Recombinant Dictyostelium discoideum ATP synthase subunit 9, mitochondrial (atp9)

What is ATP synthase subunit 9 in Dictyostelium discoideum?

ATP synthase subunit 9 in D. discoideum is a component of the mitochondrial ATP synthase complex that plays a crucial role in oxidative phosphorylation. It forms part of the F₀ sector embedded in the inner mitochondrial membrane, creating a ring structure that functions as a proton channel. This structure facilitates the conversion of the proton gradient energy into the mechanical energy required for ATP synthesis. In D. discoideum, atp9 appears to be encoded by a nuclear gene, unlike some other organisms where it is encoded by mitochondrial DNA. The protein likely contains a mitochondrial import sequence at its N-terminus to direct it to the mitochondria after synthesis in the cytoplasm .

How is the atp9 gene organized in the D. discoideum genome?

Based on comparative analysis with other organisms, the atp9 gene in D. discoideum is located in the nuclear genome rather than in mitochondrial DNA. This is similar to what has been observed in Trypanosoma brucei, where the ATPase subunit 9 gene was identified in the nuclear genome and shown not to be present in kinetoplast DNA (kDNA) . The gene likely contains regions encoding a mitochondrial targeting sequence at the 5' end, which directs the protein to mitochondria after translation. The nuclear localization of atp9 in D. discoideum represents an evolutionary transfer of this gene from the mitochondrial genome to the nucleus, a phenomenon observed in various organisms throughout evolution.

How conserved is atp9 across different species?

ATP synthase subunit 9 shows significant conservation across diverse species, reflecting its essential role in cellular energy metabolism. In T. brucei, the ATPase subunit 9 gene has been reported to have between 40 and 60% identity with subunit 9 from a variety of organisms . This level of conservation suggests strong selective pressure to maintain the protein's structure and function. The conservation is particularly evident in functional domains involved in proton transport and interaction with other ATP synthase subunits.

In the case of ATP synthase assembly, studies in yeast have revealed that it is a complex of 28 subunits of 17 different types, with subunits 6, 8, and 9 encoded by mitochondrial genes . The conservation of this complex assembly across species highlights the fundamental importance of this machinery for cellular function. Although D. discoideum-specific conservation data is not explicitly provided in the available literature, the protein likely shows similar conservation patterns given its crucial role in energy production.

How is atp9 expression regulated during D. discoideum development?

The regulation of atp9 expression during D. discoideum development appears to be complex and developmentally controlled. Drawing parallels from studies in T. brucei, where ATP synthase is developmentally regulated throughout the life cycle, we can infer similar mechanisms may operate in D. discoideum. In T. brucei, Northern analysis and quantitative RT-PCR revealed that atp9 transcript levels are 10-14-fold higher in the procyclic form than in early and late bloodstream forms .

In D. discoideum, which undergoes a unique developmental cycle transitioning from unicellular amoebae to multicellular structures, the expression of mitochondrial components likely changes to accommodate shifting energy requirements. During starvation-induced development, there may be significant alterations in mitochondrial function and energy metabolism necessitating adjustments in ATP synthase expression, including atp9. Proteomic studies have shown that in certain ATG knockout strains of D. discoideum (specifically ATG9‾/16‾), subunit 9 of ATP synthase is increased along with other components involved in oxidative phosphorylation, suggesting an up-regulation of the respiratory chain in these conditions .

What techniques are most effective for studying atp9 expression patterns?

Several complementary techniques can be employed to comprehensively study atp9 expression patterns in D. discoideum:

RNA-level analysis:

• Northern blotting can detect atp9 mRNA and assess its size and abundance, as demonstrated in studies of ATP synthase subunits in T. brucei .

• Quantitative reverse transcriptase-PCR (RT-qPCR) provides a more sensitive and quantitative assessment of transcript levels. In T. brucei studies, this method revealed significant differences in expression between life cycle stages .

• RNA sequencing (RNA-Seq) offers a comprehensive view of the transcriptome, including atp9 expression across different conditions or developmental stages .

• Deep sequencing of co-immunoprecipitated RNAs can identify RNAs associated with specific proteins, which may be relevant for studying post-transcriptional regulation of atp9 .

Protein-level analysis:

• Western blotting using specific antibodies can detect and quantify atp9 protein in cell lysates or purified mitochondria. For optimal results, this technique typically employs polyclonal peptide antibodies at appropriate dilutions, as described for other D. discoideum proteins .

• Mass spectrometry approaches allow for identification and quantification of atp9 in complex samples, as demonstrated in proteomic studies of D. discoideum .

• Blue Native PAGE can be used to analyze the incorporation of atp9 into ATP synthase complexes .

For developmental studies, these techniques should be applied across different stages of the D. discoideum life cycle to capture temporal changes in expression patterns.

How does starvation affect atp9 expression and ATP synthase activity?

Starvation is a key trigger for development in D. discoideum, causing the transition from single-cell amoebae to multicellular structures. This transition involves substantial changes in metabolism and gene expression patterns that likely affect ATP synthase components, including atp9.

Research on D. discoideum early development indicates that mitochondrial processes undergo significant changes during starvation-induced development. The rate of oxygen uptake in D. discoideum cells can be measured using a Clark-type O₂ electrode to estimate the functional states of mitochondria at different developmental stages. Through this approach, researchers can calculate mitochondrial coupling capacity and efficiency based on basal respiration, state 4 (resting state), state 3 (phosphorylating state), and maximal respiration .

In ATG knockout strains (specifically ATG9‾/16‾), an increase in ATP synthase subunit 9 and other proteins involved in oxidative phosphorylation has been observed . This suggests that under certain stress conditions, cells may upregulate components of the respiratory chain to enhance ATP production. While direct evidence specifically for starvation effects on atp9 in wild-type D. discoideum is not explicitly outlined in the available literature, the developmental regulation observed in related organisms suggests that atp9 expression likely responds to the metabolic shifts occurring during starvation-induced development.

How can recombinant D. discoideum atp9 protein be produced for experimental studies?

Generating recombinant D. discoideum atp9 protein involves several key steps, from gene cloning to protein purification:

Cloning the atp9 gene

• PCR amplification of the atp9 coding sequence from D. discoideum genomic DNA or cDNA

• Design primers to include appropriate restriction sites for cloning into expression vectors

• Example PCR components: 1× Taq buffer with (NH₄)₂SO₄, 2 mM MgCl₂, 0.2 mM dNTP mixture, 1 μM primers, genomic DNA template, and Taq polymerase

Expression vector construction

• Clone the PCR product into a suitable expression vector, such as:

  • pET vectors for E. coli expression with His₆ tags

  • pGEX vectors for GST-tagged proteins

  • pEXP5-CT/TOPO vector (used successfully for other D. discoideum proteins)

Expression system selection

• E. coli (DE3) Rosetta pLysS cells are commonly used for heterologous protein expression

• Induction conditions: typically 1 mM IPTG at OD₆₀₀ of 0.6, with temperature optimization (22-37°C)

Protein purification

• For His₆-tagged proteins:

  • Lyse cells by sonication in appropriate buffer (e.g., 30 mM KPi pH 7.0, 300 mM KCl, 10% glycerol, 10 mM imidazole, 0.1 mM DTT with protease inhibitors)

  • Incubate cleared lysate with Ni-NTA resin

  • Wash and elute with imidazole-containing buffer

  • Dialyze to remove imidazole and stabilize protein

Special considerations for atp9

• As a membrane protein, atp9 may require detergents for proper folding and stability

• Consider expressing only the mature form without the mitochondrial targeting sequence

• For structural studies, coexpression with stabilizing partners might be necessary

Protein quality can be assessed by SDS-PAGE, Western blotting with specific antibodies, and functional assays to ensure the recombinant protein maintains its native activity.

What approaches can be used to study atp9 protein-protein interactions?

Several complementary approaches can be employed to study protein-protein interactions involving atp9 in D. discoideum:

Yeast Two-Hybrid (Y2H) screening

• This method has been used successfully to study protein-protein interactions in D. discoideum, particularly for the autophagy pathway

• For membrane proteins like atp9, a modified split-ubiquitin Y2H system may be more appropriate

• This approach allows for initial screening of potential interaction partners

Co-immunoprecipitation (Co-IP)

• Express tagged versions of atp9 in D. discoideum cells

• Lyse cells under conditions that preserve protein-protein interactions

• Immunoprecipitate atp9 using tag-specific antibodies

• Identify co-precipitated proteins by Western blotting or mass spectrometry

Pull-down assays

• Express recombinant atp9 with an affinity tag (GST, His)

• Immobilize the protein on an appropriate matrix

• Incubate with D. discoideum cell lysates

• Wash and elute bound proteins for identification

• This approach has been used successfully for analyzing protein-protein interactions in D. discoideum

Blue Native PAGE

• This technique preserves native protein complexes during electrophoresis

• Can be used to analyze the incorporation of atp9 into ATP synthase complexes

• Has been applied successfully to study protein complexes in D. discoideum

Crosslinking combined with mass spectrometry

• Treat intact cells or isolated mitochondria with crosslinking agents

• Purify atp9-containing complexes

• Analyze by mass spectrometry to identify crosslinked peptides

• This reveals spatial relationships between proteins in the complex

Sucrose density gradient sedimentation

• This method separates protein complexes based on size and density

• Has been used to analyze protein complexes in D. discoideum

• Can provide information about the native state of atp9 within ATP synthase

These techniques can be combined to build a comprehensive understanding of how atp9 interacts with other proteins within the ATP synthase complex and potentially with other cellular components.

What are effective methods for studying atp9 function in vivo?

Studying atp9 function in vivo in D. discoideum requires approaches that can link molecular alterations to physiological effects. Several effective methods include:

Genetic manipulation approaches

• Creation of knockout or knockdown strains: While complete deletion of atp9 may be lethal due to its essential function, conditional knockouts or RNA interference approaches can reduce expression levels

• Site-directed mutagenesis: Introduction of specific mutations can target key functional residues in atp9

• Expression of tagged versions: Fluorescent or epitope tags can facilitate localization and interaction studies without completely disrupting function

Mitochondrial function assays

• Oxygen consumption measurements: Using a Clark-type O₂ electrode to assess respiratory capacity in cells with altered atp9 expression or structure

• ATP synthesis assays: Measuring ATP production rates in isolated mitochondria

• Membrane potential analysis: Using fluorescent dyes to assess the proton gradient across the inner mitochondrial membrane

Developmental phenotype analysis

• Growth rate determination: Measuring the impact of atp9 modifications on cell proliferation

• Development monitoring: Assessing progression through the D. discoideum developmental cycle

• Spore formation and viability analysis: Similar to approaches used in other D. discoideum studies where genetic modifications affected spore formation and viability

Mitochondrial isolation and analysis

• Isolation of mitochondria from D. discoideum cells at different developmental stages

• Analysis of ATP synthase assembly and activity in isolated mitochondria

• The technique has been described for investigating mitochondrial processes during early development of D. discoideum

Live cell imaging

• Expression of fluorescently tagged atp9 to monitor localization and dynamics

• Super-resolution microscopy to visualize ATP synthase complex assembly

• FRET-based approaches to detect protein-protein interactions in living cells

These methods collectively provide a comprehensive toolkit for investigating atp9 function in the context of living D. discoideum cells, linking molecular details to physiological outcomes across different developmental stages.

What is known about the structure of D. discoideum ATP synthase containing subunit 9?

Recent structural studies have begun to shed light on the architecture of D. discoideum ATP synthase, including subunit 9. A bioRxiv preprint from March 2025 describes using AlphaFold 3 to predict the structure of D. discoideum ATP synthase, which was then docked into refined cryo-EM density . While specific details about subunit 9 are not extensively discussed, the model incorporated three α-subunits, three β-subunits, and one γ-subunit to represent the natural assembly.

ATP synthase subunit 9 (also known as the c subunit or proteolipid) typically forms a ring structure in the F₀ sector of ATP synthase, embedded in the inner mitochondrial membrane. In the general model of ATP synthase, multiple copies of subunit 9 (usually 8-15, depending on the species) assemble into a ring, creating a central pore through which protons can flow.

Interestingly, the bioRxiv preprint notes that mass spectrometry analysis of D. discoideum identified only the β-subunit of ATP synthase (specifically, the AtpB protein), with no evidence for either the α- or γ-subunits . The authors discuss the possibility of homo-hexameric β rings in ATP synthase homologs, although they acknowledge that such structures have not been previously documented for D. discoideum.

The visualization of the predicted ATP synthase model within the cryo-EM map suggests a hexameric structure with a central density feature where the γ-subunit is expected to reside . This structural arrangement aligns with the general architecture of ATP synthases across species, though with potentially interesting D. discoideum-specific features.

How does atp9 contribute to ATP synthase function in D. discoideum?

ATP synthase subunit 9 plays a crucial role in the function of the ATP synthase complex in D. discoideum, particularly in the process of energy conversion through oxidative phosphorylation:

Proton transport and energy conversion

• Subunit 9 forms a ring structure in the F₀ portion of ATP synthase embedded in the inner mitochondrial membrane

• This ring contains proton-binding sites that facilitate the movement of protons across the membrane

• The proton flow through the c-ring drives the rotation of the central stalk (γ subunit) of ATP synthase

• This rotation induces conformational changes in the F₁ catalytic domain, leading to ATP synthesis

Integration with cellular metabolism

• The ATP synthase complex, including subunit 9, represents a crucial link between the electron transport chain and ATP production

• Studies in D. discoideum have examined mitochondrial processes during early development , highlighting the importance of mitochondrial function during this critical period

• Proteomic studies have shown that in certain ATG knockout strains of D. discoideum, subunit 9 of ATP synthase is increased along with other components involved in oxidative phosphorylation, suggesting an up-regulation of the respiratory chain

Developmental regulation

• Like in T. brucei , atp9 expression in D. discoideum is likely developmentally regulated, reflecting changing energy demands throughout the life cycle

• The expression pattern may change during the transition from single-cell amoebae to multicellular structures during the D. discoideum life cycle

Response to cellular stress

• Changes in ATP synthase activity, potentially involving regulation of subunit 9, might be part of the cellular response to stressors like starvation

• The increased levels of subunit 9 and other oxidative phosphorylation components observed in certain mutant strains could represent a compensatory mechanism to maintain ATP production under stress conditions

Understanding the specific contributions of atp9 to ATP synthase function in D. discoideum provides insights into both conserved aspects of mitochondrial energy production and potentially unique features related to the organism's complex life cycle and developmental regulation.

How does D. discoideum atp9 compare with its counterparts in other model organisms?

A comparative analysis of D. discoideum atp9 with its counterparts in other model organisms reveals both conserved features and potentially unique aspects:

Genomic organization

• Based on the comparison with T. brucei , D. discoideum atp9 is likely encoded in the nuclear genome, not in the mitochondrial DNA

• This differs from organisms like yeast (S. cerevisiae), where atp9 is encoded by the mitochondrial genome

• In plants like Petunia, there can be both nuclear and mitochondrial copies of atp9, with evidence of recombination between them

Expression and regulation

• In T. brucei, atp9 shows significant developmental regulation with 10-14-fold higher expression in the procyclic form compared to bloodstream forms

• D. discoideum, with its complex life cycle, likely shows similar developmental regulation of atp9

• The specific regulatory mechanisms may differ between species

Assembly and interactions

• In yeast, the assembly of subunit 9 into the ATP synthase complex involves specific factors that regulate translation and incorporation of the protein

• Studies in yeast have revealed that ATP synthase assembly is a complex process, with specific mechanisms for the translation and incorporation of subunit 9

• The assembly process in D. discoideum may involve similar factors, though potentially with organism-specific variations

Table 1: Comparative Features of ATP Synthase Subunit 9 Across Species

The comparative analysis highlights that while the fundamental function of atp9 in ATP synthesis is conserved across species, significant variations exist in genomic organization, expression patterns, and potentially in the specific mechanisms of complex assembly and regulation. These differences likely reflect adaptations to the specific life cycles and metabolic requirements of each organism.

How does atp9 assemble into the ATP synthase complex in D. discoideum?

The assembly of atp9 into the ATP synthase complex in D. discoideum likely involves a coordinated series of steps, although the specific details for D. discoideum are not fully characterized in the available literature. Based on studies in other organisms, particularly yeast, and general principles of ATP synthase assembly, we can propose the following model:

Initial c-ring formation

• Multiple atp9 monomers must interact to form the c-ring structure

• This assembly likely occurs within the inner mitochondrial membrane

• If atp9 is nuclear-encoded in D. discoideum (as inferred from comparison with T. brucei ), the protein must first be imported into mitochondria and inserted into the inner membrane

Integration with other F₀ components

• The c-ring associates with other membrane-embedded components, particularly subunits a and b

• This assembly creates the complete F₀ sector capable of proton translocation

Assembly factors and chaperones

• In yeast, specific factors regulate atp9 translation and incorporation into ATP synthase

• These include proteins involved in mRNA stability/processing (e.g., Atp25, Aep1, Aep2) and translation activation

• D. discoideum likely possesses homologs of these assembly factors, although they have not been specifically characterized

Connection with F₁ sector

• The F₀ sector, including the c-ring, must associate with the F₁ sector to form the complete ATP synthase

• This involves interactions between the c-ring and the central stalk components

Assembly regulation

• Studies in yeast have revealed assembly-dependent feedback loops affecting the translation of some ATP synthase subunits

• Similar regulatory mechanisms might exist in D. discoideum

Research in yeast has shown that translation of subunits 6 and 9 is enhanced in mutant strains with specific defects in the assembly of these proteins, suggesting complex regulatory mechanisms . Additionally, the assembly process appears to involve specific interactions between assembly intermediates and regulatory sequences that control gene expression.

To fully characterize the assembly pathway in D. discoideum, approaches such as pulse-chase experiments, analysis of assembly intermediates using Blue Native PAGE, and identification of assembly factors through genetic screens would be necessary.

What are the challenges in studying atp9 structure-function relationships?

Studying the structure-function relationship of D. discoideum atp9 presents several significant challenges that researchers must address:

Membrane protein nature

• ATP synthase subunit 9 is a highly hydrophobic membrane protein, making it difficult to express, purify, and study using traditional structural biology techniques

• Obtaining sufficient quantities of properly folded protein for structural studies requires specialized expression systems

• The hydrophobic nature of the protein complicates both expression and subsequent structural analyses

Integration into a multiprotein complex

• Atp9 functions as part of the large ATP synthase complex

• Studying its properties in isolation may not reflect its true functional characteristics within the complex

• The assembly process itself may influence the structural and functional properties of atp9

Functional assays

• Assessing the functional impact of structural alterations requires sensitive and specific assays

• Measuring ATP synthase activity involves complex procedures with isolated mitochondria

• Methods for measuring oxygen uptake and ATP synthase activity in isolated mitochondria have been described , but applying these to specific atp9 modifications presents technical challenges

Developmental regulation

• If atp9 expression in D. discoideum is developmentally regulated (similar to T. brucei ), this adds complexity to functional studies

• Experiments must account for developmental variables when interpreting results

• The protein's role may vary across different developmental stages

Genetic manipulation

• Creating specific mutations or deletions in atp9 may have lethal effects if the protein is essential

• Conditional expression systems may be necessary but add complexity to experimental design

Integration with cellular metabolism

• ATP synthase function is intimately connected to other metabolic pathways

• Interpreting atp9 function requires considering its role in the broader context of cellular metabolism

• Perturbations to atp9 may have indirect effects on multiple cellular processes

Addressing these challenges requires an integrated approach combining structural biology, biochemistry, genetics, and systems biology. Recent advances in techniques like cryo-electron microscopy (as suggested by the work mentioned in search result ), in situ structural studies, and artificial intelligence-based structure prediction offer promising avenues for overcoming some of these obstacles.

How can CRISPR-Cas9 technology be applied to study atp9 in D. discoideum?

CRISPR-Cas9 technology offers powerful approaches for studying atp9 function in D. discoideum, enabling precise genetic modifications that were previously difficult to achieve. While implementation in D. discoideum may require optimization, the following strategies show significant promise:

Gene knockout studies

• Create complete deletion of atp9 to determine its essentiality

• If atp9 is essential (likely, given its role in ATP synthesis), develop conditional knockout approaches:

  • Use an inducible CRISPR system

  • Place the endogenous atp9 under an inducible promoter before knockout

• Methodology:

  • Design guide RNAs (gRNAs) targeting the atp9 coding sequence

  • Introduce a donor template with homology arms flanking a selection marker

  • Select transformants and verify deletion by PCR and sequencing

Structure-function analysis through precise mutations

• Introduce specific point mutations to study key residues:

  • Mutations in the putative proton-binding site

  • Alterations to residues involved in c-ring formation

  • Changes to potential interaction surfaces with other ATP synthase subunits

• Methodology:

  • Design gRNAs targeting specific regions of atp9

  • Provide donor templates containing the desired mutations

  • Screen transformants using sequencing or restriction enzyme analysis

Tagging for localization and interaction studies

• Add reporter tags (GFP, mCherry) to study subcellular localization

• Introduce affinity tags (FLAG, HA) for purification and interaction studies

• Methodology:

  • Design gRNAs targeting the N- or C-terminus of atp9

  • Provide donor templates containing the tag sequence with homology arms

  • Verify correct integration and expression of the tagged protein

Promoter replacement for expression studies

• Replace the endogenous atp9 promoter with controlled promoters to:

  • Study the effects of over- or under-expression

  • Decouple atp9 expression from its normal regulatory mechanisms

• Methodology:

  • Design gRNAs targeting the region upstream of the atp9 coding sequence

  • Introduce a donor template containing the new promoter

  • Validate altered expression patterns using RT-qPCR or Western blotting

Investigating regulatory elements

• Create deletions or mutations in potential regulatory regions of the atp9 gene

• Assess the impact on atp9 expression and ATP synthase function

Table 2: CRISPR-Cas9 Applications for atp9 Research in D. discoideum