The His-tag facilitates purification, while the recombinant design preserves functional domains critical for proton transport .
Protein-Protein Interaction Studies: Investigates ATP synthase assembly mechanisms .
Functional Assays: Tests proton transport efficiency in reconstituted systems .
ATP9 is central to mitochondrial bioenergetics, particularly under osmotic stress. In D. hansenii, mitochondrial ATP synthase activity is linked to alternative oxidase (Aox) regulation, enabling adaptation to high salinity .
In yeast models, ATP9 (and subunit 6) translation is regulated by assembly intermediates. Defects in ATP synthase assembly enhance translation rates to restore subunit stoichiometry, preventing proton leakage . This feedback mechanism ensures efficient energy production while mitigating oxidative stress .
Under hyperosmotic conditions (e.g., NaCl, KCl), D. hansenii upregulates Aox, a cyanide-insensitive oxidase, to maintain mitochondrial membrane potential. ATP9-mediated proton transport likely supports this adaptive response .
Source | Species | Tag | Purity | Sequence Length |
---|---|---|---|---|
Creative BioMart | D. hansenii | N-terminal His | >90% | 1–75 aa |
Cusabio | D. hansenii | N-terminal His | N/A | 1–76 aa |
MyBioSource | D. hansenii | N-terminal His | N/A | N/A |
Variations in sequence length (1–75 vs. 1–76 aa) may reflect truncation or alternative splicing .
KEGG: dha:ATP9
Debaryomyces hansenii is a non-conventional yeast species that has garnered significant attention in biotechnology due to its exceptional tolerance to harsh environmental conditions. The organism demonstrates remarkable resistance to high salt concentrations, various fermentation inhibitors (including furfural, vanillin, and organic acids), and compatibility with a wide substrate range . These characteristics make D. hansenii particularly valuable as a potential cell factory for biotechnological applications, especially those involving industrial side-streams and complex feedstocks.
The yeast's halotolerance enables it to grow in environments that would inhibit other microorganisms, allowing for open (non-sterile) cultivations in certain salt-rich conditions. This feature significantly reduces production costs and simplifies bioprocessing requirements. Research has demonstrated D. hansenii's ability to grow on various industrial by-products, particularly those rich in salt, and successfully produce recombinant proteins without requiring nutritional supplements or freshwater . This combination of robustness and versatility positions D. hansenii as a superior candidate for sustainable bioprocessing applications.
ATP9 encodes subunit 9 (also known as subunit c) of the mitochondrial ATP synthase, a crucial component of the cellular energy generation machinery. This protein has only two transmembrane segments yet is extremely hydrophobic and classified as a proteolipid due to its ability to be easily extracted from mitochondria using organic solvents . The functional significance of ATP9 derives from its structural role in the F₀ domain of ATP synthase.
Subunit 9 forms a ring structure composed of multiple copies (ten in yeast) that constitutes an essential component of the ATP synthase proton-translocating domain (F₀). During oxidative phosphorylation, this subunit 9-ring rotates in response to proton movement across the inner mitochondrial membrane. This rotation induces conformational changes in the catalytic head of ATP synthase (F₁), facilitating ATP production and its subsequent release into the mitochondrial matrix . Due to its central role in energy metabolism, ATP9 is essential for respiratory growth and mitochondrial function.
Organism Type | ATP9 Genetic Location | Examples |
---|---|---|
Most yeasts | Mitochondrial genome | Saccharomyces cerevisiae |
Filamentous fungi | Nuclear genome | Podospora anserina |
Most animals | Nuclear genome | Humans, mammals |
Plants | Primarily mitochondrial | Arabidopsis thaliana |
This natural variation demonstrates that while there is no insurmountable barrier to the functional relocation of ATP9 from mitochondria to the nucleus, significant adaptations are typically required for successful expression from the nuclear genome . The nuclear versions of ATP9 generally feature reduced hydrophobicity and appropriate mitochondrial targeting sequences to ensure proper localization of the protein after cytoplasmic synthesis.
The development of a plasmid-based CRISPR-CUG/Cas9 system represents a significant breakthrough for D. hansenii transformation. This tool facilitates efficient gene editing even in prototrophic strains by utilizing a dominant marker rather than relying on auxotrophic markers . The system allows for the quick assembly of vectors expressing Cas9 and single or multiple single-guide RNAs (sgRNAs), enabling multiplex gene engineering capabilities.
For highly efficient genomic modification, researchers have developed NHEJ-deficient D. hansenii strains that enhance homologous recombination-based editing. These strains support the introduction of point mutations and single/double gene deletions with remarkable efficiency . Additionally, investigators have demonstrated that 90-nucleotide single-stranded DNA oligonucleotides are sufficient for direct repair of CRISPR-induced DNA breaks, resulting in precise edits with efficiency rates approaching 100% .
For in vivo DNA assembly, D. hansenii can successfully perform the fusion of up to three different DNA fragments containing 30-bp homologous overlapping overhangs when co-transformed into the yeast. This technique has been utilized to screen various genetic elements including promoters, terminators, and signal peptides to enhance recombinant protein production .
Relocating the ATP9 gene from the mitochondrial to the nuclear genome represents a significant challenge due to several factors including differences in genetic code, protein import requirements, and the extreme hydrophobicity of the encoded protein. Research has identified several critical strategies for successful relocation:
This multi-faceted approach has enabled the first successful relocation of ATP9 in experimental systems, providing valuable insights for mitochondrial gene transfer studies and potential therapeutic applications for mitochondrial diseases.
Optimizing CRISPR-Cas9 for D. hansenii requires addressing several species-specific considerations:
Codon adaptation for CUG-clade organisms: D. hansenii belongs to the CUG clade of yeasts, where the CUG codon is translated as serine instead of leucine. Therefore, a specialized CRISPR-CUG/Cas9 system with appropriate codon adaptation is required for efficient expression .
Plasmid-based expression system: A plasmid-based CRISPR-CUG/Cas9 method utilizing dominant selection markers rather than auxotrophic markers allows for transformation of prototrophic (wild-type) D. hansenii strains .
sgRNA design and expression: The system should facilitate the assembly of vectors expressing Cas9 and single or multiple sgRNAs for potential multiplex gene editing. Selection of appropriate RNA polymerase III promoters for sgRNA expression is critical .
NHEJ modification: Since D. hansenii preferentially uses non-homologous end-joining for DNA damage repair, construction of NHEJ-deficient strains (such as ku70 knockout strains) significantly improves the efficiency of homology-directed repair .
Repair template optimization: For precise editing, 90-nucleotide single-stranded DNA oligonucleotides have proven sufficient as repair templates, achieving editing efficiencies up to 100% when combined with the above optimizations .
Implementation of these optimizations has enabled highly efficient gene editing in D. hansenii, greatly advancing both basic and applied research with this organism.
The formulation of growth media significantly impacts the expression of recombinant proteins in D. hansenii, particularly for hydrophobic mitochondrial proteins like ATP9. Researchers should consider the following evidence-based approaches:
Salt-rich industrial by-products: D. hansenii exhibits optimal growth and protein expression in media containing elevated salt concentrations. Research has demonstrated successful cultivation and recombinant protein production using salt-rich industrial by-products from the dairy and pharmaceutical industries without requiring additional nutritional supplements or freshwater .
Open cultivation potential: The high salt concentration in certain industrial by-products creates selective conditions favoring D. hansenii's metabolism while inhibiting contaminating non-halotolerant microorganisms. This enables open (non-sterile) cultivations at various laboratory scales (1.5 mL, 500 mL, and 1 L), significantly reducing processing complexity and costs .
Scaling considerations: When designing expression experiments, researchers should evaluate performance across different scales. Studies have confirmed consistent results from microwell plates to liter-scale bioreactors, indicating robust scalability of D. hansenii cultivation systems .
Promoter-media compatibility: The selection of promoters should be matched to media composition. For salt-rich industrial by-products, the TEF1 promoter from Arxula adeninivorans combined with the CYC1 terminator has demonstrated superior performance for recombinant protein production .
These considerations enable researchers to design sustainable and economical bioprocesses while achieving high-level expression of recombinant proteins, including challenging targets like ATP9.
Verifying the successful integration of nuclear-encoded ATP9 into mitochondrial ATP synthase requires a multi-faceted experimental approach addressing protein localization, processing, assembly, and functionality:
Complementation analysis: The most definitive evidence of functional integration comes from complementation experiments using Δatp9 strains. These strains are typically generated by replacing the mitochondrial ATP9 gene with a selectable marker (such as ARG8m). Successful complementation is indicated by restoration of growth on non-fermentable carbon sources like glycerol .
Protein localization and processing: Western blot analysis using antibodies against ATP9 and/or epitope tags can verify both the presence of the protein in mitochondrial fractions and proper processing of the mitochondrial targeting sequence. Comparison of protein size before and after mitochondrial import provides evidence of MTS cleavage .
Assembly analysis: Blue Native PAGE (BN-PAGE) followed by Western blotting can demonstrate incorporation of the recombinant ATP9 into assembled ATP synthase complexes. This technique separates native protein complexes and can detect:
Functional assays: Measurement of ATP synthase activity in isolated mitochondria provides functional verification. This can include:
Respiratory chain analysis: Oxygen consumption measurements using different substrates and inhibitors can confirm integration of ATP9 into functional respiratory chain complexes.
This comprehensive verification approach ensures that nuclear-expressed ATP9 not only reaches mitochondria but also integrates correctly into functional ATP synthase complexes.
When comparing strains with mitochondrial-encoded versus nuclear-encoded ATP9, researchers should implement a structured analytical approach that addresses multiple phenotypic dimensions:
This multifaceted approach enables researchers to comprehensively characterize the consequences of ATP9 gene relocation beyond simple growth complementation.
Codon optimization for nuclear expression of the ATP9 gene requires careful consideration of several factors beyond simple conversion to preferred codons:
CUG codon handling: D. hansenii belongs to the CUG clade where this codon encodes serine instead of the standard leucine. This critical difference must be addressed during codon optimization to prevent mistranslation . The genetic code differences between mitochondrial and nuclear systems must be reconciled.
Balance between optimal codons and translation efficiency: While using the most frequent codons might seem ideal, research suggests that a mixture of optimal and non-optimal codons can enhance proper protein folding by modulating translation speed. This is particularly important for membrane proteins like ATP9 where co-translational folding is critical .
GC content and secondary structure: Optimized sequences should avoid:
Extreme GC content
Strong secondary structures in mRNA
Cryptic splice sites
Internal ribosome binding sites
These elements can significantly impact expression efficiency regardless of codon optimization.
Context-dependent effects: Codon usage should consider:
Neighboring nucleotides (codon context)
Avoidance of rare codon clusters
Distribution of optimal and non-optimal codons
Empirical validation: Multiple codon-optimized variants should be tested experimentally. Research has shown that theoretical codon optimization doesn't always yield optimal expression, particularly for complex membrane proteins like ATP9.
The successful nuclear expression of ATP9 in yeast was achieved using a version specifically codon-optimized for nuclear expression, combined with the mitochondrial targeting sequence from a naturally nuclear version of the gene from Podospora anserina . This empirical approach proved more effective than theoretical optimization alone.
The selection and optimization of mitochondrial targeting sequences (MTS) significantly impacts the import efficiency of hydrophobic proteins like ATP9. Researchers should consider the following evidence-based observations:
Natural versus synthetic MTS performance: Naturally occurring MTS from nuclear-encoded mitochondrial proteins typically outperform synthetic variants for challenging hydrophobic proteins. In the case of ATP9, the MTS from naturally nuclear ATP9 genes (such as from Podospora anserina) has demonstrated superior performance .
Species-specific considerations: The effectiveness of an MTS can vary across species. Research has shown that while the Podospora anserina ATP9 MTS worked effectively in Saccharomyces cerevisiae, not all cross-species MTS transfers maintain functionality . The MTS from PaAtp9-7 in P. anserina was effective in yeast, as evidenced by successful complementation experiments.
Length and charge distribution factors: The length and physicochemical properties of the MTS influence import efficiency:
Longer MTS sequences (>20 amino acids) typically improve import of highly hydrophobic proteins
A net positive charge facilitates interaction with the negatively charged mitochondrial membrane
Amphiphilic helical structure enhances recognition by import machinery
Context-dependent efficiency: The same MTS can exhibit different import efficiencies depending on the cargo protein. For example, the P. anserina MTS that successfully facilitated ATP9 import also showed high efficiency when tested with a recoded yeast ATP8 gene .
Processing considerations: Proper cleavage of the MTS by mitochondrial processing peptidase is essential for protein maturation. Inefficient processing can result in compromised protein function even if import occurs.
Researchers should empirically test multiple MTS variants when designing nuclear expression systems for mitochondrial proteins, particularly for challenging hydrophobic proteins like ATP9.
Investigating ATP synthase assembly dynamics with nuclear-encoded ATP9 requires specialized techniques that can capture both spatial and temporal aspects of the process:
Time-resolved proteomics: Pulse-chase experiments combined with immunoprecipitation and mass spectrometry can track the incorporation kinetics of newly synthesized ATP9 into assembling ATP synthase complexes. This approach can identify potential assembly intermediates and rate-limiting steps that may differ between mitochondrial and nuclear-encoded ATP9 .
Inducible expression systems: Establishing regulated expression systems for nuclear ATP9 enables controlled initiation of protein synthesis and subsequent assembly tracking. Options include:
Tetracycline-responsive promoters
Galactose-inducible systems
Degron-based protein stabilization systems
Native gel electrophoresis techniques: Blue Native PAGE combined with second-dimension SDS-PAGE or liquid chromatography-mass spectrometry (LC-MS) provides snapshots of assembly intermediates. Specific techniques include:
Clear Native PAGE for preserving labile interactions
Complexome profiling for comprehensive analysis of all assembly stages
Antibody shift assays to verify specific protein interactions
Super-resolution microscopy: Techniques such as photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) with appropriate fluorescent tags can visualize the spatiotemporal dynamics of ATP9 import and assembly in living cells.
Genetic interaction analysis: Combining nuclear ATP9 expression with mutations in known assembly factors can reveal differential dependencies or synthetic interactions that illuminate assembly pathway alterations.
Chemical crosslinking mass spectrometry: This technique captures transient protein-protein interactions during the assembly process, potentially identifying unique interaction patterns for nuclear-expressed ATP9.
These approaches collectively enable researchers to develop comprehensive models of ATP synthase assembly pathways when ATP9 originates from nuclear rather than mitochondrial expression, potentially revealing adaptations required for efficient integration.
The successful nuclear expression of mitochondrial ATP9 has profound implications across multiple research domains:
Evolutionary biology insights: The experimental relocation of ATP9 provides a model for studying mitochondrial gene transfer, a critical process in eukaryotic evolution. The adaptations required for functional nuclear expression (codon optimization, reduced hydrophobicity, effective targeting) illuminate the constraints and mechanisms that shaped the endosymbiotic transfer of genes during evolution .
Mitochondrial disease therapeutics: Mitochondrial diseases caused by mutations in mitochondrial genes currently have few treatment options. The demonstration that essential mitochondrial genes like ATP9 can be functionally expressed from the nucleus opens potential avenues for allotopic expression therapies for mitochondrial disorders .
Synthetic biology applications: The successful expression of highly hydrophobic membrane proteins from nuclear genes expands the toolkit for synthetic biology applications, enabling more complex engineering of energy metabolism in microorganisms.
Industrial biotechnology advancements: The combination of successful nuclear ATP9 expression with D. hansenii's exceptional stress tolerance creates opportunities for robust industrial bioprocesses in challenging conditions, such as high-salt or inhibitor-rich environments using industrial side-streams .
Mitochondrial import mechanism understanding: The process of optimizing ATP9 for nuclear expression has revealed insights into the constraints and requirements of the mitochondrial protein import machinery, particularly for challenging hydrophobic proteins.
These diverse implications highlight how fundamental research on mitochondrial gene expression can connect to therapeutic development, industrial applications, and our understanding of evolutionary processes.
The methodologies and findings from D. hansenii ATP9 research provide valuable frameworks that can be applied to similar studies in other organisms:
Adaptation of CRISPR systems for non-conventional organisms: The development of the CRISPR-CUG/Cas9an tailored for D. hansenii provides a template for adapting gene editing technologies to other non-conventional organisms, particularly those in the CUG clade of yeasts . The strategies for optimizing sgRNA design, Cas9 expression, and repair template delivery can inform similar adaptations for other challenging species.
Methodology for mitochondrial gene transfer: The successful approach of utilizing naturally nuclear versions of mitochondrial genes from other organisms represents a novel strategy that could be applied to relocate other mitochondrial genes or to address similar challenges in different species . This methodology circumvents some of the difficulties encountered with direct recoding of mitochondrial genes.
Optimization framework for hydrophobic protein expression: The systematic approach to enabling functional expression of the highly hydrophobic ATP9 protein provides a roadmap for addressing similar challenges with other membrane proteins across various expression systems.
Industrial applications of halotolerant organisms: The demonstration of D. hansenii's ability to utilize salt-rich industrial by-products for recombinant protein production establishes a model for developing similar bioprocesses in other extremophilic organisms .
Comparative mitochondrial biology: The successful nuclear expression of ATP9 enables comparative studies of mitochondrial function when key components are encoded in different genomic compartments, potentially revealing subtle regulatory mechanisms that coordinate nuclear and mitochondrial gene expression.
By leveraging these translatable approaches, researchers working with diverse organisms can build upon the foundations established in D. hansenii to address similar challenges in their systems.