Recombinant Pisum sativum ATP synthase subunit 9, mitochondrial (ATP9)

What is the basic structure and function of ATP9 in the mitochondrial ATP synthase complex?

ATP9 (also referred to as subunit 9 or subunit c) forms a critical oligomeric ring structure within the membrane domain (F₀) of the ATP synthase complex. In yeast, this ring consists of 10 identical ATP9 subunits that collectively form an integral proton channel along with subunit 6 (ATP6) . The ATP9 ring is responsible for the rotational movement that occurs during proton translocation across the mitochondrial inner membrane, which ultimately drives conformational changes in the F₁ domain to catalyze ATP synthesis .

In Pisum sativum, ATP9 serves a similar fundamental function, although specific structural details might vary slightly from the well-characterized yeast model. The protein is encoded by the mitochondrial genome and contributes to the energy conservation process during oxidative phosphorylation, which is essential for plant growth and development .

How does ATP9 contribute to phosphorus use efficiency in Pisum sativum?

Field pea, like other legumes, requires significant phosphorus (P) inputs for optimal growth and nodule formation . The ATP synthase complex, including ATP9, is integral to energy transformation processes that support phosphorus acquisition and utilization. When P becomes limited, ATP synthase function may be compromised, affecting energy production necessary for nodule development and nitrogen fixation .

P uptake is regulated by high and low affinity transporters throughout the plant's vascular system, with initial absorption occurring at root hairs via P-type H⁺-ATPase pumps. This process maintains a strict Pi concentration of approximately 5-10 mM in the cytoplasm . The ATP synthase complex, including ATP9, provides the ATP necessary to power these transport mechanisms, making it indirectly crucial for phosphorus use efficiency (PUE) .

What is the genetic origin of ATP9 in Pisum sativum mitochondria?

In plants including Pisum sativum, ATP9 is encoded by the mitochondrial genome . This mitochondrial origin has important implications for inheritance patterns and gene expression regulation. Unlike nuclear-encoded components of the ATP synthase complex, mitochondrially-encoded ATP9 is subject to organelle-specific gene expression mechanisms and assembly processes .

The dual genetic origin of ATP synthase components (with some subunits encoded by nuclear genes and others by mitochondrial genes) necessitates coordinated expression and assembly to maintain proper stoichiometry . This coordination involves complex regulatory mechanisms that ensure appropriate levels of each subunit are produced despite being synthesized in different cellular compartments.

What is known about ATP9 ring assembly mechanisms in plant mitochondria?

In yeast, a complex called Atco, comprising mitochondrially-encoded ATP9 and nuclear-encoded Cox6 (a structural subunit of cytochrome oxidase), serves as an assembly intermediate for ATP synthase . Pulse-chase experiments demonstrated that newly translated ATP9 present in Atco is converted to the ring form, which is then incorporated into the ATP synthase with kinetics characteristic of a precursor-product relationship . This indicates that Atco is the exclusive source of ATP9 for ATP synthase assembly in yeast.

While specific assembly mechanisms in Pisum sativum have not been as thoroughly characterized, similar principles likely apply. The assembly process would involve coordination between mitochondrially-synthesized ATP9 and nuclear-encoded ATP synthase components to ensure proper complex formation and function.

How is ATP9 expression coordinated with other ATP synthase components in plants?

The coordination of ATP9 expression with other ATP synthase components involves sophisticated regulatory mechanisms. Research in yeast has shown that the translation rate of ATP9 is enhanced in strains with mutations leading to specific defects in the assembly of this protein . These translation modifications involve assembly intermediates interacting with ATP9 within the final enzyme complex and cis-regulatory sequences that control gene expression in the organelle .

This suggests the existence of assembly-dependent feedback loops that regulate the production of ATP9 to match the availability of other ATP synthase components . Such regulatory mechanisms ensure appropriate stoichiometry despite the dual genetic origin of the complex components.

In Pisum sativum, similar coordination mechanisms likely exist, though they may have plant-specific adaptations. The assembly process would need to account for tissue-specific energy demands and environmental responses that affect mitochondrial function.

What role do assembly intermediates play in ATP9 incorporation into the ATP synthase complex?

Assembly intermediates play crucial roles in facilitating the orderly incorporation of ATP9 into the complete ATP synthase complex. In yeast, the Atco complex (composed of ATP9 and Cox6) serves as a key assembly intermediate . Even though Atco does not contain the ring form of ATP9, cross-linking experiments indicate that ATP9 within this complex is oligomeric, with inter-subunit interactions similar to those in the functional ring .

These assembly intermediates not only facilitate the proper folding and oligomerization of ATP9 but also coordinate this process with the assembly of other ATP synthase modules. By providing ATP9 for ATP synthase biogenesis, these intermediates may also free up other components (such as Cox6 in yeast) for assembly of other respiratory complexes, suggesting a role in coordinating the assembly and maintaining proper stoichiometry of different oxidative phosphorylation enzymes .

What methods are most effective for studying recombinant ATP9 expression and assembly?

Several experimental approaches have proven effective for studying ATP9 expression and assembly:

• Pulse-chase experiments: These are invaluable for tracking the fate of newly synthesized ATP9 and determining precursor-product relationships during assembly. This technique involves briefly labeling newly synthesized proteins (pulse) followed by a period with unlabeled amino acids (chase) to observe the incorporation of labeled proteins into complexes over time .

• Cross-linking experiments: Chemical cross-linking can reveal interactions between ATP9 subunits and with other proteins. This approach has been used to demonstrate that ATP9 in assembly intermediates exhibits inter-subunit interactions similar to those in the functional ring .

• Blue native polyacrylamide gel electrophoresis (BN-PAGE): This technique separates intact protein complexes based on size while maintaining native protein-protein interactions, making it useful for studying ATP synthase assembly intermediates.

• Import assays: For studying the import and assembly of nuclear-encoded ATP synthase components that interact with ATP9.

• Mitochondrial translation assays: These can reveal how mutations in ATP9 or associated assembly factors affect translation rates and assembly efficiency .

What genetic modification approaches can be used to study ATP9 function in Pisum sativum?

• Somatic hybridization: This technique involves the fusion of protoplasts from different plant lines, allowing recombination between mitochondrial genomes. This approach has successfully generated novel ATP9 genes through intergenomic recombination, as demonstrated in Petunia .

• RNA editing analysis: Studying RNA editing of mitochondrial transcripts, including ATP9, can provide insights into post-transcriptional regulation.

• Nuclear transformation with mitochondria-targeted proteins: While not directly modifying ATP9, expressing nuclear-encoded proteins targeted to mitochondria can affect ATP9 assembly and function.

• CRISPR/Cas9 with mitochondrial targeting: Emerging approaches aim to adapt CRISPR systems for mitochondrial genome editing, though these techniques remain challenging in plants.

• Mutant screening: Screening for nuclear mutations affecting mitochondrial gene expression or ATP synthase assembly can provide insights into ATP9 function and regulation.

How can researchers isolate and purify functional ATP9 for biochemical studies?

Isolating and purifying functional ATP9 for biochemical studies requires specialized techniques due to its hydrophobic nature and tendency to form oligomeric structures:

• Detergent solubilization: Appropriate detergents must be selected to extract ATP9 from mitochondrial membranes while maintaining its native structure.

• Affinity chromatography: When working with tagged versions of ATP9 or associated proteins, affinity purification can be used to isolate specific complexes.

• Size exclusion chromatography: This technique can separate different assembly states of ATP9-containing complexes based on their molecular weight.

• Density gradient centrifugation: This approach can separate ATP9-containing complexes based on their density.

• Two-dimensional gel electrophoresis: Combining techniques like BN-PAGE with SDS-PAGE can resolve ATP9-containing complexes and their constituents.

For studies requiring recombinant ATP9, heterologous expression systems must be carefully selected and optimized considering the hydrophobic nature of ATP9 and potential toxicity when overexpressed.

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

Mutations in ATP9 can have profound effects on ATP synthase function and consequently on plant phenotypes. These effects may include:

• Reduced ATP synthesis: Mutations disrupting the proton channel function can decrease ATP production, affecting energy-dependent processes throughout the plant.

• Altered proton gradient: Some mutations may affect the proton-translocating properties of the ATP9 ring, potentially uncoupling electron transport from ATP synthesis.

• Assembly defects: Mutations may disrupt the assembly of the ATP9 ring or its incorporation into the complete ATP synthase complex.

• Growth and developmental abnormalities: Severe ATP synthase defects typically lead to reduced growth rates, developmental abnormalities, and decreased stress tolerance.

• Phosphorus use efficiency impacts: Since phosphorus is essential for ATP synthesis and utilization, ATP9 mutations may affect the plant's ability to efficiently use available phosphorus, particularly important in field pea which requires significant P inputs for nodule formation and function .

What is the relationship between ATP9 function and phosphorus metabolism in Pisum sativum?

The relationship between ATP9 function and phosphorus metabolism in Pisum sativum is multifaceted:

• Energy for P acquisition: ATP9, as part of the ATP synthase complex, contributes to ATP production necessary for P uptake through energized transport systems .

• Nodule formation: Field pea requires significant P inputs for nodule formation, which is essential for nitrogen fixation. ATP9 function supports the energy-intensive processes of nodule development and maintenance .

• P allocation: Under P-limited conditions, plants must efficiently allocate available P to various tissues and processes. ATP production via ATP synthase is crucial for this allocation process .

• Metabolic adaptations: When P is limiting, plants may adjust their metabolism to conserve P. These adaptations can influence ATP synthase expression and function, including ATP9.

When P becomes limited throughout the plant, vacuolar Pi will efflux into the cell cytoplasm and be allocated to vital tissues, such as legume nodules . ATP9 function influences this process by contributing to the energy production necessary for P transport and utilization.

How does ATP9 expression respond to environmental stresses in Pisum sativum?

ATP9 expression in Pisum sativum likely responds to various environmental stresses, though specific responses in pea have not been thoroughly characterized in the provided search results. Based on studies in other plant species, potential responses may include:

How has ATP9 evolved across different plant species?

ATP9 has undergone evolutionary changes across different plant species while maintaining its core function in ATP synthesis. Several evolutionary patterns are notable:

• Sequence conservation: The functional domains of ATP9 involved in proton transport and ring formation tend to be conserved across species.

• Genomic location: In most plants, ATP9 is encoded in the mitochondrial genome, though there are examples of transfer to the nuclear genome in some lineages.

• Recombination events: Intergenomic recombination between ATP9 genes from different sources can generate novel variants, as demonstrated in Petunia somatic hybrids . These recombination events contribute to mitochondrial genome diversity.

• Copy number variation: Some plant species contain multiple copies of ATP9, which may provide redundancy or specialized functions.

• RNA editing patterns: The extent and sites of RNA editing in ATP9 transcripts vary across plant species, potentially affecting protein structure and function.

What can comparative studies tell us about ATP9 function across different organisms?

Comparative studies of ATP9 across different organisms provide valuable insights into its function and evolution:

• Structural conservation: The ATP9 ring structure is remarkably conserved across diverse organisms, from bacteria to plants and animals, highlighting its fundamental importance in ATP synthesis.

• Stoichiometric variations: The number of ATP9 subunits in the ring varies between species (e.g., 10 in yeast , 8 in bovine mitochondria), potentially affecting the energetics of ATP synthesis.

• Assembly mechanisms: Studies in yeast have revealed assembly intermediates like Atco , which might have analogs in plants. Comparative studies can identify conserved and divergent assembly mechanisms.

• Regulatory mechanisms: Translation and assembly regulation of ATP9 appear to involve feedback loops in yeast , and comparative studies could reveal whether similar mechanisms operate in plants.

• Functional adaptations: Comparative analyses can reveal adaptations in ATP9 that might correlate with specific metabolic demands or environmental conditions faced by different species.

What insights from yeast ATP9 research can be applied to understanding Pisum sativum ATP9?

Yeast has served as an important model system for understanding mitochondrial ATP synthase, including ATP9. Several insights from yeast research can be applied to Pisum sativum:

• Assembly intermediates: The discovery of the Atco complex in yeast, comprising ATP9 and Cox6, suggests that similar assembly intermediates might exist in pea mitochondria . These intermediates could coordinate the assembly of respiratory complexes in plant mitochondria as well.

• Translation regulation: The finding that ATP9 translation in yeast is enhanced in strains with specific assembly defects suggests that similar feedback regulation might operate in peas . This could involve assembly-dependent translation regulation mediated by specific factors.

• Ring formation mechanism: Cross-linking studies in yeast have shown that ATP9 in assembly intermediates exhibits interactions similar to those in the final ring structure . Similar approaches could reveal the mechanisms of ring formation in pea mitochondria.

• Precursor-product relationships: Pulse-chase experiments in yeast demonstrated that ATP9 in the Atco complex serves as a precursor for the ATP synthase ring . Similar experimental approaches could track ATP9 assembly in pea mitochondria.

• Coordination of nuclear and mitochondrial gene expression: Yeast studies have provided insights into how the expression of nuclear and mitochondrial genes for ATP synthase is coordinated , which is also a critical aspect of ATP synthase biogenesis in plants.

ATP9 Expression Patterns and Regulation Data

ATP9 Structural and Functional Relationships

Phosphorus-Related Functions in Pisum sativum ATP Synthase