Recombinant Oryza sativa subsp. japonica ATP synthase subunit 9, mitochondrial (ATP9)

What is ATP synthase subunit 9 in Oryza sativa and how is it characterized?

ATP synthase subunit 9 in Oryza sativa (rice) is a small mitochondrial protein (74 amino acids) that forms part of the nonenzymatic membrane component (F0) of mitochondrial ATP synthase (Complex V). This protein constitutes one of the critical chains in the proton channel of the enzyme complex. The protein is encoded by the mitochondrial genome, specifically by the ATP9 gene, and is alternatively known as a lipid-binding protein due to its membrane-embedded nature .

ATP9 is characterized by its high hydrophobicity, which allows it to be embedded within the inner mitochondrial membrane. Structurally, it contains two transmembrane alpha-helices connected by a polar loop region. Multiple ATP9 subunits assemble into a ring structure (c-ring) that plays a crucial role in the rotational mechanism of ATP synthase. The protein can be studied through various biochemical and biophysical techniques, including SDS-PAGE for purity assessment, where recombinant preparations typically show >85% purity .

What is the functional significance of ATP9 in mitochondrial energy production?

ATP9 plays a critical role in cellular energy metabolism by constituting a key component of the proton channel in ATP synthase. Within the F0 domain of the enzyme, ATP9 forms a ring of 10 identical subunits (the c-ring) that, together with subunit 6, facilitates proton translocation across the mitochondrial inner membrane . This proton movement is coupled to ATP synthesis in the extramembranous F1 domain of the enzyme complex.

The proton translocation mechanism involves the rotation of the c-ring as protons pass through the channel. This rotational motion is mechanically coupled to conformational changes in the F1 domain, where ADP and inorganic phosphate are converted to ATP. The precise arrangement of ATP9 subunits in the c-ring is essential for this process, as it creates the path through which protons flow down their electrochemical gradient . Dysfunction of ATP9 can severely compromise mitochondrial ATP production, highlighting its fundamental importance in cellular bioenergetics.

How does ATP9 integrate into the complete ATP synthase complex?

The integration of ATP9 into the complete ATP synthase complex follows a precise assembly pathway that involves both separate module formation and coordinated assembly steps. Current research suggests that ATP synthase assembly involves multiple modules that converge in the final stages of complex formation .

In the assembly process, ATP9 subunits first form the c-ring structure, which is one of the earliest assembly intermediates. According to studies in yeast (which have homologous systems to rice), this c-ring then associates with the F1 domain module. The peripheral stalk components are added subsequently, providing stability to the c-ring/F1 complex. The final steps involve the addition of mitochondrially-encoded subunits a (ATP6) and A6L (ATP8) .

Recent evidence suggests that ATP synthase is formed from three different modules: the c-ring (containing ATP9), the F1 domain, and the ATP6/ATP8 complex. The proper integration of these modules is essential for the formation of functional ATP synthase. The assembly process is carefully regulated, with translation of ATP9 being enhanced under specific conditions related to the assembly state of the complex .

What expression systems yield functional recombinant ATP9 protein?

Expression of recombinant ATP9 from Oryza sativa presents significant challenges due to its hydrophobic nature and the requirements for proper folding and assembly. Based on available research data, yeast expression systems have shown considerable success for producing functional recombinant ATP9 . Yeast systems offer advantages for mitochondrial proteins because they possess similar compartmentalization and processing machinery to plant mitochondria.

For optimal expression, researchers should consider the following methodological approaches:

• Codon optimization of the ATP9 gene sequence for the selected expression host

• Addition of appropriate tags (typically N-terminal) that do not interfere with protein folding

• Use of inducible promoters to control expression levels and minimize toxicity

• Growth at lower temperatures (typically 20-25°C) during induction to enhance proper folding

• Supplementation with specific lipids that assist membrane protein folding

The expression yield can be monitored through Western blot analysis using antibodies against either ATP9 or the fusion tag. Initial screening of multiple constructs with variations in tags, linkers, and promoters is recommended to identify optimal expression conditions .

What are the optimal purification strategies for recombinant ATP9?

Purification of recombinant ATP9 requires specialized approaches due to its hydrophobic nature and membrane association. Based on research protocols for similar proteins, a multi-step purification strategy is recommended:

• Cell lysis using methods that preserve protein structure (e.g., French press or sonication with protease inhibitors)

• Membrane fraction isolation through differential centrifugation

• Solubilization using mild detergents such as n-dodecyl-β-D-maltoside (DDM) or digitonin

• Affinity chromatography using the fusion tag (if present)

• Size exclusion chromatography for further purification and buffer exchange

The choice of detergent is critical for maintaining protein stability and function. Typically, a detergent screening panel is recommended to identify optimal solubilization conditions. The purification process should be monitored by SDS-PAGE, with target purity exceeding 85% for most research applications .

For functional studies, it is important to maintain the protein in a native-like environment, which may involve reconstitution into liposomes or nanodiscs after purification. The storage buffer composition significantly impacts stability, with typical formulations including 20-50 mM phosphate or Tris buffer, 100-150 mM NaCl, 5-10% glycerol, and detergent at concentrations slightly above the critical micelle concentration.

How can the functionality of purified recombinant ATP9 be verified experimentally?

Verifying the functionality of purified recombinant ATP9 is essential before using it in downstream applications. Several complementary approaches can be employed:

• Proton transport assays: Reconstitute ATP9 into liposomes containing pH-sensitive fluorescent dyes (e.g., ACMA or pyranine) and measure fluorescence changes upon addition of ionophores or establishment of pH gradients.

• Binding studies: Assess the interaction of purified ATP9 with known partners (e.g., F1 subunits or other F0 components) using techniques such as surface plasmon resonance, microscale thermophoresis, or co-immunoprecipitation.

• Structural integrity analysis: Employ circular dichroism spectroscopy to verify the secondary structure content, which should show predominantly alpha-helical characteristics.

• ATP synthase reconstitution: Combine purified ATP9 with other purified subunits to reconstitute partial or complete ATP synthase complexes, followed by ATP synthesis activity measurements.

• Complementation assays: Test whether the recombinant protein can restore function in ATP9-deficient systems, such as modified yeast strains or isolated mitochondria with depleted ATP9.

These functional assays should be performed alongside appropriate controls, including denatured protein samples and known functional reference proteins. The specific methodology must be optimized based on the exact experimental questions being addressed and the available equipment .

How can recombinant ATP9 advance structural studies of ATP synthase?

Recombinant ATP9 from Oryza sativa provides valuable opportunities for structural studies of ATP synthase, particularly for understanding species-specific aspects of enzyme architecture and function. Methodological approaches include:

• Cryo-electron microscopy (cryo-EM): Purified recombinant ATP9 can be reconstituted with other subunits to form partial or complete ATP synthase complexes suitable for high-resolution structural analysis. This technique has revolutionized our understanding of membrane protein complexes and can reveal detailed interactions between ATP9 and other subunits.

• X-ray crystallography: While challenging for membrane proteins, crystallographic studies of the c-ring formed by ATP9 subunits can provide atomic-level insights into proton-binding sites and rotational mechanisms. This approach requires highly pure, homogeneous protein samples typically obtained through specialized crystallization techniques for membrane proteins.

• NMR spectroscopy: Solution or solid-state NMR can provide information about ATP9 dynamics and interactions in membrane environments. Isotopic labeling of recombinant ATP9 (typically with 15N, 13C, or 2H) is necessary for these studies.

• Cross-linking mass spectrometry: Chemical cross-linking combined with mass spectrometry can identify interaction interfaces between ATP9 and neighboring subunits, providing constraints for structural modeling.

These structural studies can specifically address questions about the c-ring stoichiometry in rice ATP synthase, the precise arrangement of transmembrane helices, and species-specific adaptations in ATP9 structure that may relate to environmental adaptations or regulatory mechanisms specific to rice .

What techniques effectively investigate ATP9 assembly into the c-ring?

Investigating the assembly of ATP9 into the c-ring requires specialized techniques that can capture intermediate states and protein-protein interactions. Recommended methodological approaches include:

• Blue Native PAGE (BN-PAGE): This technique separates intact protein complexes and can resolve assembly intermediates containing ATP9. Sequential immunoblotting with antibodies against ATP9 and other subunits can track the incorporation of ATP9 into larger complexes.

• Pulse-chase labeling: In systems where protein synthesis can be monitored (e.g., isolated mitochondria or cell-free translation systems), radioactive labeling of newly synthesized ATP9 followed by chase periods allows tracking of its incorporation into the c-ring and larger assemblies.

• Proximity labeling: Techniques such as BioID or APEX2 proximity labeling, where ATP9 is fused to a biotin ligase, can identify proteins that interact with ATP9 during the assembly process.

• Real-time fluorescence microscopy: Using fluorescently tagged ATP9 in combination with other tagged subunits allows visualization of assembly processes in living cells, particularly when combined with techniques like Förster resonance energy transfer (FRET).

Recent research suggests that ATP9 assembly may be more complex than previously thought, with evidence indicating that the translation of ATP9 is enhanced in strains with mutations affecting its assembly. This suggests the existence of assembly-dependent feedback mechanisms that regulate ATP9 production .

What methods can reveal ATP9 contribution to proton translocation?

Investigating ATP9's role in proton translocation requires specialized biophysical and biochemical techniques. The following methodological approaches are particularly valuable:

• Site-directed mutagenesis: Creating specific mutations in the proton-binding residues of ATP9 (typically conserved acidic amino acids) followed by functional analysis can reveal their contribution to proton transport. This approach can be combined with complementation assays in model systems.

• Proton transport assays: Reconstitution of purified ATP9 (alone or with other subunits) into liposomes containing pH-sensitive fluorescent dyes allows direct measurement of proton movement. This can be extended to patch-clamp electrophysiology for single-channel recordings.

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS): This technique can identify regions of ATP9 that undergo conformational changes during the proton translocation cycle by measuring the rate of hydrogen/deuterium exchange in different functional states.

• Computational molecular dynamics: Simulations based on structural data can model proton movement through the c-ring and identify key residues and water molecules involved in the translocation pathway.

• Spectroscopic approaches: Techniques such as Fourier-transform infrared spectroscopy (FTIR) or electron paramagnetic resonance (EPR) with appropriate probes can track protonation states of key residues during catalytic cycles.

These methods should ideally be combined to build a comprehensive understanding of how ATP9 contributes to the proton translocation mechanism that drives ATP synthesis .

How does ATP9 contribute to ATP synthase dimerization and oligomerization?

ATP synthase forms dimers and higher oligomers in the mitochondrial inner membrane, and this supramolecular organization is functionally significant. The contribution of ATP9 to these higher-order structures can be investigated through several methodological approaches:

• Cryo-electron tomography: This technique can visualize the arrangement of ATP synthase dimers and oligomers in the membrane, revealing the position of the c-ring (formed by ATP9) in these structures.

• Cross-linking studies: Chemical cross-linking combined with mass spectrometry can identify specific residues of ATP9 that interact with neighboring ATP synthase monomers in dimers or oligomers.

• Blue Native PAGE: Varying the detergent type and concentration during solubilization can preserve different oligomeric states of ATP synthase, allowing the investigation of conditions that promote or disrupt oligomerization.

• Mutagenesis: Targeted mutations in specific regions of ATP9 followed by assessment of oligomerization can identify residues critical for higher-order interactions.

What is known about assembly-dependent translation regulation of ATP9?

Research in yeast systems has revealed fascinating insights into the assembly-dependent regulation of mitochondrial-encoded ATP synthase subunits, including ATP9 homologs. These findings provide a framework for investigating similar mechanisms in rice:

• Translational activation: Studies have shown that translation of ATP9 can be enhanced in strains with mutations leading to specific defects in ATP9 assembly. This suggests the existence of feedback mechanisms that coordinate ATP9 synthesis with its assembly into the complete complex .

• Regulatory factors: Several proteins have been identified that influence ATP9 expression, including Aep1, Aep2, and the N-terminal fragment of Atp25. These factors may mediate the assembly-dependent regulation by monitoring the assembly state and adjusting translation accordingly .

• Transcript stability: The stability of ATP9 mRNA is dependent on specific factors, such as a 35 kDa C-terminal cleavage fragment of Atp25. This represents another layer of regulation that could respond to assembly status .

• cis-regulatory elements: Assembly-dependent translation involves cis-regulatory sequences within the ATP9 gene or its untranslated regions. These sequences may serve as binding sites for regulatory factors that sense assembly state .

Methodologically, this regulation can be studied through:

• Pulse-labeling experiments to measure ATP9 synthesis rates under different assembly conditions

• RNA binding assays to identify factors that interact with ATP9 mRNA

• Genetic approaches to identify and characterize assembly-monitoring factors

• Reporter constructs to map regulatory elements within the ATP9 gene and transcripts

These regulatory mechanisms ensure balanced production of mitochondrial and nuclear-encoded subunits, which is essential for efficient assembly of the complete ATP synthase complex .

How do mutations in ATP9 affect ATP synthase function and cellular phenotypes?

Investigating the consequences of ATP9 mutations provides valuable insights into structure-function relationships and potential disease mechanisms. Methodological approaches include:

Mutations in ATP9 can affect ATP synthase function through several mechanisms:

• Disruption of c-ring assembly

• Alteration of proton-binding sites

• Impaired rotation of the c-ring

• Destabilization of interactions with other subunits

• Compromised dimerization or oligomerization

These functional defects can manifest as reduced ATP synthesis capacity, altered mitochondrial morphology, or increased production of reactive oxygen species, ultimately affecting plant growth, development, and stress responses .

What are the optimal conditions for storing recombinant ATP9?

Proper storage of recombinant ATP9 is critical for maintaining its structural integrity and functionality. Based on established protocols for membrane proteins, the following guidelines are recommended:

• Temperature considerations:

  • Liquid form: Store at -20°C or -80°C for up to 6 months

  • Lyophilized form: Store at -20°C or -80°C for up to 12 months

• Buffer composition:

  • pH: Maintain between 7.0-7.5 (typically phosphate or Tris buffer)

  • Salt: Include 100-150 mM NaCl to maintain ionic strength

  • Stabilizers: 5-10% glycerol or sucrose can enhance stability

  • Detergent: Maintain at concentrations slightly above critical micelle concentration

  • Reducing agents: Consider adding DTT or β-mercaptoethanol to prevent oxidation

• Storage format:

  • Prepare small working aliquots to avoid repeated freeze-thaw cycles

  • For short-term use, samples can be stored at 4°C for up to one week

  • For membrane-reconstituted protein, storage in liposomes or nanodiscs may provide better stability

• Quality control:

  • Perform functional and structural integrity tests after storage periods

  • Monitor protein aggregation through techniques such as dynamic light scattering

  • Establish quality thresholds for specific applications

Repeated freezing and thawing should be strictly avoided as it significantly decreases protein stability and activity . For long-term storage beyond the recommended periods, it is advisable to reassess protein functionality before use in critical experiments.

How can researchers troubleshoot issues with ATP9 stability and functionality?

Troubleshooting stability and functionality issues with recombinant ATP9 requires systematic investigation of multiple factors. The following methodological approach is recommended:

• Protein aggregation issues:

  • Verify protein solubility in the working buffer through centrifugation or light scattering

  • Screen different detergents or lipid environments that better mimic the native membrane

  • Consider using fusion partners that enhance solubility

  • Optimize protein concentration to avoid concentration-dependent aggregation

• Loss of functional activity:

  • Check pH and ionic conditions, as proton-binding function is pH-sensitive

  • Verify the presence of essential post-translational modifications

  • Ensure complete removal of potentially inhibitory purification reagents

  • Examine the oligomeric state to confirm proper c-ring formation

• Poor expression yields:

  • Optimize codon usage for the expression host

  • Test different expression temperatures, induction times, and inducer concentrations

  • Consider using specialized strains designed for membrane protein expression

  • Evaluate different fusion tags and their positions (N- or C-terminal)

• Reconstitution problems:

  • Screen lipid compositions that better match the native mitochondrial membrane

  • Optimize protein-to-lipid ratios for reconstitution

  • Test different reconstitution methods (detergent dialysis, direct incorporation, etc.)

  • Verify orientation in membrane systems using protease accessibility assays

For each troubleshooting step, it is important to include appropriate controls and systematic variation of parameters. Documentation of all conditions and results will facilitate identification of critical factors affecting ATP9 stability and functionality .

What controls should be included when working with recombinant ATP9?

Rigorous experimental design requires appropriate controls when working with recombinant ATP9. The following controls should be considered for different experimental contexts:

• Expression and purification controls:

  • Empty vector control (processed identically to the ATP9 expression construct)

  • Known well-expressing membrane protein as a positive control

  • SDS-PAGE standards for molecular weight verification

  • Western blot controls with antibodies against the tag and, if available, ATP9 itself

• Functional assay controls:

  • Heat-denatured ATP9 (negative control)

  • Commercial ATP synthase preparations (positive control where applicable)

  • Buffer-only controls for background measurements

  • Known inhibitors of ATP synthase (e.g., oligomycin) to confirm specificity

• Interaction studies controls:

  • Tag-only protein to identify tag-mediated interactions

  • Unrelated membrane protein to control for nonspecific hydrophobic interactions

  • Competition experiments with unlabeled proteins to verify specificity

  • Reversed experimental setup (e.g., pulling down interaction partners with different tags)

• Structural analysis controls:

  • Samples at different concentrations to identify concentration-dependent effects

  • Measurements under denaturing conditions as reference

  • Well-characterized membrane proteins with known structural features

• Reconstitution controls:

  • Empty liposomes or nanodiscs

  • Liposomes containing unrelated membrane proteins

  • Various lipid compositions to identify lipid-specific effects

These controls help identify artifacts, nonspecific effects, and technical issues, ensuring that experimental observations truly reflect the properties and functions of ATP9 .