Recombinant Debaryomyces hansenii ATP-dependent RNA helicase DHH1 (DHH1), partial

What is the function of DHH1 in Debaryomyces hansenii?

DHH1 in D. hansenii, similar to its homologs in other yeasts like Saccharomyces cerevisiae, is likely a DEAD-box protein implicated in mRNA regulation processes. Based on conservation among DEAD-box proteins, DHH1 would function in cytoplasmic foci called processing bodies (P-bodies) and play critical roles in mRNA decay pathways and translational repression . In S. cerevisiae, DHH1 has been characterized as having RNA-dependent ATPase activity, though weaker than other DEAD-box helicases . The protein contains conserved helicase motifs found in the superfamily 2 (Sf2) of DEX/D/H-box proteins, which are essential for its function .

To investigate its specific role in D. hansenii, researchers should employ the recently developed gene disruption methods based on homologous recombination, similar to the approach used for disrupting other genes in this yeast . A histidine auxotrophic recipient strain and the DhHIS4 gene as a selectable marker can serve as the foundation for genetic manipulation studies .

How conserved is the DHH1 sequence across yeast species?

DHH1 is highly conserved across yeast species, including Saccharomyces cerevisiae, Debaryomyces hansenii, and other fungi. The protein belongs to the DEAD-box family characterized by the presence of at least nine conserved motifs, including the signature DEAD-box (Asp-Glu-Ala-Asp) sequence .

Most DEAD-box proteins share two RecA-like domains that are responsible for ATP binding and hydrolysis. The N-terminal domain typically contains the conserved motifs I, Ia, Ib, II, and III, while the C-terminal domain includes motifs IV, V, and VI . Comparative sequence analysis would reveal that DHH1 in D. hansenii likely maintains these core functional domains while potentially having unique features related to the halotolerant nature of this organism.

What expression systems are available for recombinant D. hansenii DHH1 production?

For expression of recombinant D. hansenii DHH1, several systems can be employed:

• Homologous expression in D. hansenii: The development of transformation vectors with autonomous replication sequences (ARS) for D. hansenii now enables homologous expression . This system uses a histidine auxotrophic recipient strain and the DhHIS4 gene as a selectable marker, achieving transformation efficiencies of >1.5 × 10^5 transformants per μg of DNA .

• Heterologous expression systems: Similar to other recombinant proteins, D. hansenii DHH1 can be expressed in:

  • E. coli (bacterial system)

  • Other yeasts (e.g., S. cerevisiae)

  • Baculovirus-infected insect cells

  • Mammalian cell systems

For heterologous expression, codon optimization might be necessary given the distinct codon usage bias of D. hansenii as a halophilic yeast. When selecting an expression system, consider the following comparison table:

How can I assess the ATP hydrolysis activity of recombinant D. hansenii DHH1?

Assessment of ATP hydrolysis activity of recombinant D. hansenii DHH1 requires careful experimental design. Based on studies with other DEAD-box helicases, the following methodological approach is recommended:

• Purification of recombinant protein: Express DHH1 with an affinity tag (His-tag or GST) and purify using affinity chromatography followed by size exclusion chromatography to ensure high purity (≥85%) .

• ATP hydrolysis assay: The standard method involves measuring the release of inorganic phosphate from ATP. This can be done using:

  • Malachite green assay for colorimetric detection

  • Thin-layer chromatography with [γ-32P]ATP

  • Coupled enzyme assay using pyruvate kinase and lactate dehydrogenase to monitor NADH oxidation

• Critical controls:

  • RNA-dependent activity assessment: Compare activity with and without RNA substrates

  • Negative control: ATP hydrolysis-deficient mutant (e.g., mutation in the DEAD motif)

  • Positive control: Well-characterized DEAD-box protein (e.g., eIF4A)

Note that DHH1 from S. cerevisiae has been shown to have weaker ATPase activity compared to other DEAD-box helicases, which is restricted by interdomain interactions between the N- and C-terminal RecA-like domains . Similar behavior might be expected for D. hansenii DHH1.

What experimental approaches can be used to study DHH1's role in P-body formation in D. hansenii?

To investigate DHH1's role in P-body formation in D. hansenii, several complementary approaches can be employed:

• Fluorescent tagging and microscopy:

  • Create a DHH1-GFP fusion construct using the recently developed methods for tagging open reading frames with GFP in D. hansenii

  • Visualize P-body formation under various stress conditions (e.g., glucose deprivation, osmotic stress)

  • Perform co-localization studies with other known P-body components

• Genetic manipulation:

  • Generate dhh1Δ knockout strains using homologous recombination techniques established for D. hansenii

  • Create DHH1 mutants with alterations in key domains (DEAD-box motif, RNA-binding regions)

  • Examine P-body formation in these genetic backgrounds

• Biochemical approaches:

  • Perform RNA immunoprecipitation (RIP) to identify RNAs associated with DHH1

  • Conduct protein-protein interaction studies to map the P-body interactome in D. hansenii

• Functional assays:

  • Measure mRNA decay rates in wild-type vs. dhh1Δ strains

  • Assess translation efficiency under various stress conditions

One interesting aspect to investigate would be whether the ATPase activity of DHH1 is required for its recruitment into P-bodies in D. hansenii, as studies in other organisms have shown that this activity is not required for recruitment but regulates DHH1's association with RNA in vivo .

How do mutations in the DEAD-box motif affect DHH1 function in D. hansenii?

The DEAD-box motif (Asp-Glu-Ala-Asp) is crucial for ATP hydrolysis in DEAD-box helicases. Based on studies in other organisms, mutations in this motif would likely have the following effects on DHH1 function in D. hansenii:

• DEAD → DQAD mutation:

  • Impairs ATP hydrolysis while retaining ATP binding

  • May act as a dominant negative by trapping RNA substrates

  • Likely to impair mRNA decay functions

• DEAD → AAAD mutation:

  • Disrupts both ATP binding and hydrolysis

  • Complete loss of enzymatic activity

  • May still allow protein-protein interactions in P-bodies

To study these effects experimentally, researchers should:

• Generate the mutants using site-directed mutagenesis

• Express them in dhh1Δ strains for complementation studies

• Assess protein localization, RNA binding, and functional outcomes

The following table summarizes expected phenotypes based on studies in other organisms:

These predictions should be experimentally verified in D. hansenii, as the halotolerant nature of this yeast might influence protein behavior.

How does the halotolerant nature of D. hansenii affect DHH1 protein function compared to other yeasts?

D. hansenii is known for its extreme osmotolerance and halotolerance, capable of growing in high salt concentrations . This unique physiological characteristic may influence DHH1 function in several ways:

• Protein stability and conformation: The ionic environment in D. hansenii cells might affect the interdomain interactions of DHH1, potentially altering its ATPase activity. Studies have shown that in S. cerevisiae, interdomain interactions between the N- and C-terminal RecA-like domains restrict DHH1's ATPase activity . In D. hansenii, these interactions might be differently regulated due to adaptation to high salt conditions.

• P-body dynamics: Salt stress is known to induce P-body formation in yeasts. D. hansenii may have evolved distinctive P-body regulation mechanisms involving DHH1 to cope with its natural high-salt environment.

• RNA substrate specificity: DHH1 in D. hansenii might have evolved to recognize RNA substrates with different stability characteristics, as RNAs in halophilic organisms often have adaptations for stability under high salt conditions.

To investigate these differences experimentally:

• Compare the biochemical properties (ATP hydrolysis, RNA binding) of purified DHH1 from D. hansenii and S. cerevisiae under various salt concentrations

• Examine P-body formation and DHH1 localization under salt stress in both yeasts

• Perform complementation studies by expressing D. hansenii DHH1 in S. cerevisiae dhh1Δ strains and vice versa

What methods are available for gene disruption of DHH1 in D. hansenii?

Gene disruption of DHH1 in D. hansenii can be achieved using homologous recombination techniques that have been specifically developed for this yeast. The following methodology is recommended:

• Design of disruption cassette:

  • Amplify the genomic region containing the DHH1 gene from D. hansenii

  • Remove an essential part of the DHH1 ORF and replace it with a selectable marker such as DhHIS4

  • Ensure sufficient homology arms (optimally 90-100 bp) for efficient targeting

• Transformation protocol:

  • Use a histidine auxotrophic recipient strain (e.g., equivalent to DBH9 described for other genes)

  • Transform with the linearized disruption cassette using a high-efficiency transformation protocol

  • For optimal transformation efficiency, use sorbitol as a stabilizer

• Verification of disruption:

  • PCR confirmation of correct integration

  • Sequencing of integration junctions

  • Phenotypic analysis (e.g., growth defects, P-body formation)

This approach has been successful for disrupting other genes in D. hansenii with transformation efficiencies reaching >1.5 × 10^5 transformants per μg of DNA . For DHH1 specifically, researchers should be aware that complete deletion might be lethal if the gene is essential in D. hansenii, in which case conditional expression systems might be necessary.

What are the key controls needed when examining DHH1's RNA binding properties?

When examining DHH1's RNA binding properties, several controls are essential to ensure reliable and interpretable results:

• Protein quality controls:

  • Purity assessment (≥85% by SDS-PAGE)

  • Proper folding verification (circular dichroism spectroscopy)

  • Batch-to-batch consistency checks

• Binding specificity controls:

  • Comparison with known RNA-binding mutants (e.g., mutations in RNA-binding motifs)

  • Competition assays with specific vs. non-specific RNAs

  • Negative control: unrelated RNA-binding protein

• Methodological controls:

  • For electrophoretic mobility shift assays (EMSA): non-specific competitor (e.g., tRNA, heparin)

  • For filter binding: pre-equilibration of filters, non-specific binding controls

  • For fluorescence anisotropy: fluorophore-only controls, buffer effects

• Biological relevance controls:

  • Correlation with in vivo RNA targets (e.g., RIP-seq data)

  • Comparison of binding to functional vs. non-functional RNA targets

  • Assessment of binding under physiologically relevant salt and pH conditions

The last point is particularly important for D. hansenii DHH1, as this halotolerant yeast naturally experiences higher ionic strengths, which may affect RNA-protein interactions.

How can interdomain interactions in DHH1 be experimentally manipulated?

Based on studies in S. cerevisiae, interdomain interactions between the N- and C-terminal RecA-like domains of DHH1 restrict its ATPase activity . Manipulating these interactions could provide valuable insights into DHH1 function in D. hansenii. Here are methodological approaches:

• Targeted mutagenesis:

  • Identify residues at the domain interface using homology modeling based on known structures

  • Introduce charge-reversal or hydrophobicity-altering mutations at key interaction points

  • Create a library of mutants with varying degrees of interdomain interaction disruption

• Domain swapping experiments:

  • Create chimeric constructs with domains from DHH1 orthologs (e.g., S. cerevisiae Dhh1, human DDX6)

  • Assess how domain swapping affects protein function and regulation

• Small molecule modulators:

  • Screen for compounds that stabilize or disrupt interdomain interactions

  • Use these as tools to probe the functional consequences of altering these interactions

• Analytical approaches to measure interdomain interactions:

  • FRET-based assays with fluorophores in each domain

  • Hydrogen-deuterium exchange mass spectrometry

  • Disulfide crosslinking of strategically placed cysteine residues

The following table summarizes potential mutation strategies and their expected effects:

Studies in S. cerevisiae have shown that mutations disrupting interdomain interactions enhanced ATP hydrolysis, mRNA turnover, RNA binding, and recruitment into cytoplasmic foci . Similar effects might be expected in D. hansenii, potentially with unique characteristics related to its halotolerant physiology.

What is the relationship between DHH1 activity and lipid metabolism in D. hansenii?

D. hansenii is known for its ability to accumulate lipids to over 50% of its biomass, making it valuable for biotechnological applications . The relationship between DHH1 activity and lipid metabolism presents an intriguing research direction:

• Potential regulatory mechanisms:

  • DHH1 may regulate the expression of key lipid metabolism genes at the post-transcriptional level

  • Stress conditions that trigger lipid accumulation likely also affect DHH1 activity and P-body formation

  • mRNAs encoding lipid metabolism enzymes might be specific targets of DHH1-mediated regulation

• Experimental approaches:

  • Transcriptome analysis comparing wild-type and dhh1Δ strains under lipid-accumulating conditions

  • Lipid profiling using techniques such as thin-layer chromatography or mass spectrometry

  • RNA immunoprecipitation to identify lipid metabolism-related mRNAs associated with DHH1

  • Polysome profiling to assess translation efficiency of lipid metabolism genes

• Integration with fatty acid β-oxidation pathway:

  • Recent studies have explored the fatty acid β-oxidation pathway in D. hansenii

  • Investigation of whether DHH1 plays a role in regulating this pathway could be valuable

  • The relationship between peroxisomal NAD(H) homeostasis and DHH1 function is worth exploring

This research could provide insights not only into basic RNA biology but also into biotechnological applications for enhanced lipid production in D. hansenii.

How can DHH1 be used as a tool to study the halotolerant nature of D. hansenii?

DHH1 can serve as a molecular tool to investigate the halotolerant mechanisms of D. hansenii through several experimental approaches:

• Comparative studies with non-halotolerant yeasts:

  • Express fluorescently tagged DHH1 from D. hansenii in S. cerevisiae

  • Compare P-body dynamics and stress responses under salt stress

  • Assess whether D. hansenii DHH1 confers any salt tolerance advantages

• Identification of salt-responsive mRNA targets:

  • Perform DHH1 RNA immunoprecipitation under normal and high-salt conditions

  • Identify differentially bound mRNAs that may contribute to halotolerance

  • Investigate whether these targets are regulated differently in halotolerant versus non-halotolerant yeasts

• Structure-function analysis in salt environments:

  • Examine how salt concentrations affect DHH1 enzymatic activities

  • Investigate potential salt-specific conformational changes

  • Determine if D. hansenii DHH1 has evolved specific adaptations for function in high ionic strength environments

These studies could reveal novel aspects of post-transcriptional regulation in extremophilic organisms and potentially identify strategies for engineering salt tolerance in other yeasts or crops.