Recombinant Vicia faba ATP synthase subunit 9, mitochondrial (ATP9)

What is ATP9 and what role does it play in Vicia faba mitochondrial energy production?

ATP9 (also referred to as subunit 9 or subunit c) is a critical component of the mitochondrial ATP synthase (Complex V) in Vicia faba. This protein forms an oligomeric ring structure consisting of 10 identical subunits (9₁₀-ring) within the membrane-embedded F₀ domain of ATP synthase. In conjunction with subunit 6, the ATP9 ring constitutes the essential proton channel that spans the inner mitochondrial membrane. During oxidative phosphorylation, the proton gradient generated by the electron transport chain drives protons through this channel, causing the ATP9 ring to rotate. This rotational motion is mechanically coupled to conformational changes in the F₁ domain, ultimately facilitating ATP synthesis from ADP and inorganic phosphate .

The functional significance of ATP9 is highlighted by its high conservation across species and its essential role in the proton-motive force conversion to chemical energy. Experimental analysis of newly synthesized ATP9 shows it is initially associated with a complex called Atco before being incorporated into the complete ATP synthase structure, suggesting a regulated assembly pathway that may be critical for proper function .

How is ATP9 encoded and organized in the Vicia faba mitochondrial genome?

ATP9 is encoded by the mitochondrial gene ATP9 in the Vicia faba genome. According to sequencing data, the V. faba circular mitochondrial master chromosome (cultivar Broad Windsor) spans approximately 588,000 bp with a genome complexity of 387,745 bp. Within this genome, 52 conservative mitochondrial genes have been identified, including 32 protein-encoding genes, 3 rRNA genes, and 17 tRNA genes .

The ATP9 gene is one of the conserved protein-coding genes found in the V. faba mitochondrial genome. Interestingly, the gene appears to be duplicated, as mentioned in the context of other duplicated genes like nad9, atp6, atp9, ccmC, rpl16, rps3, and tatC . This duplication may have functional significance for regulation or expression levels.

The organization of the gene within the mitochondrial genome reflects the complex evolutionary history of plant mitochondrial genomes, with evidence of gene duplication events and possible horizontal gene transfer contributing to the current genomic architecture.

How is ATP9 expression regulated in mitochondria?

The expression of ATP9 in Vicia faba mitochondria involves sophisticated regulatory mechanisms operating at multiple levels. At the transcript level, the stability of the ATP9 mRNA depends critically on a 35 kDa C-terminal cleavage fragment of the nuclear-encoded protein Atp25. This fragment is released in the mitochondrial matrix by the mitochondrial processing peptidase and plays an essential role in maintaining ATP9 transcript integrity .

Translation of ATP9 is subject to assembly-dependent regulation. Research has demonstrated that the rate of ATP9 translation is enhanced in mutant strains with specific defects in its assembly. This suggests the existence of a feedback mechanism where assembly intermediates interact with ATP9 within the final enzyme complex. These interactions involve cis-regulatory sequences that control gene expression within the organelle .

This assembly-dependent translation regulation serves as an elegant solution to the challenge of coordinating the production of ATP synthase subunits encoded by two different genomes (nuclear and mitochondrial). It helps ensure proper stoichiometry despite the physically separated translation machineries.

What methodologies can be used to study ATP9 translation in experimental settings?

To investigate ATP9 translation dynamics, researchers can employ several methodologies:

• Pulse-chase radiolabeling: This technique allows tracking of newly synthesized ATP9 through the assembly process. Experiments using 35S-methionine/cysteine labeling of mitochondria followed by blue native gel electrophoresis (BN-PAGE) in the first dimension and SDS-PAGE in the second dimension have revealed that a large fraction of nascent ATP9 associates with Atco complexes initially. This approach demonstrated that newly translated ATP9 in Atco is converted to the ring structure incorporated into ATP synthase with kinetics characteristic of a precursor-product relationship .

• Cross-linking experiments: These have been valuable in demonstrating that ATP9 in Atco is oligomeric with inter-subunit interactions similar to those in the mature ring, despite not yet forming the complete ring structure .

• Genetic manipulation: Creating specific mutants with defects in ATP synthase assembly has shown that ATP9 translation is enhanced in response to assembly defects, suggesting feedback regulation .

• Ribosome profiling: Though not explicitly mentioned in the search results, this technique could provide genome-wide insights into translation efficiency of mitochondrial genes including ATP9.

The combination of these approaches provides a comprehensive view of ATP9 translation and its regulation in response to assembly status.

How does ATP9 assemble into the functional ATP synthase complex?

The assembly of ATP9 into the functional ATP synthase complex involves a precise sequence of events that challenges previous models. Contrary to the generally accepted view that the ATP9 ring (9₁₀-ring) forms separately and independently of other ATP synthase components, recent evidence suggests a more integrated assembly pathway .

Research reveals that newly translated ATP9 first associates with a complex called Atco, which also contains Cox6, a subunit of cytochrome c oxidase. Through pulse-chase experiments, it has been demonstrated that ATP9 in Atco serves as a precursor for the formation of the ATP9 ring that is subsequently incorporated into the complete ATP synthase. This indicates a precursor-product relationship between Atco-associated ATP9 and the ATP9 ring in the mature enzyme .

Interestingly, while ATP9 in Atco has not yet formed the complete ring structure, cross-linking experiments indicate that it is already oligomeric and exhibits inter-subunit interactions similar to those found in the mature ring. This suggests that Atco may function as a scaffold for the initial oligomerization of ATP9 subunits prior to their incorporation into the final ring structure .

The assembly process appears to be coordinated with other ATP synthase components. In yeast, it was proposed that ATP synthase assembly involves two separate pathways (F₁/Atp9p and Atp6p/Atp8p/stator subunits/Atp10p chaperone) that converge at the end stage . The final addition of mitochondrially encoded subunits like ATP9 is translationally regulated, allowing for balanced output between nuclear-encoded and mitochondrially encoded components.

What is the Atco complex and how does it contribute to ATP9 function?

The Atco complex represents a critical intermediate in ATP synthase assembly that contains newly translated ATP9 along with Cox6, a subunit typically associated with cytochrome c oxidase (COX). This complex serves multiple important functions:

• ATP9 assembly platform: Atco provides a scaffold for the initial association of ATP9 subunits. Although ATP9 in Atco has not yet formed the complete ring structure characteristic of mature ATP synthase, cross-linking experiments indicate that it is already oligomeric with inter-subunit interactions similar to those in the functional ring .

• Precursor reservoir: Pulse-chase experiments demonstrate that ATP9 in Atco is converted to the ring structure incorporated into ATP synthase with kinetics characteristic of a precursor-product relationship. This suggests that Atco functions as a reservoir of ATP9 subunits available for ATP synthase assembly .

• Coordination of respiratory complex assembly: By temporarily sequestering Cox6, a component of cytochrome c oxidase, Atco may play a role in coordinating the assembly of multiple respiratory complexes. When ATP9 is released from Atco for incorporation into ATP synthase, Cox6 becomes available for assembly into COX. This mechanism could help maintain proper stoichiometry between these two oxidative phosphorylation enzymes .

The table below summarizes the key characteristics of ATP9 in different states:

What techniques are most effective for isolating and studying recombinant Vicia faba ATP9?

When working with recombinant Vicia faba ATP9, researchers should consider the following methodological approaches:

• Two-dimensional gel electrophoresis: The combination of blue native gel electrophoresis (BN-PAGE) in the first dimension with SDS-PAGE in the second dimension has proven effective for separating and visualizing ATP9 in different complex states. This technique successfully distinguished between ATP9 in Atco complexes, independent ATP9 rings, and ATP9 incorporated into assembled ATP synthase .

• Pulse-chase radiolabeling: To study the dynamics of ATP9 assembly, pulse-chase experiments using 35S-methionine/cysteine labeling of mitochondria provide valuable insights into the precursor-product relationship between newly synthesized ATP9 and its incorporation into mature ATP synthase .

• Cross-linking studies: Chemical cross-linking approaches have been instrumental in demonstrating that ATP9 in Atco complexes is oligomeric with inter-subunit interactions similar to those in the mature ring, despite not yet forming the complete ring structure .

• Mitochondrial isolation: For studying native ATP9, isolation of intact mitochondria is essential. Established protocols for plant mitochondrial isolation, including differential centrifugation and density gradient purification, can be adapted for Vicia faba tissues.

• Computational analysis: Sequence analysis tools have been valuable in identifying and characterizing ATP9 in the mitochondrial genome. The Vicia faba mitochondrial genome has been analyzed using NCBI tools, with the master chromosome sequence submitted to the GenBank database (accession number KC189947) .

For expression of recombinant ATP9, researchers must consider the hydrophobic nature of this protein and the challenges associated with membrane protein expression and purification.

How can researchers verify the proper folding and assembly of recombinant ATP9?

Verifying the proper folding and assembly of recombinant Vicia faba ATP9 presents unique challenges due to its membrane-embedded nature and tendency to form oligomeric structures. Several complementary approaches can be employed:

• Functional assays: The ultimate test of proper folding and assembly is functional activity. For ATP9, this involves incorporation into a functional ATP synthase complex capable of ATP synthesis. In reconstituted systems, proton transport capability can be assessed using pH-sensitive fluorescent dyes.

• Structural analysis: Techniques such as circular dichroism (CD) spectroscopy can provide information about secondary structure content, which is particularly useful for confirming the alpha-helical structure characteristic of ATP9. More advanced structural methods like cryo-electron microscopy can visualize the ring formation when sufficient material is available.

• Cross-linking studies: Chemical cross-linking approaches, similar to those used with native ATP9, can confirm the formation of appropriate inter-subunit interactions in recombinant ATP9 oligomers. These experiments have been valuable in demonstrating that ATP9 in Atco complexes exhibits interactions similar to those in the mature ring .

• Detergent solubility profiles: The behavior of recombinant ATP9 in different detergents can provide indirect evidence of proper folding, as correctly folded membrane proteins typically show characteristic detergent solubility profiles.

• Binding studies: The ability of recombinant ATP9 to interact with known binding partners, such as other ATP synthase subunits, can indicate proper folding and functional capacity.

These complementary approaches provide a comprehensive assessment of recombinant ATP9 quality and functionality.

How does Vicia faba ATP9 compare to homologous proteins in other species?

Comparative analysis of Vicia faba ATP9 with homologous proteins in other species reveals both conservation and divergence that reflect evolutionary relationships and functional constraints:

In mitochondrial genomes across plant species, ATP9 shows significant conservation reflecting its essential role in energy production. The Vicia faba mitochondrial genome analysis revealed that the ATP9 gene is among the 35 mitochondrial genes encoding conserved proteins found in the master chromosome .

The functional assembly of ATP9 into the proton channel of ATP synthase appears to be conserved across species. In yeast, ATP synthase contains a ring of 10 identical subunits 9 that, together with subunit 6, forms the proton channel responsible for coupling proton transport to ATP synthesis . This fundamental structural arrangement is likely conserved in Vicia faba as well.

Interestingly, the assembly pathway of ATP9 may show species-specific variations. Recent research challenges the previously accepted view that the ATP9 ring forms separately and independently of other ATP synthase components , suggesting that assembly pathways may have evolved differently across lineages.

What can we learn from studying mitochondrial genome evolution through ATP9?

The study of ATP9 in the context of mitochondrial genome evolution provides valuable insights into several key evolutionary processes:

• Gene transfer between organelles and nucleus: The analysis of Vicia faba mitochondrial DNA revealed significant homology (approximately 45%) with the Medicago truncatula nuclear genome . While not specifically mentioned for ATP9, this pattern suggests active genetic exchange between mitochondrial and nuclear genomes during evolution. This phenomenon, known as endosymbiotic gene transfer, has been a major force in shaping mitochondrial genomes.

• Conservation of essential functions: Despite evolutionary divergence, the fundamental role of ATP9 in forming the proton channel of ATP synthase remains conserved, reflecting strong selective pressure on this essential function. The conservation of this protein across diverse lineages provides a window into core mitochondrial functions that have remained unchanged over evolutionary time.

• Regulatory evolution: The complex regulation of ATP9 expression, involving assembly-dependent translation and interaction with specific chaperones and assembly factors , illustrates how regulatory mechanisms have evolved to coordinate the expression of genes encoded in different cellular compartments.

• Structural adaptations: Comparative analysis of ATP9 structure and assembly across species can reveal adaptations to different environmental conditions or metabolic demands. Variations in the number of subunits in the ATP9 ring or in the specific interactions with other ATP synthase components may reflect such adaptations.

• Horizontal gene transfer: The Vicia faba mitochondrial genome contains unique open reading frames (ORFs) with sequence homologies to diverse plant species including Beta vulgaris, Nicotiana tabacum, Vitis vinifera, and even monocots like Oryza sativa and Zea mays . This suggests that horizontal gene transfer has played a role in mitochondrial genome evolution, potentially affecting the genetic context in which ATP9 functions.

How can researchers use ATP9 to study bioenergetic coupling in plant mitochondria?

Recombinant Vicia faba ATP9 provides a valuable tool for investigating bioenergetic coupling in plant mitochondria through several sophisticated experimental approaches:

These approaches collectively provide a comprehensive understanding of ATP9's role in the complex process of converting the proton-motive force into chemical energy in the form of ATP.

What are the conflicting models of ATP9 assembly and how can researchers address these contradictions?

The assembly pathway of ATP9 into the functional ATP synthase complex has been the subject of conflicting models, presenting an opportunity for researchers to address fundamental questions in mitochondrial biogenesis:

Contradicting Models:

• Classical Independent Assembly Model: The traditionally accepted view holds that the ATP9 ring forms separately and independently of other ATP synthase components before being incorporated into the complete complex. This model suggests a modular assembly process where different parts of the ATP synthase are built separately before final integration .

• Integrated Assembly Model: Recent evidence challenges this view, suggesting that ATP9 assembly is integrated with that of other ATP synthase components. This model proposes that newly translated ATP9 first associates with the Atco complex before being incorporated into the ATP synthase, contradicting the idea of independent ring formation .

Methodological Approaches to Resolve These Contradictions:

• Time-resolved structural studies: Utilizing cryo-electron microscopy at different stages of assembly could provide direct visualization of intermediate structures, helping to distinguish between competing models.

• Genetic manipulation: Creating specific mutations in genes encoding ATP synthase assembly factors could help identify the proteins involved in each step of ATP9 assembly. The effects of these mutations on the formation of Atco and subsequent incorporation of ATP9 into ATP synthase would provide valuable insights.

• In vitro reconstitution: Developing in vitro systems that recapitulate ATP9 assembly under controlled conditions would allow systematic testing of factors proposed to be involved in each model.

• Pulse-chase experiments: Extending the pulse-chase approach used to demonstrate the precursor-product relationship between ATP9 in Atco and in ATP synthase could further clarify the temporal sequence of assembly events.

• Comparative studies across species: Investigating whether the Atco-mediated assembly pathway is conserved across species could help determine if it represents a fundamental mechanism or a species-specific variation.

By combining these approaches, researchers can work toward a unified model of ATP9 assembly that reconciles the apparent contradictions in the current literature.

What novel experimental techniques are emerging for studying ATP9 function and assembly?

The study of ATP9 function and assembly is benefiting from several emerging experimental techniques that promise to provide unprecedented insights:

• Cryo-electron tomography: This technique allows visualization of macromolecular complexes in their native cellular environment without the need for purification. Applied to mitochondria, it could reveal the spatial organization of ATP9 assembly intermediates in situ, providing context that is lost in traditional biochemical approaches.

• Mass spectrometry-based proteomics: Advanced proteomics approaches, including cross-linking mass spectrometry and thermal proteome profiling, can map protein-protein interactions and conformational changes in ATP9 during assembly and function. These techniques could identify transient interactions that are difficult to capture with traditional methods.

• Single-molecule fluorescence microscopy: By labeling individual ATP9 molecules with fluorescent tags, researchers can track their movement and incorporation into larger complexes in real-time. This approach could resolve the temporal sequence of assembly events with unprecedented precision.

• Genome editing with CRISPR-Cas9: Precise modification of mitochondrial genes, though technically challenging, is becoming more feasible. This would allow introduction of specific mutations or tags into the endogenous ATP9 gene, enabling study of the protein under physiological conditions.

• Organelle-specific ribosome profiling: This technique provides genome-wide information on translation efficiency specifically within mitochondria. It could reveal how ATP9 translation is coordinated with that of other mitochondrial and nuclear-encoded ATP synthase components.

• In silico molecular dynamics simulations: Computational approaches can model the behavior of ATP9 within the membrane and its interactions with other subunits at an atomic level. These simulations can generate hypotheses about proton transport mechanisms and conformational changes that can be tested experimentally.

These emerging techniques, used in combination, have the potential to resolve longstanding questions about ATP9 function and assembly.

How might understanding ATP9 contribute to advances in bioenergetics and synthetic biology?

Deep understanding of ATP9 structure, function, and assembly has far-reaching implications for both fundamental bioenergetics and applied synthetic biology:

• Biomimetic energy conversion systems: The ATP9 ring represents one of nature's most efficient rotary motors. Detailed understanding of its structure and mechanism could inspire the design of artificial molecular motors for nanoscale energy conversion devices. The precise arrangement of proton-binding sites in ATP9 that enables efficient energy coupling could inform the development of synthetic proton pumps.

• Engineering enhanced photosynthetic efficiency: In plants, mitochondrial ATP synthase works alongside the chloroplast ATP synthase to maintain cellular energy balance. Insights into how ATP9 contributes to mitochondrial ATP synthesis efficiency could inform strategies for engineering plants with improved photosynthetic performance by optimizing the coordination between these two energy-producing organelles.

• Minimal synthetic cells: Efforts to create minimal synthetic cells require essential energy-generating components. A detailed understanding of ATP9 assembly and function could help in designing simplified ATP synthase complexes for incorporation into synthetic cellular systems with predictable energetic properties.

• Mitochondrial disease models and therapies: Though not directly related to plant ATP9, insights from plant systems could inform understanding of human mitochondrial diseases. The assembly pathways and regulatory mechanisms discovered in plant ATP9 might have parallels in human systems, potentially revealing new therapeutic targets.

• Biosensors and bioelectronic devices: The proton-translocating capability of ATP9-containing complexes could be harnessed in the development of biosensors for pH, membrane potential, or ATP levels. Such biosensors could find applications in environmental monitoring or medical diagnostics.

• Cross-species compatibility testing: Understanding the species-specific features of ATP9 could help predict compatibility issues in synthetic biology applications involving components from different organisms, guiding the design of hybrid systems with optimal functionality.

The fundamental knowledge gained from studying ATP9 thus serves as a foundation for diverse applications that bridge basic science and technological innovation.

What are the most significant unresolved questions about Vicia faba ATP9?

Despite considerable progress in understanding Vicia faba ATP9, several significant questions remain unresolved:

• Complete assembly pathway: While recent evidence challenges the traditional view of independent ATP9 ring formation , the complete sequence of events in ATP9 assembly, from translation to incorporation into functional ATP synthase, remains to be fully elucidated. The precise mechanisms by which ATP9 transitions from the Atco complex to the mature ring require further investigation.

• Regulatory crosstalk: The coordination between nuclear and mitochondrial genomes in regulating ATP9 expression and assembly is incompletely understood. How signals about assembly status are communicated between these compartments and integrated with other cellular processes remains unclear.

• Evolutionary origins: The relationship between ATP9 in Vicia faba and homologous proteins in other species, both closely and distantly related, could provide insights into the evolutionary history of this essential component of energy metabolism. How the assembly pathway involving Atco evolved and whether it represents a conserved or species-specific mechanism remains to be determined.

• Structural details: High-resolution structural information about ATP9 in different states (in Atco, in the mature ring, and during proton translocation) would provide valuable insights into its function and assembly. Such structural details could help explain how proton movement through ATP9 drives rotation of the ring.

• Functional significance of duplication: The duplication of the ATP9 gene observed in the Vicia faba mitochondrial genome raises questions about the functional significance of this genomic arrangement. Whether both copies are expressed and assembled into functional protein, and whether they play different roles, remains to be determined.

Addressing these questions will require integration of multiple experimental approaches and could significantly advance our understanding of mitochondrial bioenergetics and genome evolution.

How can researchers contribute to advancing knowledge of ATP9 in plant bioenergetics?

Researchers interested in advancing knowledge of ATP9 in plant bioenergetics can contribute through several strategic approaches:

• Develop improved expression systems: Creating efficient systems for recombinant expression of Vicia faba ATP9 would facilitate structural and functional studies. Given the hydrophobic nature of ATP9, this might involve specialized expression hosts or fusion protein strategies that enhance solubility while maintaining native-like structure.

• Apply integrative structural biology: Combining complementary structural techniques such as cryo-electron microscopy, NMR spectroscopy, and computational modeling could provide a comprehensive view of ATP9 in different functional states and assembly intermediates. This integrative approach is particularly valuable for membrane proteins like ATP9 that are challenging to study by any single method.

• Investigate species-specific variations: Comparative studies of ATP9 across plant species could reveal adaptations to different environmental conditions or metabolic demands. Such studies might identify natural variations in ATP9 structure or assembly that correlate with physiological differences between species.

• Explore interactions with assembly factors: The identification and characterization of proteins involved in ATP9 assembly, beyond those already known like Atco , could provide insights into the mechanisms that ensure proper incorporation of this protein into functional ATP synthase.

• Investigate the role of lipids: The specific lipid environment of ATP9 likely influences its structure, assembly, and function. Studies exploring these lipid-protein interactions could reveal additional factors affecting ATP synthase efficiency.

• Develop in vivo imaging approaches: Methods for visualizing ATP9 assembly and dynamics in living cells would provide valuable insights into its behavior under physiological conditions. This might involve fluorescent protein fusions or other labeling strategies compatible with live-cell imaging.

By pursuing these approaches, researchers can contribute to a more complete understanding of ATP9's role in plant bioenergetics, with potential implications for fields ranging from agriculture to synthetic biology.