counterpart in Arabidopsis?

Comparison between chloroplast and mitochondrial ATP synthase subunit 9 in Arabidopsis reveals:

• Structural similarities and differences:

  • Both form part of the proton-translocating channel in their respective ATP synthases

  • Both participate in ring structures, though the exact stoichiometry may differ

  • Sequence divergence reflects adaptation to the specific environment of each organelle

• Functional specializations:

  • Chloroplast ATP synthase operates primarily in ATP synthesis mode in light conditions

  • Mitochondrial ATP synthase functions continuously in respiration

  • Chloroplast ATP synthase exhibits redox regulation not present in the mitochondrial counterpart

• Assembly pathways:

  • Different assembly factors are involved in each organelle

  • Some assembly factors like Atp11 may be dual-localized and function in both organelles

  • Chloroplast assembly involves specific factors like PAB, BFA1, and BFA3

How is ATP9 expression regulated in Arabidopsis mitochondria?

ATP9 expression in Arabidopsis mitochondria is regulated at multiple levels:

• Transcriptional regulation:

  • Mitochondrial RNA polymerase activity affects ATP9 transcript levels

  • Promoter elements and transcription start sites specific to mitochondrial genes influence expression

• Post-transcriptional mechanisms:

  • RNA stabilization factors, including pentatricopeptide repeat (PPR) proteins, affect ATP9 mRNA stability

  • RNA processing and maturation are critical steps in gene expression

  • Translation activation involves specific factors that recognize mRNA features

• Maternal regulation:

  • Evidence suggests that accumulation of mitochondrially encoded proteins like ATP9 is under maternal control during early development

  • This control involves proteins like LETM1 and LETM2, which affect translation efficiency

What post-translational modifications affect ATP9 function in the ATP synthase complex?

Post-translational modifications of ATP9 and their effects include:

• N-terminal processing:

  • ATP9 may undergo N-terminal processing during import or assembly

  • This processing can affect protein stability and integration into the complex

• Other modifications:

  • Acetylation sites have been identified in ATP synthase subunits, though specific data on ATP9 acetylation is limited

  • Phosphorylation is relatively rare in ATP synthase subunits (∼0.2%)

  • The abundance of PTMs at modified sites is generally low, suggesting they may not be as critical for regulation as in other photosynthetic complexes

• Regulatory impact:

  • PTMs may influence protein stability, complex assembly, and protein-protein interactions

  • PTMs could affect proton translocation efficiency or coupling with the F1 domain

  • Some modifications may respond to physiological conditions or stress responses

What are the main challenges in producing functional recombinant ATP9 protein?

Producing functional recombinant ATP9 presents several challenges:

• Hydrophobicity issues:

  • ATP9 is highly hydrophobic with multiple transmembrane domains

  • This property makes expression, solubilization, and purification technically demanding

  • Specialized expression systems and detergents are often required

• Proper folding:

  • Ensuring correct folding of the recombinant protein in heterologous systems

  • Maintaining the native conformation during purification

  • Avoiding aggregation of the hydrophobic regions

• Functional assessment:

  • Recombinant ATP9 must be reconstituted into lipid environments to assess functionality

  • Proving that the recombinant protein retains native characteristics

  • Developing assays for isolated ATP9 function separate from the complete complex

How can researchers distinguish between direct effects of ATP9 manipulation and secondary consequences in experimental systems?

Distinguishing direct from secondary effects of ATP9 manipulation requires multiple approaches:

• Complementation strategies:

  • Re-express wild-type ATP9 in depleted backgrounds

  • Use tissue-specific or inducible systems to control timing and extent of ATP9 expression

  • Create dose-response relationships by varying ATP9 levels

• Temporal analysis:

  • Monitor changes immediately following ATP9 depletion

  • Track progression of phenotypes over time

  • Early effects are more likely to be direct consequences

• Multi-omics approaches:

  • Combine transcriptomics, proteomics, and metabolomics

  • Identify pathway changes that occur earliest

  • Use network analysis to distinguish primary from secondary effects

• Specific experimental controls:

  • Compare effects of ATP9 manipulation with manipulation of other ATP synthase subunits

  • Use targeted inhibitors of ATP synthase (oligomycin) versus general mitochondrial inhibitors

  • Design experiments that can isolate specific functions of ATP9

What are emerging technologies that could advance ATP9 research in Arabidopsis?

Emerging technologies with potential to advance ATP9 research include:

• Cryo-electron microscopy (Cryo-EM):

  • High-resolution structural analysis of ATP9 within the intact ATP synthase complex

  • Visualization of dynamic states during catalysis

  • Detection of conformational changes associated with mutations or modifications

• CRISPR-based approaches:

  • Base editing for precise modifications of ATP9

  • Prime editing for controlled alterations

  • RNA-targeting CRISPR systems for transcript modulation without permanent genetic changes

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) to study ATP9 movements

  • Optical tweezers to measure forces involved in ATP9 function

  • Real-time imaging of ATP synthase assembly in living cells

• Synthetic biology approaches:

  • Designer ATP9 variants with modified properties

  • Engineering altered stoichiometry of the c-ring

  • Creating minimal functional versions for biotechnological applications

How might understanding ATP9 function contribute to improving plant stress tolerance or bioenergy applications?

ATP9 research could contribute to improved plant traits through:

• Stress tolerance enhancement:

  • Modifying ATP9 to optimize ATP synthase efficiency under stress conditions

  • Engineering plants with improved energy balance during environmental challenges

  • Using knowledge of ATP9 regulation to maintain energy homeostasis during stress

• Bioenergy applications:

  • Optimizing ATP production efficiency in plants grown for bioenergy

  • Engineering ATP synthase with altered proton:ATP ratios for improved biomass production

  • Creating plants with enhanced carbon fixation through optimized energy generation

• Synthetic applications:

  • Using ATP9 and ATP synthase engineering for creating artificial energy systems

  • Developing plant bioreactors with enhanced ATP production capabilities

  • Creating biohybrid systems that couple biological ATP production to synthetic applications

• Translational research:

  • Applying insights from plant ATP9 to address mitochondrial disorders in other organisms

  • Using knowledge of assembly pathways to enhance biotechnological production systems

  • Developing strategies to modulate ATP production in crops under changing climate conditions