Recombinant Debaryomyces hansenii Uncharacterized protein YAE1 (YAE1)

What is the YAE1 protein and what are its known functions in other organisms?

The YAE1 protein has been well-characterized in Saccharomyces cerevisiae, where it functions in complex with Lto1 as an adaptor recruiting specific Fe-S cluster targets to the cytosolic Fe-S protein assembly (CIA) machinery. Specifically, Yae1-Lto1 functions as a target-specific adaptor that recruits apo-Rli1 (an essential ribosome-associated ABC protein) to the CIA machinery . This process is critical for the maturation of cytosolic and nuclear iron-sulfur proteins involved in essential pathways including translation and DNA maintenance .

The human homolog YAE1D1 can functionally replace the yeast counterpart, demonstrating evolutionary conservation of this protein across diverse eukaryotic lineages . In S. cerevisiae, the Yae1-Lto1 complex formation utilizes conserved deca-GX3 motifs, and Lto1 employs its C-terminal tryptophan for binding to the CIA targeting complex .

In D. hansenii, YAE1 remains largely uncharacterized, though based on evolutionary conservation, it likely performs similar functions in Fe-S protein maturation.

Why is Debaryomyces hansenii an important model organism for studying stress adaptation?

D. hansenii has emerged as a valuable model organism for several reasons:

• High halotolerance: Can grow in environments with high salt concentrations, making it ideal for studying osmotic adaptations and salt tolerance mechanisms

• Stress tolerance: Demonstrates resistance to various stress conditions, qualifying it as a resilient eukaryotic model system

• Oleaginous properties: Capable of accumulating lipids, relevant for biotechnological applications

• Metabolic versatility: Shows high respiratory and low fermentative activity, with the ability to utilize various carbon sources

• Killer toxin production: Produces toxins effective against pathogenic Candida species, with potential antifungal applications

• Genetic capacity: Possesses one of the highest coding capacities among yeasts, allowing for diverse metabolic functions

• Ecological distribution: Colonizes diverse microhabitats, indicating adaptive potential through chromosomal polymorphism

What genetic manipulation tools are currently available for studying proteins in D. hansenii?

Recent advances have significantly improved genetic engineering capabilities for D. hansenii:

The PCR-based method is particularly valuable as it allows gene targeting at high efficiency in wild-type isolates without requiring strains with auxotrophic markers . The technique uses simple PCR-based amplification that extends heterologous selectable markers with 50 bp flanks identical to the target genomic site .

How can researchers predict the potential function of uncharacterized YAE1 in D. hansenii based on homology studies?

Functional prediction of uncharacterized proteins like YAE1 in D. hansenii can be approached through:

• Sequence-based homology analysis: Comparing amino acid sequences with characterized homologs from S. cerevisiae and humans to identify conserved domains and motifs, particularly the deca-GX3 motifs critical for Yae1-Lto1 complex formation

• Structural prediction: Using computational tools to model protein structure based on homologs with known structures

• Phylogenetic analysis: Examining evolutionary relationships to infer functional conservation

• Complementation studies: Testing whether D. hansenii YAE1 can rescue phenotypes of yeast Yae1 deletion mutants, similar to how human YAE1D1 can functionally replace yeast Yae1

• Interaction prediction: Identifying potential binding partners based on known interactors in other species, particularly investigating if D. hansenii possesses a Lto1 homolog that might form a complex with YAE1

What are the optimal protocols for recombinant expression and purification of YAE1 in D. hansenii?

Based on recent advances in D. hansenii engineering, the following methodological approach is recommended:

Expression optimization:

• Promoter selection: The TEF1 promoter (from Arxula adeninivorans) has shown high efficiency for recombinant protein expression in D. hansenii

• Terminator selection: The CYC1 terminator has proven effective for protein expression

• Signal peptide screening: Test multiple signal peptides to enhance secretion and production

• Growth conditions: Leverage D. hansenii's halotolerance by using salty-rich media, which both enhances growth and inhibits contaminating microorganisms

• Integration site selection: Utilize safe chromosomal harbor sites for stable expression

Purification strategy:

• Affinity tag selection: Incorporate hexahistidine or other affinity tags compatible with high-salt purification conditions

• Buffer optimization: Adjust salt concentrations to maintain protein stability while enabling purification

• Validation: Confirm protein identity and purity through mass spectrometry and activity assays

How can researchers design efficient gene targeting experiments to characterize YAE1 function in D. hansenii?

An effective gene targeting strategy would include:

• Target selection: Design experiments to either disrupt, tag, or modify the endogenous YAE1 gene

• PCR-based targeting construct design:

  • Design primers with 50 bp homology arms flanking the YAE1 locus

  • Amplify heterologous selectable markers (Hygromycin B or G418 resistance cassettes)

  • For tagging experiments, ensure in-frame fusion with fluorescent or affinity tags

• Transformation protocol:

  • Optimize transformation conditions specific to D. hansenii strains

  • Screen transformants using the appropriate selective media

  • Verify integration through PCR verification and sequencing

• Phenotypic analysis:

  • Assess growth under various stress conditions (salt, pH, oxidative stress)

  • Evaluate Fe-S protein maturation, particularly focusing on Rli1 homologs

  • Perform comparative analyses with wild-type strains

A key advantage of the PCR-based method is its high efficiency (>75%) in integrating constructs through homologous recombination, allowing for rapid generation of transformants .

What experimental approaches can determine if YAE1 in D. hansenii functions in the iron-sulfur cluster assembly pathway?

To investigate YAE1's potential role in Fe-S cluster assembly in D. hansenii:

• Comparative analysis with known systems:

  • In S. cerevisiae, depletion of Yae1 specifically affects Fe-S maturation of Rli1 but not other tested targets

  • Identify D. hansenii Rli1 homolog and assess its dependence on YAE1

• Protein interaction studies:

  • Perform co-immunoprecipitation to determine if D. hansenii YAE1 interacts with:
    a) CIA targeting complex components
    b) A potential Lto1 homolog
    c) Specific target proteins like Rli1

• Conditional expression systems:

  • Generate strains with regulated YAE1 expression

  • Analyze Fe-S protein activity under depletion conditions

  • Assess growth in media with varying iron availability

• Domain function analysis:

  • Create mutations in conserved deca-GX3 motifs and evaluate functional consequences

  • Test if the human YAE1D1 can complement D. hansenii YAE1 deletion

• Biochemical assays:

  • Develop in vitro Fe-S cluster transfer assays using purified components

  • Measure iron binding and Fe-S cluster coordination

How can transcriptomic and proteomic approaches be integrated to understand YAE1 function in D. hansenii?

An integrated -omics approach, similar to the "DebaryOmics" study , would provide comprehensive insights:

• Experimental design:

  • Compare wild-type and YAE1 mutant strains under controlled conditions

  • Utilize chemostat cultivations to maintain precise growth parameters

  • Implement salt stress conditions to investigate stress-specific responses

• Multi-omics data collection:

  • Transcriptomics: RNA-seq to identify differentially expressed genes

  • Proteomics: Quantitative protein profiling

  • Phosphoproteomics: Assess changes in signaling networks

  • Metabolomics: Analyze metabolic consequences of YAE1 disruption

• Integrated analysis framework:

  • Pathway enrichment analysis across multiple data types

  • Correlation networks to identify functional modules

  • Comparative analysis with known YAE1-dependent processes in other organisms

• Validation experiments:

  • Targeted gene expression analysis

  • Protein-protein interaction verification

  • Phenotypic assays for predicted functions

This multi-layered approach would reveal both direct targets and broader cellular responses to YAE1 function in D. hansenii's unique physiological context.

What are the potential challenges in distinguishing YAE1 functions related to Fe-S cluster assembly from those potentially involved in salt tolerance?

This represents a fundamental research challenge that requires sophisticated experimental design:

Potential overlapping functions:

• Fe-S cluster assembly may be affected by salt stress

• YAE1 might have evolved additional functions in D. hansenii related to halotolerance

• Iron homeostasis and salt stress response pathways may interact

Methodological solutions:

• Genetic separation of function:

  • Generate point mutations that specifically disrupt either Fe-S cluster-related interactions or potential salt stress-specific functions

  • Create chimeric proteins combining domains from halotolerant and non-halotolerant species

• Condition-specific phenotyping:

  • Systematically vary salt concentration and iron availability independently

  • Implement matrix-based phenotyping under multiple stress conditions

  • Monitor specific Fe-S dependent enzymes under varying salt conditions

• Comparative genomics approach:

  • Compare YAE1 sequences from halotolerant and non-halotolerant yeasts

  • Identify D. hansenii-specific sequence features that might correlate with salt tolerance

• Protein localization studies:

  • Track YAE1 subcellular localization under normal and high-salt conditions

  • Determine if salt stress alters YAE1 interaction partners

How should researchers interpret contradictory results when comparing YAE1 function across different yeast species?

When facing contradictory findings across species, consider:

• Species-specific adaptations:

  • D. hansenii's evolutionary adaptation to high-salt environments may have altered canonical pathways

  • Unlike S. cerevisiae, D. hansenii shows chromosomal polymorphism and strain variability

• Experimental variables:

  • Standardize growth conditions, accounting for D. hansenii's unique physiological requirements

  • Consider that some D. hansenii isolates may retain wild-type gene copies even after disruption attempts

  • Use multiple independent techniques to verify findings

• Functional redundancy:

  • Investigate potential compensatory mechanisms or redundant proteins

  • Search for D. hansenii-specific gene duplications or paralogs

• Analytical framework:

  • Develop clear criteria for distinguishing conserved vs. species-specific functions

  • Use statistical approaches that account for strain-specific variations

  • Implement rescue experiments with cross-species complementation

What strategies can overcome technical obstacles in studying protein-protein interactions involving YAE1 in D. hansenii?

Protein interaction studies in D. hansenii present unique challenges that require adapted methodologies:

• Salt-compatible interaction assays:

  • Modify traditional yeast two-hybrid (Y2H) systems to function under high salt conditions

  • Develop split-protein complementation assays optimized for D. hansenii's physiology

  • Adjust co-immunoprecipitation protocols to maintain native osmotic conditions

• In vivo interaction mapping:

  • Implement proximity-labeling methods (BioID, APEX) adapted for D. hansenii

  • Develop fluorescence-based interaction assays (FRET, BiFC) with D. hansenii-optimized fluorescent proteins

  • Use crosslinking mass spectrometry (XL-MS) to capture transient interactions

• Comparative interaction profiling:

  • Compare YAE1 interactomes across multiple conditions (varying salt, iron availability)

  • Identify interaction changes following stress exposure

  • Look for condition-specific interaction partners

• Technical adaptations:

  • As observed with Nbp35 depletion in S. cerevisiae, interaction detection may be enhanced when cytosolic Fe-S protein maturation is impaired

  • Consider that YAE1 interactions with CIA components may be strengthened under conditions that impair normal function

How might YAE1 research in D. hansenii contribute to developing improved stress-tolerant yeast strains for biotechnology?

Understanding YAE1's role in D. hansenii could advance biotechnological applications:

• Engineering stress tolerance:

  • If YAE1 contributes to halotolerance, its overexpression or modification might enhance salt resistance in other yeasts

  • Potential to create yeast strains capable of utilizing industrial side-streams and complex feedstock

• Improved recombinant protein production:

  • Insights into YAE1's role in protein synthesis (through Fe-S clusters in translation factors) could optimize expression systems

  • D. hansenii's high coding capacity and ability to grow in extreme conditions offers advantages for industrial applications

• Novel antifungal strategies:

  • Understanding YAE1's essential functions could identify targets for selective inhibition of pathogenic fungi

  • D. hansenii produces killer toxins effective against pathogenic Candida species , and YAE1 research might reveal novel antimicrobial mechanisms

• Metabolic engineering applications:

  • If YAE1 influences D. hansenii's distinctive metabolism, this knowledge could be leveraged for:
    a) Enhanced production of valuable compounds (xylitol, lipases, exopeptidases)
    b) Improved utilization of industrial by-products
    c) Development of robust cell factories for the green transition

What evolutionary insights might be gained by comparing YAE1 function across yeasts with varying halotolerance?

Evolutionary analysis of YAE1 across diverse yeasts could reveal:

• Adaptation signatures:

  • Identification of sequence changes correlated with increased halotolerance

  • Potential positive selection signatures in specific protein domains

  • Co-evolution patterns with interaction partners (like Lto1)

• Functional diversification:

  • Whether YAE1 has acquired new functions in halotolerant species

  • If the specificity for target proteins (like Rli1) has changed

  • Evolution of regulatory mechanisms controlling YAE1 expression

• Convergent evolution:

  • Comparison across phylogenetically distant halotolerant species

  • Identification of convergent adaptations in the Fe-S cluster assembly pathway

  • Correlation between YAE1 sequence features and ecological niches

• Evolutionary trade-offs:

  • Investigation of whether specialization for salt tolerance affects other functions

  • Analysis of YAE1 conservation in relation to metabolic capabilities across species

How might structural biology approaches advance understanding of YAE1 function in D. hansenii?

Structural studies would provide mechanistic insights:

• Structure determination priorities:

  • Crystal or cryo-EM structure of D. hansenii YAE1

  • Complex structures with interacting partners (particularly Lto1 homolog)

  • Comparative analysis with S. cerevisiae Yae1 and human YAE1D1

• Functional implications:

  • Identification of binding interfaces for protein-protein interactions

  • Characterization of the deca-GX3 motifs' structural role

  • Potential salt-adaptation features in protein structure

• Structure-guided engineering:

  • Rational design of mutations to test functional hypotheses

  • Engineering modified YAE1 variants with enhanced or altered functions

  • Development of specific inhibitors for functional studies

• Dynamic analyses:

  • Molecular dynamics simulations under varying salt conditions

  • Conformational changes associated with binding partners

  • Effects of osmolytes on protein stability and interactions

What statistical approaches are recommended for analyzing differential expression of YAE1 and associated genes under varying conditions?

For robust statistical analysis of YAE1-related expression data:

• Experimental design considerations:

  • Include biological replicates (minimum n=3) for each condition

  • Implement time-course studies to capture dynamic responses

  • Include appropriate controls for salt concentration effects

• Normalization strategies:

  • For RNA-seq: Utilize variance stabilizing transformation or regularized log transformation

  • For proteomics: Consider salt-specific normalization methods to account for matrix effects

  • Implement spike-in controls for cross-condition normalization

• Statistical testing framework:

  • Employ generalized linear models that account for multiple experimental factors

  • Apply multiple testing correction (Benjamini-Hochberg FDR)

  • Consider Bayesian approaches for improved estimation with limited replicates

• Integration with functional data:

  • Correlation analysis between YAE1 expression and phenotypic measurements

  • Gene set enrichment analysis to identify coordinated pathway responses

  • Network-based approaches to contextualize YAE1 within broader stress response systems

How can researchers accurately interpret phenotypic effects when YAE1 is disrupted in different D. hansenii isolates?

Interpreting variable phenotypes requires careful consideration:

• Strain diversity awareness:

  • D. hansenii demonstrates significant chromosomal polymorphism and adaptation to specific microhabitats

  • Some isolates may show no phenotypic effects from gene disruptions despite successful targeting

  • Wild-type gene copies may persist alongside disrupted copies in some strains

• Comprehensive verification:

  • Confirm disruption through multiple methods (PCR, sequencing, expression analysis)

  • Screen multiple transformants to account for integration variability

  • Consider whole genome sequencing to identify potential compensatory mutations

• Systematic phenotyping:

  • Implement quantitative growth assays under multiple conditions

  • Measure specific biochemical activities related to Fe-S proteins

  • Conduct comparative analysis across multiple independently derived mutants

• Genetic background solutions:

  • When consistent disruption proves challenging, utilize safe harbor sites for heterologous gene expression

  • Consider creating hybrid strains with well-characterized genetic backgrounds

  • Implement controlled expression systems rather than complete disruption

What approaches can distinguish between direct and indirect effects of YAE1 perturbation in D. hansenii?

Differentiating direct from indirect effects requires:

• Temporal analysis:

  • Implement time-resolved studies following YAE1 perturbation

  • Early effects are more likely to represent direct consequences

  • Use inducible systems for controlled YAE1 depletion

• Direct binding evidence:

  • Identify physical interaction partners through techniques like crosslinking-MS

  • Map binding interfaces through mutagenesis studies

  • Compare interactomes across conditions to identify core vs. condition-specific partners

• Specificity controls:

  • Test effects of disrupting known YAE1 functions (such as Yae1-Lto1 interaction)

  • Compare with phenotypes from disrupting other CIA components

  • Perform rescue experiments with targeted mutations affecting specific functions

• Pathway reconstruction:

  • Systematically build models of YAE1-dependent pathways

  • Test predictions through targeted perturbations

  • Implement Bayesian network analysis to infer causal relationships

The investigation of YAE1 in D. hansenii represents an opportunity to advance understanding of both fundamental iron-sulfur cluster assembly mechanisms and specialized adaptations enabling extreme stress tolerance in eukaryotes.

Citations Duran. "Establishing Debaryomyces hansenii as a superior cell factory for the green transition: optimizing the use of industrial side-streams and complex feedstock for biotech applications." PhD Thesis. "Efficient PCR-based gene targeting in isolates of the non-conventional yeast Debaryomyces hansenii." bioRxiv, 2023. "The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion." eLife, 2015. "DebaryOmics: an integrative –omics study to understand the halotolerance of Debaryomyces hansenii." 2024. Banjara, N. "Debaryomyces hansenii: a foodborne yeast that produces anti-Candida killer toxin." MS Thesis, 2014. "Debaryomyces hansenii Is a Real Tool to Improve a Diversity of Characteristics in Biotechnology." 2021.