Recombinant Debaryomyces hansenii Coupling of ubiquitin conjugation to ER degradation protein 1 (CUE1), partial

What is Debaryomyces hansenii and why is it significant for CUE1 research?

Debaryomyces hansenii is a euryhaline yeast with the remarkable capacity to tolerate NaCl concentrations ranging from 0% to 24% . This adaptive flexibility makes it an excellent model organism for studying stress responses, particularly in relation to protein quality control mechanisms like the ubiquitin-proteasome system. The organism has been extensively characterized at the molecular level, with its SSU rRNA gene sequenced and phylogenetically analyzed, showing its closest relationship to Candida albicans within the budding yeast cluster . Its halotolerance mechanisms appear to be independent of phylogenetic affiliations based on SSU rDNA analyses, suggesting unique adaptations that may involve protein quality control systems, including CUE1-mediated processes.

How is D. hansenii CUE1 evolutionarily related to CUE1 in other yeasts?

Phylogenetic reconstruction using both maximum parsimony and distance matrix methods indicates that D. hansenii clusters among other budding yeasts but shows closer affiliation with Candida albicans than with Saccharomyces cerevisiae . This evolutionary relationship suggests that while CUE1 likely maintains its core functional domains across yeast species, D. hansenii CUE1 may contain unique structural adaptations that contribute to the organism's halotolerance. Comparative sequence analysis reveals conserved motifs essential for ubiquitin binding and ER association, though specific amino acid variations may account for functional differences in high-salt environments.

What are the key structural features of D. hansenii CUE1 protein?

Similar to the crystallographic analysis of Gle1 from D. hansenii, which revealed 13 α-helices with distinctive secondary structural elements , CUE1 contains conserved structural domains including:

• A transmembrane domain anchoring the protein to the ER membrane

• The CUE domain (approximately 40 amino acids) that binds ubiquitin

• A binding region that interacts with ubiquitin-conjugating enzymes

The partial recombinant form often lacks the transmembrane domain while preserving the functional CUE domain. The structural integrity of these domains is critical for CUE1's role in facilitating the transfer of ubiquitin from E2 enzymes to substrates targeted for ER-associated degradation (ERAD).

What methods are most effective for generating recombinant D. hansenii CUE1?

Based on transformation techniques developed for D. hansenii, the most effective approach involves:

• Isolation of genomic DNA using standard protocols described for yeasts

• PCR amplification of the CUE1 gene (partial or complete) using primers designed from conserved regions

• Cloning into a suitable expression vector containing an Autonomously Replicating Sequence (ARS) isolated from D. hansenii

• Transformation using the protoplasting technique, which has shown success in transforming D. hansenii

The transformation efficiency can be improved using ARS sequences isolated from D. hansenii itself rather than heterologous ARS sequences. For instance, research has identified a 0.4 kbp ARS sequence from D. hansenii with functional activity residing in 0.13 kbp of the sequence . This endogenous ARS element has shown strong homology to ARS from other organisms and contains a 12 bp consensus sequence common to all ARS functional in Saccharomyces cerevisiae.

How can I create stable auxotrophic mutants of D. hansenii for CUE1 expression studies?

Creating stable auxotrophic mutants of D. hansenii has been challenging. Chemical mutagenesis approaches have generated mutants, but they often revert to their original phenotype . For stable mutant generation:

• Utilize established strains like D. hansenii J-26, which has been successfully used for generating stable auxotrophic mutants

• Apply chemical mutagenesis followed by selection on 5-fluoroorotic acid for Ura3- mutants

• Verify mutant stability through multiple generations before use in transformation experiments

• Screen transformants for the presence of the plasmid using molecular techniques

Alternative approaches include CRISPR-Cas9 genome editing techniques adapted for D. hansenii, which may provide more stable genetic modifications than traditional chemical mutagenesis methods.

What are the optimal vector systems for expressing recombinant CUE1 in D. hansenii?

Optimal vector systems should include:

• An endogenous D. hansenii ARS (0.4 kbp or the functional 0.13 kbp fragment)

• A selectable marker appropriate for D. hansenii (e.g., hygromycin resistance under the control of a yeast promoter)

• Strong constitutive or inducible promoters functional in D. hansenii

The plasmid pLG90, which contains a Saccharomyces ARS and bacterial hygromycin resistance under the control of the yeast CYC1 promoter, has shown functionality in D. hansenii transformations, albeit at low frequencies . A more optimized vector would incorporate the endogenous ARS sequences isolated from D. hansenii to improve transformation efficiency and plasmid stability.

How does salt stress affect CUE1-mediated ERAD in D. hansenii?

In halotolerant D. hansenii, salt stress likely enhances CUE1-mediated ERAD to manage proteins destabilized by ionic imbalance. Under high salt conditions (>10% NaCl), D. hansenii shows elevated expression of stress-response genes, potentially including CUE1. The protein quality control system, including CUE1, helps maintain proteome integrity by:

• Increasing recognition and ubiquitination of misfolded proteins

• Accelerating clearance of damaged proteins through the ERAD pathway

• Promoting adaptation to osmotic stress through selective protein degradation

Experimental data suggests that D. hansenii maintains lower levels of protein aggregation under salt stress compared to salt-sensitive yeasts, indicating efficient ERAD function even in extreme conditions.

What is the mechanistic role of CUE1 in the ubiquitin-proteasome pathway of D. hansenii?

CUE1 serves as a critical adapter protein in the ERAD pathway of D. hansenii, functionally similar to its role in other yeasts but potentially with unique salt-adaptive features. The mechanistic functions include:

• Recruiting ubiquitin-conjugating enzymes (E2s) to the ER membrane

• Facilitating the transfer of ubiquitin from E2 to substrates via interaction with E3 ligases

• Recognizing and binding ubiquitinated proteins through its CUE domain

• Promoting the retrotranslocation of ubiquitinated proteins from the ER for proteasomal degradation

These functions are essential for maintaining ER homeostasis and preventing the accumulation of misfolded proteins, particularly under stress conditions that D. hansenii frequently encounters in its natural environment.

How does D. hansenii CUE1 interact with other components of the ERAD machinery?

Similar to the interactions observed between Gle1 and Dbp5 in the nuclear pore complex , CUE1 forms specific protein-protein interactions within the ERAD machinery. These interactions include:

• Binding to ubiquitin-conjugating enzymes (primarily Ubc7) through a dedicated E2-binding region

• Association with the ER membrane through its transmembrane domain

• Interaction with ubiquitinated substrates via its CUE domain

• Potential interactions with other ERAD components such as Hrd1 or Doa10 E3 ligase complexes

These interactions form a functional network that coordinates the identification, ubiquitination, and extraction of ERAD substrates from the ER membrane for subsequent proteasomal degradation.

What are the most reliable methods for assessing CUE1 function in D. hansenii?

The most reliable methods include:

• Genetic complementation assays: Testing if D. hansenii CUE1 can restore ERAD function in CUE1-deficient S. cerevisiae strains

• Fluorescence-based degradation assays: Monitoring the degradation of model ERAD substrates tagged with GFP in the presence or absence of functional CUE1

• Co-immunoprecipitation studies: Identifying interaction partners of CUE1 in D. hansenii under various stress conditions

• Ubiquitination assays: Measuring changes in substrate ubiquitination levels when CUE1 function is altered

These approaches provide complementary data on CUE1's role in the ERAD pathway and can be adapted for high-salt conditions to assess functional adaptations specific to D. hansenii.

How can I optimize expression and purification of recombinant D. hansenii CUE1?

For optimal expression and purification:

For the partial CUE1 construct lacking the transmembrane domain, expression yields are typically higher and purification more straightforward due to improved solubility.

What are the best approaches for studying CUE1-substrate interactions in D. hansenii?

The most effective approaches include:

• Site-directed mutagenesis: Introducing specific mutations in the CUE domain to identify critical residues for ubiquitin binding

• Surface plasmon resonance (SPR): Measuring binding kinetics between purified CUE1 and ubiquitin/ubiquitinated substrates

• NMR spectroscopy: Characterizing structural changes in CUE1 upon binding to ubiquitin or ubiquitinated proteins

• Cross-linking mass spectrometry: Identifying interaction interfaces between CUE1 and its binding partners

These methods can be combined with salt stress conditions to determine how D. hansenii CUE1-substrate interactions may be adapted to function in high-salt environments, potentially revealing unique structural or functional adaptations not present in mesophilic yeasts.

How can comparative analysis of CUE1 across halotolerant and halophilic yeasts inform our understanding of protein quality control adaptation?

Comparative analysis of CUE1 from D. hansenii (tolerating up to 24% NaCl) with orthologs from non-halotolerant yeasts reveals evolutionary adaptations in protein quality control systems. Key research approaches include:

• Sequence comparison: Identifying conserved and divergent motifs that correlate with halotolerance

• Structural analysis: Determining if halotolerant yeasts share specific structural adaptations in CUE1

• Functional complementation: Testing if CUE1 from halotolerant species can function under high salt in non-halotolerant backgrounds

Analysis of SSU rDNA sequences has shown that D. hansenii's halotolerance is independent of phylogenetic affiliations , suggesting that adaptations in protein quality control systems like CUE1 may have evolved independently multiple times in response to environmental pressures.

What is the significance of post-translational modifications on D. hansenii CUE1 function?

Post-translational modifications (PTMs) of CUE1 likely regulate its activity in response to changing environmental conditions. Research approaches to study these include:

• Mass spectrometry: Identifying PTMs present on CUE1 isolated from D. hansenii grown under various salt conditions

• Site-directed mutagenesis: Creating non-modifiable variants of putative modification sites

• Phosphorylation-specific antibodies: Monitoring phosphorylation states under different stress conditions

• Kinase/phosphatase inhibitors: Determining effects on CUE1 function and ERAD efficiency

Preliminary studies suggest that phosphorylation of CUE1 increases under salt stress, potentially enhancing its ability to recruit ubiquitin-conjugating enzymes to the ER membrane and accelerating ERAD to manage damaged proteins.

How do structural features of D. hansenii CUE1 contribute to its function in high-salt environments?

Similar to structural studies on Gle1 from D. hansenii that revealed 13 α-helices with distinct secondary structural elements , CUE1's adaptation to high salt likely involves:

• Increased surface negative charge: Enhancing protein solubility and stability in high ionic strength

• Modified hydrophobic core: Maintaining structural integrity under osmotic stress

• Altered ubiquitin-binding interface: Potentially optimized to maintain interactions despite ionic interference

• Adaptive transmembrane domain: Possibly modified to function in membranes with altered lipid composition under salt stress

Structural analysis through crystallography or cryo-EM of D. hansenii CUE1 compared to mesophilic orthologs would provide valuable insights into these salt-adaptive features.

How can I address the poor expression of full-length D. hansenii CUE1 in heterologous systems?

Poor expression of full-length CUE1 is often due to the transmembrane domain. Recommended solutions include:

• Codon optimization: Adapting codons to the expression host preferences

• Fusion partners: Using MBP or SUMO tags to enhance solubility

• Expression conditions: Testing various temperatures (16-30°C) and induction strengths

• Membrane mimetics: Including detergents or lipids during purification

Expression of just the functional domains (partial CUE1) typically yields better results and is sufficient for many biochemical studies of ubiquitin binding and E2 interactions.

What strategies can improve the stability of D. hansenii transformants expressing recombinant CUE1?

Based on transformation challenges noted with D. hansenii , stability improvements include:

• Using endogenous ARS elements: Incorporating the isolated 0.4 kbp D. hansenii ARS

• Chromosomal integration: Moving from episomal to integrated expression

• Selection pressure: Maintaining appropriate antibiotic selection throughout culturing

• Stable host strains: Using D. hansenii J-26 or other strains known for genetic stability

• Optimized media composition: Including salt concentrations that maintain selective pressure for plasmid retention

These approaches address the instability issues previously observed in D. hansenii transformation experiments .

How can I distinguish between functional effects of CUE1 mutations and protein stability issues?

Differentiating functional defects from stability problems requires:

When interpreting results, a reduction in function without corresponding reduction in protein levels or localization changes suggests a specific functional defect rather than a general stability issue. This distinction is crucial for accurately characterizing the functional significance of specific CUE1 domains.