Recombinant Glycine max ATP synthase subunit 9, mitochondrial (ATP9)

What is ATP synthase subunit 9 and what role does it play in plant mitochondria?

ATP synthase subunit 9 (ATP9) is a critical component of the mitochondrial ATP synthase complex responsible for ATP production through oxidative phosphorylation. The protein functions as a proteolipid that forms part of the membrane-embedded F0 domain of ATP synthase. In Glycine max (soybean), ATP9 plays a crucial role in proton translocation across the inner mitochondrial membrane, which drives the rotational mechanism of ATP synthesis. The full-length Glycine max ATP9 consists of 74 amino acids with a sequence of MLEGAKSIGAGAATIASAGAAVGIGNVFSSLIHSVARNPSLAKQLFGYAILGFALTEAIALFALMMAFLILFVF . This highly hydrophobic sequence enables ATP9 to form oligomeric rings in the F0 portion, creating the proton-conducting pathway essential for energy production in plant mitochondria.

Understanding ATP9 structure and function provides insights into fundamental aspects of bioenergetics and potentially enables applications in improving plant energy efficiency under various stress conditions.

What is the composition and structural characteristics of recombinant Glycine max ATP9 protein?

Recombinant Glycine max ATP9 protein is typically produced with a histidine tag to facilitate purification and subsequent experimental applications. The detailed composition includes:

Structurally, ATP9 is characterized by its predominantly hydrophobic nature, which facilitates its integration into the mitochondrial membrane . This hydrophobicity presents specific challenges for researchers, as it affects protein solubility and handling during experimental procedures. The protein's structure enables it to assemble into an oligomeric ring formation within the F0 portion of ATP synthase, where it participates in proton translocation across the membrane. When working with recombinant ATP9, researchers must consider these structural characteristics when designing experimental protocols, particularly for reconstitution and functional studies.

How is the ATP9 gene organized in plant mitochondrial genomes?

The ATP9 gene (atp9) in plant mitochondrial genomes exhibits interesting organizational patterns that contribute to genetic diversity. Studies in Petunia have revealed that:

• Multiple copies or variants of the atp9 gene can exist within a single plant's mitochondrial genome.

• These variants can undergo recombination events, particularly in somatic hybrid plants.

Research on Petunia somatic hybrids identified a novel ATP synthase subunit 9 gene generated through intergenomic recombination between atp9 genes from two parental plant lines . This recombinant gene contained the 5' transcribed region from one parental line (3704) and the 3' transcribed region from another parental line (3688). Remarkably, the entire coding region was preserved in this recombinant gene, and it remained transcriptionally active .

The conservation of transcript termini with respect to parental genes resulted in the production of functional hybrid transcripts . This gene organization demonstrates the plasticity of mitochondrial genomes and provides insights into how vital bioenergetic genes can be maintained functionally despite recombination events. Understanding these organizational patterns is essential for researchers investigating mitochondrial genome evolution and the genetic basis of plant energy metabolism.

What are the optimal conditions for expression and purification of recombinant Glycine max ATP9?

Achieving high-quality recombinant ATP9 requires carefully optimized expression and purification protocols that account for the protein's membrane-associated nature. Based on experimental methodologies, the following approach has proven effective:

Expression System and Conditions:

• E. coli bacterial expression system with an N-terminal His-tag fusion

• Culture in LB medium with appropriate antibiotic selection

• IPTG induction at OD600 of 0.6-0.8

• Post-induction expression at reduced temperature (16-20°C) for 16-18 hours to improve membrane protein folding

Purification Protocol:

• Cell lysis using sonication buffer (250 mM sucrose, 50 mM NaH₂PO₄, 5 mM 6-aminocaproic acid, 1 mM EDTA, pH 7.5, protease inhibitors cocktail, 1 μM PMSF)

• Sonication: 6 cycles of 10 seconds with 10-second cooling intervals on ice

• Centrifugation at 6000 × g for 10 minutes at 4°C

• Ultracentrifugation of supernatant at approximately 268,000 × g for 1 hour

• Affinity purification using Ni-NTA agarose beads with appropriate washing and elution buffers

Storage Considerations:

• Store purified protein at -20°C/-80°C

• Aliquoting is necessary to avoid repeated freeze-thaw cycles

• Reconstitution in deionized sterile water to 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (final concentration) for long-term storage stability

Researchers should implement rigorous quality control measures, including SDS-PAGE analysis to confirm >90% purity and functional assays to verify ATP9 activity. The hydrophobic nature of ATP9 presents unique challenges that may necessitate the use of mild detergents or lipid reconstitution for maintaining protein solubility and native conformation throughout the purification process.

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

Investigating ATP9's role in ATP synthase complex assembly requires sophisticated biochemical and structural biology approaches. The following methodologies provide complementary insights into ATP9's integration and function within the complex:

1. Native Gel Electrophoresis:

• Blue Native PAGE (BN-PAGE) enables separation of intact mitochondrial complexes

• Procedure: ATP synthase complexes are isolated using 1-2% digitonin in extraction buffer (30 mM HEPES, 150 mM potassium acetate, 12% glycerol, 2 mM 6-aminocaproic acid, 1 mM EGTA, protease inhibitors, pH 7.4)

• Separation on NativePAGE™ 3-12% Bis-Tris gels allows visualization of both monomeric and dimeric ATP synthase complexes

2. Two-Dimensional Electrophoresis:

• First dimension: Native PAGE separates intact complexes

• Second dimension: SDS-PAGE resolves individual subunits

• Protocol: Cut lanes from native gel, equilibrate in SDS-PAGE running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS, pH 8.3 with 1% β-mercaptoethanol), heat briefly, and run in the second dimension

• This approach identifies ATP9's position within the complex and reveals its interactions with other subunits

3. Affinity Purification and Interaction Analysis:

• His-tagged ATP9 can be used to pull down the entire ATP synthase complex

• Mitochondria (5 mg) are processed and the complex is isolated using Ni-NTA agarose beads

• Mass spectrometry analysis of co-purified proteins identifies ATP9-interacting components

4. Recombinant Gene Analysis:

• Study of recombinant ATP9 genes provides insights into functional domains

• Sequence comparison identifies recombination points and conserved regions

• Transcript analysis confirms expression and processing of recombinant genes

5. Cryo-Electron Microscopy:

• Provides structural information about ATP9's position within the ATP synthase complex

• Reveals conformational changes and subunit interactions during different functional states

• Requires purification of intact ATP synthase complex with properly integrated ATP9

These methodologies collectively enable researchers to determine how ATP9 contributes to the assembly, stability, and function of the ATP synthase complex, providing insights into the fundamental mechanisms of cellular energy production.

How can researchers investigate protein-protein interactions involving ATP9 in mitochondrial complexes?

Investigating protein-protein interactions involving membrane-integrated proteins like ATP9 requires specialized techniques that preserve native interactions. The following methodological approaches are particularly effective:

1. Split-Ubiquitin Membrane Yeast Two-Hybrid System:

• Specifically designed for detecting interactions involving membrane proteins

• Can identify binary interactions between ATP9 and potential partner proteins

• Similar to the approach used for identifying interactions between membrane proteins and cytosolic partners in soybean

• Enables systematic screening of mitochondrial protein libraries against ATP9 as bait

2. Bimolecular Fluorescence Complementation (BiFC):

• Allows visualization of protein interactions in living cells

• ATP9 and potential interaction partners are fused to complementary fragments of a fluorescent protein

• Interaction brings fragments together, restoring fluorescence

• Can confirm the subcellular localization of interactions within mitochondria

• This technique has successfully visualized protein-protein interactions in plant systems

3. Co-Immunoprecipitation with ATP Synthase Complex:

• Using antibodies against ATP9 or its His-tag to pull down the entire complex

• Mass spectrometry analysis of co-precipitated proteins reveals interaction partners

• Requires careful solubilization of mitochondrial membranes using appropriate detergents

• Buffer composition similar to that used in ATP synthase complex isolation (containing digitonin)

4. Chemical Cross-Linking Coupled with Mass Spectrometry:

• Proteins in close proximity are covalently linked using membrane-permeable cross-linking reagents

• Cross-linked complexes are digested and analyzed by mass spectrometry

• This approach identifies direct contacts between ATP9 and other subunits within the ATP synthase complex

• Provides spatial information about protein arrangement and proximity relationships

5. Cryo-Electron Microscopy of ATP Synthase Complexes:

• High-resolution structural analysis of intact complexes containing ATP9

• Reveals precise interaction interfaces between ATP9 and adjacent subunits

• Can identify conformational changes in protein-protein interactions during different functional states

These complementary approaches provide a comprehensive understanding of ATP9's interaction network within mitochondrial complexes. Researchers should consider using multiple techniques to validate interactions and build a complete picture of ATP9's role in complex assembly and function.

What are the characteristics of recombinant ATP9 transcripts and how do they compare to native transcripts?

The characteristics of ATP9 transcripts have been extensively studied, particularly in the context of recombinant genes formed through intergenomic recombination. Research on Petunia somatic hybrids has revealed important features of ATP9 transcripts:

Transcript Characteristics of Recombinant ATP9:

• Hybrid Structure: In the Petunia somatic hybrid (13-133), a novel ATP9 transcript was identified with a chimeric structure:

  • 5' transcribed region derived from parental line 3704

  • 3' transcribed region derived from parental line 3688

• Conservation of Termini: Despite the recombination event, the 5' and 3' transcript termini remained conserved relative to the parental genes, suggesting strong selection pressure for maintaining proper transcript processing

• Functional Expression: The recombinant ATP9 gene was transcriptionally active, producing hybrid transcripts that maintained the coding potential for functional ATP9 protein

Comparison Between Native and Recombinant ATP9 Transcripts:

For analyzing ATP9 transcripts, researchers have employed RNA isolation from mitochondria , Northern blot analysis, cDNA synthesis with sequencing, and RACE (Rapid Amplification of cDNA Ends) to map precise transcript termini. These findings highlight the remarkable plasticity of plant mitochondrial genomes and the maintenance of functional gene expression despite significant recombination events.

How can ATP9 be used in studying metabolons and protein complexes in plant mitochondria?

ATP9, as an integral component of the mitochondrial ATP synthase complex, provides an excellent model system for studying metabolons (organized enzyme complexes) and protein assembly in plant mitochondria. The following methodological approaches leverage ATP9 for such studies:

1. ATP9 as a Membrane Anchor Model:

• Similar to how cytochrome P450 enzymes anchor metabolons to the endoplasmic reticulum , ATP9 serves as a model for membrane-anchored complex assembly in mitochondria

• Tagged ATP9 variants can be used to pull down associated proteins and study assembly intermediates

• This approach parallels successful protein interaction studies in soybean metabolic pathways

2. Visualization of ATP Synthase Complexes:

• Fusion of ATP9 with fluorescent proteins enables tracking of complex formation in vivo

• Bimolecular Fluorescence Complementation (BiFC) can visualize ATP9 interactions with other subunits

• This method has successfully generated "network-like intracellular patterns" for protein complexes in plant cells

• Time-lapse imaging can monitor dynamic assembly and disassembly processes

3. ATP9 as a Component of Higher-Order Structures:

• Investigation of ATP9's role in dimer formation and the organization of ATP synthase into rows

• Analysis of ATP9's interactions with supernumerary subunits (e.g., subunits e/Atp21, g/Atp20, and k/Atp19) that stabilize dimeric structures

• Comparison to other proteins that function in stabilizing large membrane protein complexes

4. Isolation of ATP Synthase Complex States:

• Purification of ATP synthase complexes using digitonin extraction (1-2% digitonin in appropriate buffer)

• Separation of monomeric and dimeric ATP synthase complexes using native electrophoresis

• Analysis of ATP9's differential interactions in various assembly states

5. Metabolic Flux Analysis Through ATP Synthase:

• Using ATP9 variants to modify ATP synthase efficiency

• Measuring changes in metabolic flux through oxidative phosphorylation

• This approach builds on the concept that metabolons "establish efficient metabolic flux" in biosynthetic pathways

These approaches leverage ATP9's dual nature as both a membrane-embedded protein and an essential component of a major metabolic complex to provide insights into the principles governing protein complex assembly and metabolon formation in plant mitochondria. The methodologies build upon successful techniques used to study protein-protein interactions in other plant metabolic pathways .

What are the key considerations when designing experiments to study ATP9 gene recombination in plant mitochondria?

When designing experiments to investigate ATP9 gene recombination in plant mitochondria, researchers should consider several critical factors based on previous successful studies:

1. Selection of Appropriate Plant Systems:

• Choose plant species with well-characterized mitochondrial genomes

• Consider somatic hybridization systems, which have proven valuable in studying mitochondrial gene recombination

• The Petunia somatic hybrid system (lines 3688 and 3704) provides an established model

2. Experimental Approaches to Generate and Detect Recombination:

• Somatic Cell Fusion: Creates conditions where mitochondrial genomes from different sources can recombine

• Tissue Culture Stress: May induce mitochondrial genome rearrangements and recombination

• Cybrid Generation: Transfer of mitochondria between species can reveal recombination patterns

3. Analytical Methods for Recombination Detection:

• Restriction Digest Analysis: Identifies novel restriction fragments arising from recombination

• Southern Hybridization: Using ATP9 probes to detect recombinant fragments

• PCR-Based Approaches: Designing primers to amplify potential recombination junctions

• DNA Sequencing: Essential for precise characterization of recombination breakpoints

4. Transcript Analysis to Confirm Functional Expression:

• Northern Blotting: Detects transcript size and abundance

• 5' and 3' RACE: Maps transcript termini to confirm conservation with parental genes

• RT-PCR and Sequencing: Verifies the expression of recombinant genes

• RNA Editing Analysis: Determines if editing patterns are affected by recombination

5. Experimental Design Considerations:

• Include appropriate parental controls for comparative analysis

• Use multiple independent lines to distinguish genuine recombination from artifacts

• Design time-course experiments to monitor recombination dynamics

• Consider heteroplasmy (multiple mitochondrial genome types) in data interpretation

These approaches provide complementary information about ATP9 gene recombination, offering insights into mitochondrial genome evolution and the generation of genetic diversity in plant energy metabolism. The experimental design should be tailored to address specific research questions while accounting for the complex and dynamic nature of plant mitochondrial genomes.

How can structural studies of ATP9 contribute to understanding mitochondrial ATP synthase function?

Structural studies of ATP9 provide crucial insights into the mechanics of mitochondrial ATP synthase function. Based on available methodologies, the following approaches can significantly advance our understanding:

1. Structural Analysis Techniques:

• Cryo-Electron Microscopy (Cryo-EM):

  • Particularly powerful for membrane protein complexes

  • Can capture ATP synthase in different functional states

  • Positions ATP9 within the context of the entire ATP synthase complex

• X-ray Crystallography:

  • Challenging for membrane proteins but provides high-resolution structures

  • May require lipidic cubic phase crystallization for hydrophobic ATP9

  • Could reveal precise arrangement of ATP9 subunits in the c-ring

• NMR Spectroscopy:

  • Suitable for determining structures of individual ATP9 proteins

  • Provides dynamic information about protein movements

  • Requires isotope labeling of recombinant ATP9

2. Structure-Function Correlations:

• Proton Channel Formation:

  • Identify critical residues forming the proton pathway

  • The ATP9 amino acid sequence (MLEGAKSIGAGAATIASAGAAVGIGNVFSSLIHSVARNPSLAKQLFGYAILGFALTEAIALFALMMAFLILFVF) contains numerous hydrophobic residues that likely contribute to membrane spanning and channel formation

  • Structural studies reveal how these residues arrange to facilitate proton translocation

• c-Ring Assembly:

  • Determine the stoichiometry of ATP9 subunits in the c-ring

  • Identify interfaces between adjacent ATP9 proteins

  • Understand how c-ring size influences ATP synthesis efficiency

• Interface with Other Subunits:

  • Characterize interactions between ATP9 and subunit a

  • Understand the coupling mechanism between proton flow and rotor movement

  • Map contacts with the central stalk (subunit γ) that drives ATP synthesis

3. Comparative Structural Biology:

• Cross-Species Analysis:

  • Compare ATP9 structures across plant species to identify conserved features

  • Correlate structural variations with functional differences

  • Study recombinant ATP9 proteins like those found in Petunia hybrids to determine if structural changes result from recombination events

These structural studies provide a foundation for understanding the fundamental mechanisms of biological energy conversion and offer potential applications in bioengineering and synthetic biology for enhanced plant energy efficiency.

What techniques are most effective for studying post-translational modifications of ATP9?

Post-translational modifications (PTMs) of ATP9 can significantly impact its function, assembly, and regulation within the ATP synthase complex. The following methodological approaches are most effective for comprehensive PTM analysis:

1. Mass Spectrometry-Based Approaches:

• Bottom-Up Proteomics:

  • Digestion of purified ATP9 with specific proteases

  • LC-MS/MS analysis of resulting peptides

  • Database searching with variable modifications

  • Effective for identifying phosphorylation, acetylation, methylation

• Top-Down Proteomics:

  • Analysis of intact ATP9 protein

  • Provides comprehensive PTM profile and combinatorial modifications

  • Preserves information about modification stoichiometry

  • Challenging for membrane proteins but potentially more informative

• Targeted MS Methods:

  • Multiple Reaction Monitoring (MRM) for specific PTMs

  • Parallel Reaction Monitoring (PRM) for increased specificity

  • Particularly useful for quantifying dynamic changes in PTMs

2. Modification-Specific Enrichment Strategies:

• Phosphorylation Analysis:

  • TiO₂ or IMAC enrichment of phosphopeptides

  • Phospho-specific antibodies for immunoprecipitation

  • ³²P labeling for in vivo phosphorylation studies

• Acetylation Analysis:

  • Anti-acetyllysine antibodies for enrichment

  • HDAC inhibitors to preserve acetylation state

  • Stable isotope labeling to track acetylation dynamics

3. Site-Directed Mutagenesis Approaches:

• Generation of recombinant ATP9 with mutations at potential PTM sites

• Expression in E. coli or yeast expression systems

• Functional assays to determine the impact of preventing specific modifications

• Integration into ATP synthase complexes to assess assembly effects

4. ATP9-Specific Considerations:

• Extraction Conditions: Use buffers that preserve PTMs (phosphatase inhibitors, deacetylase inhibitors)

• Membrane Protein Challenges: Optimize solubilization conditions that maintain PTMs

• Functional Context: Relate identified PTMs to ATP9's role in the c-ring of ATP synthase

• Comparative Analysis: Examine PTM patterns across different physiological conditions and in relation to ATP synthase assembly states (monomers vs. dimers)

These complementary approaches provide a comprehensive view of ATP9 post-translational modifications, offering insights into the regulation of this crucial component of mitochondrial energy production machinery and potential targets for biotechnological applications.