Recombinant Debaryomyces hansenii Acetate non-utilizing protein 9, mitochondrial (ACN9)

What is Debaryomyces hansenii and why is it significant for research?

Debaryomyces hansenii is a halophilic yeast that has been studied for several decades as a eukaryotic model for understanding salt and osmotic tolerance. Despite its long research history, consensus among different studies has been limited, with contradictory information often stemming from diverse experimental conditions. After a period of reduced attention, D. hansenii is regaining research momentum thanks to advanced technologies including instrumented lab-scale bioreactors, -omics approaches, and high-throughput robotics . The yeast's exceptional halotolerance makes it particularly valuable for understanding osmotic stress response mechanisms and potentially for developing robust microbial cell factories.

What is the function of ATP synthase subunit 9 in mitochondria?

ATP synthase subunit 9 (ATP9) is a critical component of the F₀ domain of ATP synthase that facilitates proton transport across the mitochondrial membrane to drive ATP production. The protein's transmembrane helices form a pore structure that couples proton movement to ATP synthesis. In D. hansenii specifically, ATP9 contributes to mitochondrial ATP synthase assembly and maintenance of the proton gradient. Research has shown that ATP9 synthesis is upregulated during F₀ domain assembly, ensuring stoichiometric balance with nuclear-encoded subunits. Additionally, ATP synthase activity in D. hansenii increases under high salinity conditions, suggesting ATP9 plays a significant role in the organism's osmotic stress response mechanisms.

How does recombinant ATP9 differ from native ATP9 in D. hansenii?

Recombinant D. hansenii ATP9 is typically produced in heterologous expression systems like E. coli using codon-optimized cDNA and includes modifications such as N-terminal affinity tags (commonly a 10×His tag) to facilitate purification via affinity chromatography. The recombinant variant retains the functional regions of the native protein while providing experimental advantages:

While the recombinant version facilitates research applications, it's important to consider that structural differences from tagging may affect certain biochemical properties.

What genetic engineering approaches are effective for D. hansenii?

Genetic engineering of D. hansenii has historically been challenging due to its preference for the non-homologous end joining (NHEJ) pathway over homologous recombination (HR). This preference makes it nearly impossible or highly inefficient to introduce genome alterations via homology-based gene targeting using conventional methods . Recent breakthroughs include:

• CRISPR-Cas9 application for gene inactivation via defective NHEJ repair, though initial attempts at homologous recombination-directed edits showed low success rates .

• Development of an efficient CRISPR-Cas9 engineering toolbox that allows precise Cas9-mediated point mutations and marker-free gene deletion via HR. This approach involved disrupting the NHEJ pathway by creating a mutant for the KU70 gene, which codes for an essential protein in NHEJ repair .

• Multiplex gene targeting systems compatible with D. hansenii that enable simultaneous modification of multiple genomic loci .

Selecting the appropriate approach depends on the specific genetic modification goals and requires consideration of D. hansenii's unique DNA repair preferences.

How can CRISPR-Cas9 be optimized for ATP9 gene manipulation in D. hansenii?

Optimizing CRISPR-Cas9 for ATP9 gene manipulation in D. hansenii requires several specialized methodological considerations:

• NHEJ pathway disruption: Create a KU70 knockout strain to significantly enhance homologous recombination efficiency. This foundational step improves precise gene targeting outcomes when working with ATP9 .

• Guide RNA design: Design specific sgRNAs targeting the ATP9 gene with minimal off-target effects, accounting for D. hansenii's unique genomic features. Multiple algorithm tools should be used to identify optimal target sites within the ATP9 coding sequence.

• Homology arm design: For ATP9 modifications, construct homology arms of at least 40-50 bp flanking the target site. Longer homology arms (>500 bp) may further improve recombination efficiency in D. hansenii .

• Transformation protocol: Optimize electroporation parameters specifically for D. hansenii (typically higher voltage compared to S. cerevisiae protocols) to achieve maximum transformation efficiency.

• Verification methods: Implement comprehensive screening approaches combining PCR genotyping, sequencing, and functional assays to verify successful ATP9 modifications, as mitochondrial gene alterations can be challenging to detect.

This optimized methodology enables precise genetic manipulation of ATP9 while minimizing off-target effects that could compromise mitochondrial function studies.

What are the key challenges in expressing recombinant D. hansenii ATP9 in heterologous systems?

Expressing recombinant D. hansenii ATP9 in heterologous systems presents several technical challenges that researchers must address:

• Codon optimization necessity: D. hansenii's codon usage differs significantly from common expression hosts like E. coli, requiring comprehensive codon optimization of the ATP9 gene to enhance expression levels.

• Membrane protein expression issues: As a mitochondrial membrane protein, ATP9 poses typical membrane protein expression challenges including toxicity to host cells, protein misfolding, and aggregation.

• Purification complexity: The hydrophobic nature of ATP9 complicates purification processes, often requiring specialized detergents and buffer systems to maintain protein stability and solubility.

• Protein stability concerns: Recombinant ATP9 requires glycerol (5–50%) during prolonged storage to prevent aggregation.

• Functional validation: Confirming that recombinantly expressed ATP9 maintains native conformational properties requires sophisticated biophysical approaches, as functionality testing outside the context of the complete ATP synthase complex is challenging.

A methodical approach addressing these challenges involves iterative optimization of expression vectors, host selection, induction conditions, and purification protocols.

How does D. hansenii regulate oxidative phosphorylation under salt stress?

D. hansenii employs sophisticated regulatory mechanisms for oxidative phosphorylation under salt stress conditions:

• Selective uncoupling strategy: Under high salt conditions, D. hansenii exhibits a selective uncoupling of complex I-dependent respiration in the stationary growth phase, reducing it to approximately 20% of original activity. This uncoupling is not due to inactivation of complex I, lack of protein expression, or differential expression of alternative oxidoreductases .

• NAD⁺ regulation mechanism: The observed decrease in complex I-dependent respiration results from NAD⁺ loss from the mitochondrial matrix, likely through the opening of a mitochondrial unspecific channel (DhMUC). Importantly, when NAD⁺ is added back to isolated mitochondria, coupled complex I activity can be recovered .

• NAD⁺ transport system: D. hansenii possesses a NAD⁺-specific transporter that facilitates NAD⁺ re-uptake independent of DhMUC opening. This transporter shows sensitivity to inhibitors including bathophenanthroline, bromocresol purple, and pyridoxal-5'-phosphate, similar to S. cerevisiae mitochondrial NAD⁺ transporters .

• ATP synthase adaptation: Under high salinity, ATP synthase activity increases, suggesting ATP9's potential role in osmotic stress response. This adaptation helps maintain energy production despite challenging osmotic conditions.

This regulatory flexibility enables D. hansenii to maintain energy metabolism while adapting to high salt environments, representing a unique physiological uncoupling mechanism distinct from those observed in other yeasts like Y. lipolytica and S. cerevisiae .

What methodologies are most effective for studying ATP9 interactions with other ATP synthase subunits?

To effectively study ATP9 interactions with other ATP synthase subunits in D. hansenii, researchers should employ a multi-faceted methodological approach:

• Cross-linking mass spectrometry (XL-MS): This technique identifies interaction interfaces between ATP9 and other subunits like ATP6, which forms a direct interface for proton transport, and AtpF, which stabilizes the F₀ rotor structure. Protocol optimization should include:

  • Selection of appropriate cross-linking reagents (DSS, EDC, or photoactivatable reagents)

  • Controlled cross-linking reaction conditions specific to membrane proteins

  • Specialized digestion and enrichment of cross-linked peptides

  • Advanced mass spectrometry analysis with optimized parameters for hydrophobic peptides

• Cryo-electron microscopy (cryo-EM): For visualizing the structural arrangement of ATP9 within the complete ATP synthase complex, with particular attention to:

  • Sample preparation techniques that preserve native membrane protein conformation

  • Data acquisition parameters optimized for membrane protein complexes

  • Computational image processing to achieve high-resolution structural information

• Site-directed mutagenesis coupled with functional assays: Systematic mutation of key residues in ATP9's transmembrane helices followed by functional analysis can reveal critical interaction points. The comprehensive approach should include:

  • Targeted mutation design based on evolutionary conservation analysis

  • Expression and incorporation of mutant ATP9 into ATP synthase complexes

  • Proton transport and ATP synthesis activity measurements

  • Membrane potential assessments using potentiometric dyes

• Blue native PAGE and immunoprecipitation: For analyzing intact ATP synthase complexes and subcomplex assemblies with modified ATP9 variants.

These methodologies, when used in combination, provide complementary insights into ATP9's structural and functional interactions within the ATP synthase complex.

How can researchers effectively investigate the role of ATP9 in mitochondrial membrane potential maintenance?

To investigate ATP9's role in mitochondrial membrane potential maintenance in D. hansenii, researchers should implement a systematic experimental approach:

• Site-directed mutagenesis strategy:

  • Create targeted mutations in key residues of ATP9's transmembrane domains that are predicted to participate in proton channeling

  • Develop a library of ATP9 variants with graduated functional impairments

  • Verify expression and proper incorporation into ATP synthase complexes using immunoblotting and BN-PAGE

• Membrane potential measurement techniques:

  • Employ fluorescent potentiometric dyes (e.g., TMRM, JC-1) calibrated specifically for D. hansenii mitochondria

  • Implement real-time confocal microscopy for spatial and temporal analysis of membrane potential fluctuations

  • Use complementary techniques such as patch-clamp electrophysiology on isolated mitochondria to directly measure proton conductance

• Functional coupling analysis:

  • Measure oxygen consumption rates in isolated mitochondria with site-specific inhibitors to distinguish ATP9-dependent effects

  • Assess ATP synthesis rates in parallel with membrane potential measurements

  • Quantify the relationship between proton gradient and ATP production efficiency in wild-type versus ATP9-modified systems

• Response to stress conditions:

  • Systematically evaluate membrane potential maintenance under varying salt concentrations (0.5-2.0M NaCl)

  • Assess recovery kinetics following controlled depolarization events

  • Compare wild-type and mutant responses to oxidative stress challenges

Research has shown that mutations in ATP9 can disrupt proton flow, leading to loss of mitochondrial membrane potential. This methodological framework enables precise characterization of the molecular mechanisms by which ATP9 contributes to membrane potential dynamics under normal and stress conditions.

What research design approaches are most suitable for studying D. hansenii ATP9's role in halotolerance?

Designing research to investigate D. hansenii ATP9's role in halotolerance requires sophisticated approaches that incorporate multiple methodological dimensions:

• Comparative genomics foundation:

  • Analyze ATP9 sequence conservation and divergence across halotolerant and non-halotolerant yeast species

  • Identify potential halotolerance-associated structural motifs in D. hansenii ATP9

  • Construct phylogenetic relationships to trace evolutionary adaptations specific to extreme environments

• Domain swapping experimental design:

  • Create chimeric ATP9 constructs by exchanging domains between D. hansenii and non-halotolerant yeasts (like S. cerevisiae)

  • Express these chimeras in ATP9-deficient strains of both species

  • Assess functional complementation and halotolerance restoration under controlled salinity gradients

• Systems biology integration:

  • Implement transcriptomic, proteomic, and metabolomic analyses of wild-type and ATP9-modified strains under varying salt conditions

  • Develop computational models incorporating ATP9's role in energy metabolism during osmotic stress

  • Validate model predictions through targeted experimental manipulations

• In vivo real-time monitoring:

  • Develop fluorescently tagged ATP9 variants that maintain functionality

  • Track ATP9 localization, dynamics, and potential redistribution during acute and chronic salt stress

  • Correlate these observations with simultaneous measurements of mitochondrial function and cellular energy status

This multifaceted research design enables comprehensive investigation of the mechanisms by which ATP9 contributes to D. hansenii's remarkable adaptation to high-salt environments, providing insights that may inform applications in bioengineering stress-resistant systems.

How can researchers effectively design experiments to investigate ATP9's assembly-dependent translation in D. hansenii?

Designing experiments to investigate ATP9's assembly-dependent translation in D. hansenii requires a sophisticated methodological framework:

• Pulse-chase labeling with synchronization protocol:

  • Establish a synchronized expression system for ATP synthase components

  • Implement radioactive or stable isotope labeling of newly synthesized ATP9

  • Chase with unlabeled amino acids at timed intervals

  • Quantify ATP9 synthesis rates in correlation with assembly stage markers

• Ribosome profiling with assembly disruption:

  • Perform ribosome profiling to capture translation dynamics of ATP9 mRNA

  • Create controlled disruptions of ATP synthase assembly using:

    • Chemical inhibitors of specific assembly factors

    • Genetic depletion of key assembly components

    • Temperature-sensitive assembly mutations

  • Analyze changes in ribosome occupancy on ATP9 mRNA under these conditions

• Fluorescent reporter system:

  • Develop a dual-fluorescent reporter system with:

    • First fluorophore fused to ATP9 to monitor synthesis

    • Second fluorophore attached to an assembly partner

  • Measure fluorescence resonance energy transfer (FRET) to correlate synthesis with assembly

  • Implement time-lapse microscopy to visualize the process in living cells

• Gradient analysis with quantitative proteomics:

  • Fractionate mitochondria on sucrose gradients to separate assembly intermediates

  • Apply quantitative proteomics to measure ATP9 levels in each fraction

  • Compare stoichiometric relationships between ATP9 and other subunits across fractions

This experimental design approach would generate comprehensive data on how ATP9 synthesis responds to assembly status, providing insights into the molecular mechanisms that ensure stoichiometric balance with nuclear-encoded subunits during F₀ domain assembly.

What advanced methodologies can be employed to screen for ATP synthase inhibitors using recombinant D. hansenii ATP9?

Screening for ATP synthase inhibitors using recombinant D. hansenii ATP9 can be accomplished through several advanced methodological approaches:

• Structure-based high-throughput virtual screening:

  • Generate high-resolution structural models of D. hansenii ATP9 within the ATP synthase complex

  • Identify binding pockets specific to D. hansenii ATP9 using computational algorithms

  • Perform in silico docking of virtual compound libraries (>1,000,000 compounds)

  • Rank compounds based on binding energy predictions and interaction profiles

  • Select top candidates (approximately 1,000) for biochemical validation

• Reconstituted proteoliposome assay system:

  • Purify recombinant ATP9 with the N-terminal 10×His tag using optimized affinity chromatography

  • Co-reconstitute with essential partner subunits (ATP6, AtpF) in artificial liposomes

  • Implement fluorescent pH indicators to monitor proton transport activity

  • Develop a miniaturized 384-well format for high-throughput screening

  • Validate with known inhibitors before screening novel compounds

• Split-luciferase complementation system:

  • Engineer ATP9 and interacting subunits with complementary luciferase fragments

  • Express in appropriate yeast host systems

  • Monitor luciferase activity as a proxy for proper complex formation

  • Screen compounds for disruption of these specific protein-protein interactions

  • Validate hits with orthogonal functional assays

• Label-free biosensor technology:

  • Immobilize purified recombinant ATP9 on biosensor surfaces

  • Monitor binding events using surface plasmon resonance or biolayer interferometry

  • Screen fragment libraries and identify binding moieties

  • Employ structure-guided fragment evolution to develop lead compounds

  • Characterize binding kinetics and thermodynamics of promising inhibitors

These methodologies provide complementary approaches to identify and characterize compounds that specifically target D. hansenii ATP9, potentially leading to the development of selective inhibitors with applications in both fundamental research and possible therapeutic interventions.

What are the future research directions for D. hansenii ATP9 studies?

Future research on D. hansenii ATP9 should focus on several promising directions:

• Structural biology advancements: Obtaining high-resolution structures of D. hansenii ATP9 within the complete ATP synthase complex under various salt concentrations would provide unprecedented insights into halotolerance mechanisms at the molecular level.

• Synthetic biology applications: Engineering D. hansenii ATP9 variants with enhanced stability or altered regulatory properties could lead to the development of more robust microbial cell factories for biotechnological applications, particularly in high-salt environments.

• Comparative mitochondrial bioenergetics: Expanding studies to compare D. hansenii ATP9 function with homologs from other extremophilic yeasts would deepen our understanding of evolutionary adaptations in mitochondrial energy production under stress conditions.

• Integrated multi-omics approaches: Combining transcriptomics, proteomics, and metabolomics to study ATP9 in the context of global cellular responses to environmental stressors would reveal regulatory networks controlling mitochondrial function.

• Advanced imaging techniques: Developing super-resolution microscopy approaches to visualize ATP9 dynamics in living D. hansenii cells under various stress conditions would connect molecular mechanisms to cellular physiology.