Recombinant Emericella nidulans ATP synthase subunit 9, mitochondrial (atp9)

What is the structure and function of ATP synthase subunit 9 in Emericella nidulans?

ATP synthase subunit 9 in Emericella nidulans (the teleomorph/sexual form of Aspergillus nidulans) is a component of the F0 domain of the mitochondrial ATP synthase complex. It is an extremely hydrophobic protein containing two transmembrane segments that forms a ring structure essential for the proton-translocating function of ATP synthase . The protein is present in multiple copies (approximately ten) in the complex, creating a rotational ring that facilitates proton transport across the inner mitochondrial membrane . This rotation results in conformational changes in the F1 catalytic head that enables ATP production.

The A. nidulans nuclear gene oliC31 encodes this subunit 9 protein, which consists of 143 amino acids comprising a pre-sequence of 62 residues and a mature protein of 81 residues . Due to its high hydrophobicity, it is classified as a proteolipid that can be easily extracted from mitochondria with organic solvents .

How is the ATP9 gene regulated in Emericella nidulans?

The oliC31 gene in A. nidulans contains an open reading frame with no introns . The promoter region is characterized by long pyrimidine-rich tracts preceding the transcription initiation sites . Multiple transcription initiation and polyadenylation sites have been identified, suggesting complex transcriptional regulation mechanisms .

ATP9 is considered a gene of primary metabolism in A. nidulans . Like other mitochondrial proteins, its expression may be influenced by carbon source availability. Genome-wide transcription analysis of A. nidulans grown on different carbon sources (glucose, glycerol, and ethanol) has shown that the expression of mitochondrial proteins, including components of the ATP synthase complex, can be significantly affected by carbon source .

What is the evolutionary significance of ATP9 gene location in Emericella nidulans?

The ATP9 gene in A. nidulans is nuclear-encoded (oliC31), which is evolutionarily significant because in many other organisms, this gene is located in the mitochondrial genome . This represents an example of gene transfer from mitochondria to the nucleus during evolution.

The relocation of genes from mitochondria to the nucleus is considered an ongoing evolutionary process. There are several hypotheses accounting for the retention of some genes in mitochondria while others have been transferred to the nucleus :

• Some genes may be confined to mitochondria due to the difficulty of transporting their protein products into the organelle, particularly for highly hydrophobic proteins like ATP9 .

• Genes may be preferentially maintained in mitochondria to adjust gene expression according to redox or metabolic states .

The natural nuclear location of ATP9 in A. nidulans and other filamentous fungi, while remaining mitochondrial in organisms like Saccharomyces cerevisiae, provides valuable insights into the evolutionary process of mitochondrial gene transfer .

How does the sequence of ATP9 in E. nidulans compare with other fungal species?

The ATP9 protein in A. nidulans shows significant sequence homology with equivalent proteins in other fungal species. The amino acid homology with the equivalent Neurospora crassa protein is 50% for the pre-sequence and 80% for the mature protein . This high conservation of the mature protein sequence reflects the critical functional importance of this subunit in the ATP synthase complex.

For comparison, studies on ATP9 from other organisms show that the gene has between 40-60% identity across various species . The conserved regions, particularly in the mature protein, are likely essential for proper protein transport, processing, and function within the ATP synthase complex .

What challenges are associated with the recombinant expression of E. nidulans ATP9?

Recombinant expression of E. nidulans ATP9 presents several significant challenges:

• Extreme hydrophobicity: The ATP9 protein contains two transmembrane segments and is highly hydrophobic, making expression and purification difficult . This hydrophobicity can cause protein aggregation and improper folding during heterologous expression.

• Mitochondrial targeting and import: When expressing recombinant ATP9, ensuring proper targeting to mitochondria is challenging. Studies have shown that the hydrophobicity of the first transmembrane segment is a critical factor affecting mitochondrial import efficiency .

• Protein degradation: Improperly imported or folded ATP9 proteins are susceptible to degradation. For example, research has shown that recombinant yeast Atp9p fused to a mitochondrial targeting sequence (MTS) can be targeted to mitochondria but is rapidly degraded by the i-AAA protease (Yme1p) in the intermembrane space if not properly imported .

• Assembly into functional complexes: Ensuring that recombinant ATP9 correctly assembles into the F0 ring structure is challenging but crucial for functional studies.

What are the implications of relocating the ATP9 gene from mitochondria to the nucleus in fungi?

Research on relocating the ATP9 gene from mitochondria to the nucleus has revealed several important implications:

• Reduced hydrophobicity requirement: Successful relocation of ATP9 requires reduced hydrophobicity, particularly in the first transmembrane segment, to enable efficient import into mitochondria .

• Complex cellular adaptations: Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates stress responses like the heat shock response .

• Evolutionary insights: Experimental relocation of ATP9 has provided insights into why this gene was only transferred to the nucleus during the evolution of multicellular organisms and not in unicellular organisms like yeast .

• Functional implications: Studies comparing strains with mitochondrially-encoded versus nuclear-encoded ATP9 show differences in respiratory capacity, ATP synthesis rates, and assembly of ATP synthase complexes, as demonstrated in the table below:

Data shows comparison of mitochondrial function in wild-type S. cerevisiae versus strains expressing nuclear-encoded ATP9 from Podospora anserina (PaAtp9-5 and PaAtp9-7) .

How can heterologous expression systems be optimized for E. nidulans ATP9 production?

Optimizing heterologous expression of E. nidulans ATP9 requires several considerations:

What protocols yield the highest purity of recombinant E. nidulans ATP9?

Purification of recombinant E. nidulans ATP9 presents challenges due to its hydrophobic nature. The following protocol strategy has been effective:

• Affinity chromatography: Expressing ATP9 with an affinity tag (such as His-tag) facilitates initial purification. Current commercial recombinant ATP9 proteins are available with N-terminal His-tags .

• Detergent solubilization: Due to ATP9's hydrophobicity, appropriate detergents are crucial for solubilization. Mild detergents like n-dodecyl β-D-maltoside (DDM) or digitonin are often effective for membrane proteins.

• Size exclusion chromatography: This helps separate properly folded ATP9 from aggregates and can be performed in the presence of appropriate detergents.

• Quality assessment: SDS-PAGE analysis can confirm protein purity (>90% is typically achievable) . Western blotting with antibodies against ATP9 or the affinity tag can verify identity.

• Storage conditions: Lyophilization with stabilizers like trehalose (6%) in Tris/PBS-based buffer at pH 8.0 has been shown to maintain protein stability . For reconstituted protein, adding glycerol (5-50%) and storing in aliquots at -20°C/-80°C minimizes freeze-thaw damage .

What techniques are most effective for analyzing ATP9 protein-protein interactions?

Several techniques have proven effective for studying ATP9 interactions within the ATP synthase complex:

• Blue Native PAGE (BN-PAGE): This technique has been successfully used to analyze the assembly of ATP synthase complexes containing ATP9. It allows visualization of both monomeric and oligomeric forms of ATP synthase .

• Co-immunoprecipitation: Using antibodies against ATP9 or other ATP synthase subunits to pull down interaction partners, followed by mass spectrometry identification.

• Chemical cross-linking coupled with mass spectrometry: This approach can identify spatial relationships between ATP9 and neighboring subunits within the complex.

• Förster Resonance Energy Transfer (FRET): By tagging ATP9 and potential interaction partners with appropriate fluorophores, FRET can detect close proximity indicative of protein-protein interactions.

• Yeast two-hybrid system modifications: Although challenging for membrane proteins, split-ubiquitin yeast two-hybrid systems modified for membrane proteins can potentially detect ATP9 interactions.

How can site-directed mutagenesis be used to study ATP9 function in E. nidulans?

Site-directed mutagenesis is a powerful approach for investigating structure-function relationships in ATP9:

• Transmembrane segment modifications: Mutations altering the hydrophobicity of transmembrane segments can provide insights into import efficiency and assembly requirements. Research has shown that reduced hydrophobicity of the first transmembrane segment improves mitochondrial import .

• Conserved residue analysis: Mutating highly conserved residues can identify amino acids critical for proton translocation or subunit interactions.

• MTS modifications: Altering the mitochondrial targeting sequence can elucidate requirements for efficient mitochondrial import and processing.

• Chimeric constructs: Creating chimeric proteins combining segments from different species' ATP9 proteins has been informative. For example, chimeric constructs between yeast and P. anserina ATP9 have revealed factors affecting mitochondrial import .

• Expression and functional testing: After creating mutants, expressing them in ATP9-null backgrounds (Δatp9) allows assessment of functional consequences through measurements of:

  • Respiratory growth capacity

  • Oxygen consumption rates

  • ATP synthesis efficiency

  • Assembly into ATP synthase complexes

  • Oligomycin sensitivity (characteristic of functional ATP synthase)

What are the optimal conditions for expressing recombinant ATP9 in heterologous systems?

Based on studies with A. nidulans and other recombinant protein expression systems, the following conditions optimize ATP9 expression:

• E. coli expression system:

  • BL21(DE3) or similar strains with reduced protease activity

  • Induction at lower temperatures (16-20°C) to reduce inclusion body formation

  • Lower IPTG concentrations (0.1-0.5 mM) for slower, more controlled expression

  • Addition of membrane-stabilizing compounds like betaine or sorbitol

• Yeast expression system:

  • Use of inducible promoters like Tet-off system

  • Multicopy plasmids (e.g., 2μ) rather than centromeric plasmids for higher expression

  • Codon optimization for the host organism

  • Appropriate MTS for mitochondrial targeting

• A. nidulans as expression host:

  • Temperature: 30-45°C (optimal range for enzyme production)

  • pH: 3.0-10.0 (varies by specific protein)

  • Incubation period: 1-11 days (shorter times minimize contamination risk)

  • Carbon sources: Complex carbon sources like wheat bran can enhance protein production

  • Nitrogen sources: Combination of organic (peptone, yeast extract) and inorganic (ammonium sulfate) nitrogen sources

What novel approaches are being developed for ATP9 functional studies?

Recent advancements in ATP9 research include:

• Cryo-electron microscopy (cryo-EM): High-resolution structures of complete ATP synthase complexes provide detailed insights into ATP9 ring organization and interaction with other subunits.

• Native relocation strategy: A novel approach using naturally nuclear versions of ATP9 from other organisms (like P. anserina) to achieve functional expression in species where ATP9 is normally mitochondrially encoded . This approach proved successful where direct recoding of mitochondrial ATP9 failed.

• Systems biology approaches: Integration of ATP9 expression data with metabolic network models of A. nidulans provides broader context for understanding its regulation in response to environmental changes .

• Comparative genomics: Analysis of ATP9 across different fungi has revealed patterns in evolutionary gene transfer from mitochondria to nucleus and adaptation mechanisms for efficient mitochondrial import .

How does ATP9 expression vary under different metabolic conditions in E. nidulans?

The expression of ATP9 and other components of the ATP synthase complex in A. nidulans is influenced by metabolic conditions:

• Carbon source effects: Genome-wide transcription analysis has shown that carbon sources significantly affect the expression of genes involved in energy metabolism, including ATP synthase components .

• Enzyme complex co-regulation: Analysis of enzyme complexes in A. nidulans revealed that for about 30% of enzyme complexes, including ATP synthase, the expression profiles of genes encoding all subunits showed similar patterns .

• Regulatory networks: Transcription factors like CreA (involved in carbon catabolite repression) influence the expression of genes involved in central metabolism, including energy production, in response to carbon source availability .

• Metabolic shifts: When A. nidulans shifts between fermentable and non-fermentable carbon sources, significant remodeling of mitochondrial energy production occurs, affecting ATP synthase expression and assembly .