Recombinant Debaryomyces hansenii NADH-ubiquinone oxidoreductase chain 4L (ND4L)

What is Debaryomyces hansenii NADH-ubiquinone oxidoreductase chain 4L (ND4L) and what is its role in cellular metabolism?

ND4L is a subunit of the mitochondrial respiratory complex I (NADH:ubiquinone oxidoreductase), which plays a crucial role in cellular energy metabolism. In D. hansenii, this protein contributes to the electron transport chain in oxidative phosphorylation, facilitating energy production through ATP synthesis. The protein is encoded by the mitochondrial genome and represents one of the essential components for respiratory function in this osmotolerant yeast.

Recent research has demonstrated growing interest in mitochondrial genes like ND4L, with studies in human genomics indicating that variants in MT-ND4L can be associated with conditions such as Alzheimer's disease . In D. hansenii, this protein may have adapted specific properties related to the organism's exceptional halotolerance and stress resistance capabilities.

How is recombinant D. hansenii ND4L typically expressed and purified for research applications?

Expression and purification of recombinant D. hansenii ND4L typically follows these methodological steps:

• Vector Selection and Construction: Utilizing CRISPR/Cas9-based gene editing tools specifically adapted for D. hansenii as developed in recent research . The CTG codon-optimized expression systems are particularly important due to D. hansenii's alternative codon usage.

• Expression System Options:

  • Homologous expression in D. hansenii using vectors containing promoters such as Dh_RNR2p or Dh_RHR2p

  • Heterologous expression in conventional systems with codon optimization

• Purification Methodology:

  • Affinity chromatography using histidine tags

  • Ion exchange chromatography exploiting the protein's charge properties

  • Size exclusion chromatography for final polishing

The selection of an appropriate expression system is particularly important as ND4L is a hydrophobic membrane protein. For optimal expression in D. hansenii, researchers have developed specialized plasmid-based CRISPR/Cas9 methods that allow for efficient gene editing and protein expression in this non-conventional yeast .

What genetic techniques are available for studying and manipulating the ND4L gene in D. hansenii?

Several genetic techniques have been developed specifically for D. hansenii, which can be applied to studying the ND4L gene:

• CRISPR/Cas9 Gene Editing: A novel plasmid-based CRISPR CUG/Cas9 method has been developed for efficient gene editing in D. hansenii, which can be applied to ND4L research. This system utilizes a dominant marker (NAT gene providing resistance to NTC drug), allowing work with prototrophic strains .

• Oligo-Mediated Gene Editing: Researchers have demonstrated that 90-nucleotide single-stranded DNA oligonucleotides are sufficient for direct repair of DNA breaks induced by sgRNA-Cas9, resulting in precise editing with up to 100% efficiency .

• Multiplex Gene Engineering: The CRISPR CUG-tRNA vector system enables the production of multiple sgRNA species, facilitating simultaneous manipulation of multiple genes .

• NHEJ-Deficient Strains: Creating NHEJ (Non-Homologous End Joining)-deficient D. hansenii strains significantly improves the efficiency of precise gene targeting, which is valuable for introducing specific mutations or deletions in the ND4L gene .

The table below summarizes key components of the genetic toolkit for D. hansenii:

How does the function of ND4L in D. hansenii contribute to its known halotolerant properties?

The relationship between ND4L and D. hansenii's halotolerance involves several interconnected mechanisms:

• Energy Metabolism Under Salt Stress: As a component of Complex I, ND4L plays a role in maintaining mitochondrial function and energy production under high salt conditions. Research has demonstrated that D. hansenii exhibits specific transcriptomic and proteomic adaptations when grown under high salt concentrations (1M NaCl or KCl) .

• Respiratory Adaptations: The expression and regulation of mitochondrial components, including ND4L, may be altered in response to salt stress, contributing to D. hansenii's ability to maintain energy homeostasis in challenging environments.

• Redox Balance: Proper functioning of the respiratory chain, including the ND4L component, is crucial for redox balance, which is particularly important under conditions where reactive oxygen species production may be elevated, such as salt stress.

• Cross-Stress Protection: Studies have shown that the presence of sodium in the medium protects D. hansenii cells against oxidative stress and additional abiotic stresses like extreme pH or high temperature . The mitochondrial function, including ND4L activity, may contribute to this cross-protection mechanism.

Integrated -omics studies have revealed that sodium and potassium trigger different responses at both expression and regulation of protein activity levels in D. hansenii, implicating specific cellular processes as key players in halotolerance .

What are the optimal experimental conditions for functional analysis of recombinant D. hansenii ND4L in vitro?

For optimal functional analysis of recombinant D. hansenii ND4L, researchers should consider the following methodological approaches:

• Membrane Protein Reconstitution:

  • Incorporation into proteoliposomes using defined lipid compositions

  • Use of nanodiscs for single-molecule studies

  • Detergent screening (typically starting with mild detergents like DDM or LMNG)

• Activity Assay Conditions:

  • Buffer composition: 50-100 mM phosphate or Tris buffer, pH 7.0-8.0

  • Salt concentration: Variable NaCl (0-2M) to assess halotolerant properties

  • Temperature range: 20-30°C (optimal growth temperature for D. hansenii)

  • Substrate concentrations: 50-200 μM NADH, 10-50 μM ubiquinone analogs

• Spectroscopic Analysis:

  • Electron transfer activity monitoring by reduction of artificial electron acceptors

  • Fluorescence-based assays for conformational changes

  • EPR spectroscopy for analysis of iron-sulfur clusters

• Control Measures:

  • Comparison with recombinant ND4L from non-halotolerant yeasts

  • Analysis under varying salt concentrations to assess salt-dependent functionality

When studying ND4L function in the context of halotolerance, researchers should consider using the chemostat cultivation conditions described in recent studies, where D. hansenii was grown in the presence of either 1M NaCl or KCl to study stress responses .

How can CRISPR/Cas9 methods be applied to investigate the role of ND4L in D. hansenii's mitochondrial function?

The recently developed CRISPR/Cas9 system for D. hansenii provides powerful tools for investigating ND4L function:

• Gene Knockout/Modification Strategy:

  • Design sgRNAs targeting the ND4L gene using the optimized CRISPR CUG/Cas9 method

  • Generate precise point mutations in ND4L using 90-nt single-stranded DNA oligonucleotides as repair templates

  • Create truncated or tagged versions of ND4L to assess domain functions

• Expression Vector Selection:

  • Use plasmids containing the Cl_TDH3 promoter, CTG codon-optimized Cas9, and appropriate terminators

  • Incorporate the NAT marker for selection in prototrophic strains

• Transformation and Screening Protocol:

  • Transform NHEJ-deficient D. hansenii strains for highest editing efficiency

  • Select transformants on NTC-containing medium

  • Verify edits by sequencing and functional assays

• Phenotypic Analysis of Mutants:

  • Assess growth under varying salt concentrations (0-2M NaCl/KCl)

  • Measure oxygen consumption and mitochondrial membrane potential

  • Analyze reactive oxygen species production under stress conditions

  • Evaluate ATP synthesis capacity in mutants versus wild-type

The CRISPR system efficiency can be optimized by using NHEJ-deficient strains (ku70 mutants), which have demonstrated editing efficiencies approaching 100% in D. hansenii .

What insights can comparative genomics provide about the evolution of ND4L in D. hansenii compared to other yeasts?

Comparative genomics approaches can reveal important insights about ND4L evolution:

• Sequence Conservation Analysis:

  • Alignment of ND4L sequences across yeast species reveals conservation patterns

  • Identification of D. hansenii-specific residues that may contribute to halotolerance

  • Analysis of selection pressure on different protein domains

• Phylogenetic Reconstruction:

  • Construction of ND4L phylogenetic trees to trace evolutionary history

  • Correlation of sequence changes with adaptation to high-salt environments

  • Identification of convergent evolution in halotolerant species

• Codon Usage Analysis:

  • Evaluation of alternative codon usage patterns in D. hansenii ND4L

  • Assessment of codon adaptation index in relation to expression levels

  • Implications for heterologous expression and protein engineering

• Structural Prediction and Comparison:

  • Computational modeling of ND4L structure across species

  • Identification of structural adaptations in halotolerant yeasts

  • Prediction of functional consequences of amino acid substitutions

D. hansenii belongs to the CUG clade of yeasts that exhibit alternative genetic code usage, which has implications for the evolution of mitochondrial genes like ND4L. The genome sequencing of D. hansenii in 2004 provides a foundation for these comparative analyses .

What are the methodological approaches for integrating transcriptomic and proteomic data to study ND4L regulation in D. hansenii?

For comprehensive multi-omics analysis of ND4L regulation, researchers should consider:

• Experimental Design for Multi-omics Integration:

  • Synchronized sampling for transcriptomics and proteomics from the same cultures

  • Chemostat cultivations in controlled bioreactors with defined salt conditions (e.g., 1M NaCl or KCl)

  • Inclusion of time-course measurements to capture regulatory dynamics

• Data Collection Methods:

  • RNA-seq for transcriptome profiling

  • Mass spectrometry-based proteomics (e.g., LC-MS/MS)

  • Phosphoproteomics to detect post-translational modifications

  • Targeted approaches for mitochondrial fraction enrichment

• Computational Integration Strategies:

  • Correlation analysis between transcript and protein abundance

  • Network analysis to identify regulatory hubs

  • Machine learning approaches for pattern recognition

  • Pathway enrichment analysis focusing on mitochondrial function

• Validation Approaches:

  • Targeted gene expression studies (RT-qPCR)

  • Western blotting for protein abundance validation

  • Activity assays to link molecular changes to functional outcomes

Recent research has successfully applied integrative -omics approaches to understand D. hansenii's responses to high salt concentrations, demonstrating that sodium and potassium trigger different responses at both expression and regulation of protein activity levels . Similar approaches could be adapted specifically for studying ND4L regulation.

How does the variation in ND4L sequence or expression contribute to phenotypic differences among D. hansenii strains?

Investigating strain-specific variations in ND4L requires:

• Comparative Genomic Approaches:

  • Sequencing of ND4L across a diverse collection of D. hansenii strains

  • Identification of natural variants and their correlation with phenotypic traits

  • Association studies linking specific polymorphisms to halotolerance levels

• Expression Analysis Methodology:

  • RT-qPCR for quantitative expression comparison across strains

  • Reporter gene assays to assess promoter activity variations

  • In situ hybridization to evaluate spatial expression patterns

• Functional Characterization Protocol:

  • Respirometric analysis of mitochondrial function in different strains

  • Growth profiling under varying salt concentrations

  • Cross-complementation studies between strains with different ND4L variants

• High-throughput Phenotyping:

  • Automated growth curve analysis under standardized conditions

  • Metabolic profiling to detect strain-specific differences

  • Stress response assays to categorize strains by resistance profiles

Recent studies have begun to explore intraspecies behavioral characteristics of novel D. hansenii strains in response to sodium and their ability to tolerate various stress conditions . Similar approaches could be applied specifically to study the role of ND4L variation in these phenotypic differences.

What are the recommended protocols for investigating protein-protein interactions involving D. hansenii ND4L?

To study protein-protein interactions involving ND4L, researchers should consider:

• In vivo Interaction Analysis:

  • Split-reporter systems (e.g., split-GFP, BRET, FRET)

  • Co-immunoprecipitation with epitope-tagged ND4L

  • Proximity-dependent biotin labeling (BioID, APEX)

  • Yeast two-hybrid adapted for mitochondrial membrane proteins

• In vitro Interaction Studies:

  • Pull-down assays with recombinant proteins

  • Surface plasmon resonance for kinetic analysis

  • Isothermal titration calorimetry for thermodynamic parameters

  • Cross-linking mass spectrometry to map interaction interfaces

• Structural Analysis Methods:

  • Cryo-electron microscopy of intact respiratory complexes

  • NMR studies of specific domains or peptides

  • X-ray crystallography of stable subcomplexes

• Bioinformatic Prediction and Validation:

  • Computational prediction of interaction networks

  • Coevolution analysis to identify interacting residues

  • Molecular docking simulations to predict binding modes

  • Conservation analysis of interaction interfaces

For optimal results, these studies should be conducted under conditions that mimic the physiological environment of D. hansenii, including appropriate salt concentrations, as the protein interactions may be influenced by the halotolerant nature of the organism.

How can metabolomic approaches be combined with ND4L genetic manipulations to understand D. hansenii's metabolic adaptations?

Integrating metabolomics with genetic studies of ND4L requires:

• Experimental Design Considerations:

  • Creation of ND4L variants using CRISPR/Cas9 technology

  • Growth of mutant and wild-type strains under identical controlled conditions

  • Sampling at multiple time points and growth phases

  • Inclusion of varying salt concentrations to probe stress responses

• Metabolite Extraction and Analysis Methods:

  • Optimized extraction protocols for polar and non-polar metabolites

  • Targeted LC-MS/MS for specific metabolic pathways

  • Untargeted metabolomics for discovery of novel metabolic signatures

  • Stable isotope labeling to track metabolic fluxes

• Data Integration Framework:

  • Correlation of metabolite levels with ND4L expression/activity

  • Pathway analysis focusing on energy metabolism

  • Flux balance analysis incorporating ND4L function

  • Machine learning approaches to identify metabolic signatures of ND4L variants

• Validation Strategies:

  • Enzyme activity assays for key metabolic reactions

  • Oxygen consumption measurements

  • ATP/ADP ratio determination

  • Redox balance assessment (NAD+/NADH, GSH/GSSG)