Recombinant Acanthamoeba castellanii ATP synthase subunit 9, mitochondrial (ATP9)

What is the structure of A. castellanii ATP synthase subunit 9?

A. castellanii ATP synthase subunit 9 is a small, hydrophobic protein classified as a proteolipid due to its extraction properties with organic solvents. Based on homology with yeast ATP synthase subunit 9, the protein likely contains two transmembrane segments that form part of the c-ring structure in the F₀ domain of ATP synthase . This protein forms a ring structure composed of multiple copies, which is essential for proton translocation across the inner mitochondrial membrane during ATP synthesis.

How does A. castellanii ATP9 contribute to ATP synthesis?

The ATP9 protein forms a critical component of the proton-translocating domain (F₀) of ATP synthase. During oxidative phosphorylation, protons flow through the c-ring structure (formed by multiple ATP9 subunits), causing it to rotate. This rotation is mechanically coupled to conformational changes in the F₁ domain, which drives ATP synthesis. The rotation of the subunit 9-ring directly facilitates the production of ATP by the catalytic head of ATP synthase and its subsequent release into the mitochondrial matrix .

How can researchers isolate functional A. castellanii mitochondria for ATP synthase studies?

Methodological approach:

• Culture A. castellanii trophozoites in PYG medium at 25°C until log phase

• Harvest cells by centrifugation (1,000 × g for 10 minutes)

• Wash cells in isolation buffer (0.25 M sucrose, 10 mM HEPES-KOH pH 7.4, 1 mM EDTA)

• Disrupt cells using a Dounce homogenizer (20-25 strokes)

• Centrifuge homogenate at 1,000 × g for 10 minutes to remove unbroken cells

• Collect supernatant and centrifuge at 10,000 × g for 15 minutes

• Resuspend mitochondrial pellet in reaction buffer

• Verify mitochondrial integrity by measuring respiratory control ratio with oxygen electrode

For optimal results, maintain all solutions and equipment at 4°C throughout the isolation procedure to preserve enzymatic activity.

What are the challenges of expressing recombinant A. castellanii ATP9?

Expression of recombinant A. castellanii ATP9 presents several challenges:

• Extreme hydrophobicity: ATP9 is highly hydrophobic, making it difficult to express in conventional systems without aggregation .

• Import difficulties: When expressed from a nuclear gene, the protein must contain appropriate targeting sequences to ensure mitochondrial import.

• Assembly challenges: The protein must correctly incorporate into the ATP synthase complex to be functional.

• Potential toxicity: Overexpression might disrupt cellular homeostasis, as observed in yeast studies where nuclear expression of ATP9 perturbed cellular morphology and activated heat shock response .

What expression systems are optimal for producing functional recombinant A. castellanii ATP9?

Recommended expression strategy:

When designing expression constructs, researchers should consider modifying the hydrophobicity of the first transmembrane segment, as this approach has proven successful for expressing P. anserina ATP9 in S. cerevisiae .

How can I assess the functional integration of recombinant A. castellanii ATP9 into ATP synthase?

To verify functional integration of recombinant A. castellanii ATP9 into the ATP synthase complex, employ a multi-faceted approach:

• Respiratory growth assessment: In complementation experiments (using yeast Δatp9 systems), assess growth on non-fermentable carbon sources like glycerol .

• Oxygen consumption measurement: Prepare mitochondria and measure state 3 respiration rates using substrates like NADH. Functional ATP9 should support oxygen consumption rates at approximately 80% of wild-type levels (as observed with other ATP9 variants) .

• BN-PAGE analysis: Use blue native polyacrylamide gel electrophoresis to visualize assembled ATP synthase complexes, followed by in-gel ATPase activity assay.

• ATP synthesis assay: Measure ATP production in isolated mitochondria using a luciferase-based assay after adding ADP and respiratory substrates.

• Proton pumping assay: Assess the proton-translocating ability using pH-sensitive fluorescent dyes.

What purification strategies are most effective for A. castellanii ATP9?

Purification of this highly hydrophobic protein requires specialized approaches:

• Detergent extraction: Solubilize mitochondrial membranes with appropriate detergents (DDM, LDAO, or Triton X-100).

• Column chromatography sequence:

  • Initial purification: Ni-NTA affinity chromatography (requires His-tagged construct)

  • Secondary purification: Ion exchange chromatography

  • Final polishing: Size exclusion chromatography in the presence of appropriate detergent

• Quality control: Assess purity by SDS-PAGE with tricine buffer system (optimal for small hydrophobic proteins) and verify identity with Western blotting or mass spectrometry.

How can structural studies of A. castellanii ATP9 be performed?

For structural analysis of A. castellanii ATP9, consider these methodological approaches:

• Cryo-electron tomography (cryoET): This technique allows visualization of ATP synthase dimers in their native membrane environment, revealing dimer angles and organization. Studies in other organisms have shown ATP synthase dimer angles ranging from 86° in yeast to 115° in mammals, with C. elegans displaying a novel average dimer angle of 105° .

• Sub-tomogram averaging: This computational approach improves the signal-to-noise ratio of tomograms and can reveal detailed structural features at the dimer interface .

• AlphaFold prediction: Computational structure prediction using AlphaFold and AlphaFold multimer can provide insights into ATP9 structure and how it interacts with other ATP synthase subunits .

• NMR spectroscopy: For detailed analysis of the transmembrane regions, solution NMR in membrane-mimetic environments can be particularly informative.

How does A. castellanii ATP9 compare with homologs from other species?

A. castellanii ATP9 can be compared with homologs from other species to understand evolutionary conservation and functional adaptation:

• Sequence comparison: Alignment with homologs from diverse species reveals conserved residues essential for proton translocation and structural integrity.

• Hydrophobicity analysis: The hydrophobicity profile, particularly of the first transmembrane segment, influences mitochondrial import efficiency. Research with P. anserina ATP9 has shown that reduced hydrophobicity in this segment enables successful mitochondrial import when expressed from a nuclear gene .

• Ring stoichiometry: The number of ATP9 subunits forming the c-ring varies across species (typically 8-10 in yeast ) and influences the bioenergetic efficiency of ATP synthesis.

Could the ATP9 gene be relocated from the mitochondrial to the nuclear genome in A. castellanii?

The relocation of ATP9 from mitochondrial to nuclear genome presents an interesting evolutionary and experimental question:

• Evolutionary context: The transfer of mitochondrial genes to the nucleus has occurred repeatedly during evolution, with ATP9 being retained in the mitochondria of unicellular organisms but transferred to the nucleus in most multicellular organisms .

• Experimental feasibility: Studies in S. cerevisiae have demonstrated that nuclear expression of P. anserina ATP9 can functionally complement a yeast Δatp9 strain, suggesting such relocation is experimentally possible .

• Required modifications:

  • Addition of a mitochondrial targeting sequence

  • Potential modification of the first transmembrane segment to reduce hydrophobicity and facilitate import

  • Codon optimization for nuclear expression

• Expected challenges: Nuclear expression of ATP9 in yeast perturbs cellular properties including morphology and activates heat shock response, indicating complex cellular adaptations are required for successful gene relocation .

How might ATP9 contribute to A. castellanii's unique metabolic capabilities?

A. castellanii possesses distinctive metabolic pathways that may influence ATP synthesis requirements:

• Dual serine metabolism: A. castellanii has both phosphorylated and non-phosphorylated pathways for serine metabolism involving D-glycerate dehydrogenase (GDH) and serine-pyruvate aminotransferase (SPAT) . These pathways may have unique energy requirements supported by ATP synthase activity.

• Cysteine biosynthesis: A. castellanii possesses cysteine synthase (CS) but lacks serine acetyltransferase (SAT), indicating a unique cysteine biosynthetic pathway . The energy requirements for this pathway could influence ATP synthase activity and potentially ATP9 regulation.

• Stress response: During oxidative stress, A. castellanii upregulates various defense mechanisms . The ATP synthase complex, including ATP9, might be regulated differently under these conditions to maintain energy production.

Could A. castellanii ATP9 serve as a potential drug target for anti-Acanthamoeba therapies?

Given that humans lack cysteine synthase (CS), which has been identified as a potential target for anti-Acanthamoeba drugs , researchers might consider ATP9 as another potential target:

• Target validation approach:

  • Comparative analysis of A. castellanii ATP9 with human homologs to identify structural differences

  • Assessment of ATP9 essentiality using RNA interference or CRISPR-based approaches

  • In vitro inhibition studies using isolated ATP synthase or reconstituted ATP9 rings

• Integration with other targeting strategies: Recent research has explored PARP inhibitors as potential anti-Acanthamoeba agents . A combined approach targeting both DNA repair (via PARP inhibition) and energy production (via ATP synthase inhibition) might increase efficacy.

• Consideration of resistance mechanisms: A. castellanii has robust stress response systems , potentially enabling adaptation to ATP synthase inhibition. Researchers should monitor for compensatory mechanisms during drug development.

How does ATP9 function influence mitochondrial morphology in A. castellanii?

Research in other organisms has revealed a relationship between ATP synthase dimer angle and cristae morphology . For A. castellanii:

How can I overcome protein aggregation issues when working with recombinant A. castellanii ATP9?

The extreme hydrophobicity of ATP9 often leads to aggregation during expression and purification. Consider these methodological solutions:

• Expression optimization:

  • Reduce expression temperature to 16-18°C

  • Use low inducer concentrations

  • Consider specialized E. coli strains (C41/C43) designed for membrane protein expression

• Solubilization strategies:

  • Test a panel of detergents (DDM, LDAO, Fos-choline, LMNG)

  • Use lipid nanodiscs or amphipols for maintaining protein stability

  • Consider fluorinated surfactants which often improve membrane protein solubility

• Co-expression approaches:

  • Co-express with ATP synthase subunits that interact directly with ATP9

  • Include chaperones like GroEL/GroES to assist proper folding

What are the most reliable methods to measure ATP synthase activity in A. castellanii mitochondria?

For accurate assessment of ATP synthase activity in A. castellanii:

• ATP synthesis assay:

  • Isolate coupled mitochondria maintaining membrane potential

  • Incubate with ADP and respiratory substrates

  • Measure ATP production using luciferase-based luminescence assay

  • Calculate synthesis rates as nmol ATP/min/mg protein

• ATP hydrolysis assay:

  • Measure phosphate release using colorimetric methods

  • Include appropriate controls (oligomycin-sensitive activity represents ATP synthase)

  • Normalize to protein concentration

• Membrane potential measurements:

  • Use fluorescent dyes (TMRM, JC-1) to assess membrane potential

  • Monitor changes upon addition of substrates, ADP, and inhibitors

  • Correlate with ATP synthesis activity