Recombinant Debaryomyces hansenii Cap-associated protein CAF20 (CAF20)

What is CAF20 and what is its function in Debaryomyces hansenii?

CAF20 is a cap-associated protein that functions as a 4E-binding protein (4E-BP). It regulates translation by binding to eukaryotic initiation factor 4E (eIF4E), preventing its interaction with eIF4G, thereby inhibiting translation initiation of specific mRNAs. Based on studies in Saccharomyces cerevisiae, CAF20 mediates both eIF4E-dependent and independent translational repression . In D. hansenii, CAF20 likely plays similar roles in translational regulation, potentially with adaptations related to the yeast's unique halotolerant nature.

How does the structure of CAF20 relate to its function?

Based on studies in S. cerevisiae, CAF20 contains a short motif within its amino-terminal region (NTD) that interacts with eIF4E. Multiple elements in the N-terminus are required for ribosome interaction . CAF20 functions as a monomer rather than a homodimer when interacting with its binding partners . The protein appears to have distinct domains responsible for:

• eIF4E binding via the N-terminal domain

• Ribosome interaction through multiple N-terminal elements

• Potential 3' UTR binding capabilities for eIF4E-independent regulation

What are effective methods for expressing recombinant CAF20 in D. hansenii?

For expressing recombinant CAF20 in D. hansenii, the following protocol is recommended:

• PCR-based gene targeting approach: Use PCR amplification that extends a heterologous selectable marker with 50 bp flanks identical to the target site in the genome. This method achieves integration through homologous recombination at high frequency (>75%) .

• Selectable markers: Use newly developed selectable marker cassettes that confer Hygromycin B or G418 resistance to D. hansenii transformants .

• Safe harbor site integration: For strains where gene disruption shows no phenotypic effects due to persistence of wild-type gene copies, use a safe genome harbor site for integrated expression of heterologous genes .

The protocol can be summarized in the following workflow:

How can I create CAF20 mutants to study specific functions in D. hansenii?

To create functional CAF20 mutants:

• eIF4E-binding mutants: Modify the N-terminal domain containing the eIF4E-binding motif, similar to the m2 mutation approach used in S. cerevisiae studies that disrupts eIF4E binding .

• Ribosome-binding mutants: Target the extended N-terminal regions required for ribosome association, which are distinct from the eIF4E-binding motif .

• Mutant verification: Confirm mutant properties through:

  • Co-immunoprecipitation with eIF4E and ribosomal proteins

  • Polysome profile analysis

  • Functional complementation assays

What methods are most effective for studying CAF20-RNA interactions?

Based on S. cerevisiae research, the following approaches are recommended:

• RNA immunoprecipitation sequencing (RIP-seq): Immunoprecipitate CAF20-FLAG and sequence associated RNAs to identify target transcripts .

• Computational motif analysis: Analyze sequence features of CAF20-bound mRNAs, particularly focusing on 3' UTR regions that may contain binding motifs for eIF4E-independent regulation .

• Reporter assays: Test the functional significance of identified RNA motifs using heterologous reporter constructs with wild-type and mutated binding sites .

• Polysome profiling: Compare the polysome association of potential target mRNAs in wild-type and CAF20 mutant strains to assess translational effects .

How does CAF20 interact with eIF4E and what methods best characterize this interaction?

In S. cerevisiae, CAF20 binds to eIF4E through a canonical binding motif in its N-terminal region. To characterize this interaction in D. hansenii:

• Co-immunoprecipitation: Express tagged versions of both proteins and perform pull-down experiments followed by western blotting .

• Mutational analysis: Create point mutations in the predicted eIF4E-binding motif and assess binding disruption .

• Functional consequences: Compare translational profiles between wild-type CAF20 and eIF4E-binding deficient mutants to identify eIF4E-dependent targets .

• Structural analysis: Use computational modeling based on known eIF4E-4E-BP structures to predict interaction interfaces specific to D. hansenii proteins.

What is known about CAF20's interaction with ribosomes?

Studies in S. cerevisiae have revealed that:

• CAF20 associates with ribosomes independently of its eIF4E-binding capability .

• This interaction requires multiple elements in the N-terminal region distinct from the eIF4E-binding motif .

• Mass spectrometry analysis identified potential ribosomal protein partners including Rps5, Rps24, Rps27, Rpl10, Rpl27, and Rpl30 .

• Crosslinking experiments suggest these ribosomal proteins are located around the interface of the 40S and 60S subunits .

To study this in D. hansenii, similar approaches could be employed, accompanied by polysome gradient analysis under various growth conditions relevant to D. hansenii's natural environment.

How does CAF20 recognize and bind to specific mRNAs?

Based on S. cerevisiae studies, CAF20 appears to recognize mRNAs through two distinct mechanisms:

• eIF4E-dependent targeting (approximately 75% of targets): CAF20 binds to eIF4E which is bound to the 5' cap of mRNAs .

• eIF4E-independent targeting (approximately 25% of targets): CAF20 binds directly to a shared motif in the 3' UTR of target mRNAs .

A core set of over 500 Caf20p-interacting mRNAs has been identified in S. cerevisiae, with targets involved in transcription and cell cycle processes . Similar mechanisms likely exist in D. hansenii, potentially with specialization for stress response pathways.

What are the characteristics of CAF20 target mRNAs?

In S. cerevisiae, CAF20 targets have the following characteristics:

Research in D. hansenii should focus on identifying whether similar target preferences exist, and whether additional targets related to osmotic and salt stress response are present.

How can the translational repression activity of CAF20 be measured?

To quantify CAF20's repression activity:

• Polysome profiling: Compare the distribution of specific mRNAs in polysome gradients between wild-type and CAF20 deletion or mutant strains .

• Reporter assays: Construct reporters containing known or suspected CAF20 target sequences (particularly 3' UTRs) fused to reporter genes and measure expression levels .

• Ribosome profiling: Perform genome-wide analysis of ribosome-protected fragments to identify differentially translated mRNAs in the presence or absence of CAF20.

• Proteomics: Use quantitative proteomics to identify proteins whose expression is altered by CAF20 deletion or mutation .

How might CAF20 contribute to D. hansenii's halotolerance?

Given D. hansenii's exceptional halotolerance, CAF20 may play specialized roles:

• Stress-specific translation regulation: CAF20 could selectively repress or enhance translation of specific mRNAs during salt stress, similar to stress-specific translational regulation observed in other yeasts.

• Integration with salt-response pathways: CAF20 may interact with D. hansenii's specialized salt-response machinery, including transporters like DhPma1, DhVma2, DhNha1, DhEna1, DhNhx1, DhKha1, DhHak1, and DhTrk1 .

• Phosphorylation-dependent regulation: Salt stress may alter CAF20 phosphorylation status, modifying its activity or target specificity, as observed with other translational regulators.

Experimental approaches to investigate these possibilities include:

• Comparing wild-type and CAF20 mutant growth under various salt conditions

• Identifying CAF20-bound mRNAs during normal and high-salt conditions

• Analyzing CAF20 phosphorylation status during salt stress

What cellular processes are likely regulated by CAF20 in D. hansenii?

Based on S. cerevisiae studies and D. hansenii's unique physiology:

• Cell cycle and growth control: CAF20 deletion in S. cerevisiae affects growth during the switch from glucose to respiratory medium, especially at low temperatures .

• Metal ion homeostasis: S. cerevisiae strains with CAF20 show increased sensitivity to clioquinol drug and excess CuSO4 , suggesting a role in metal homeostasis that may be relevant to D. hansenii's environmental adaptations.

• Stress response pathways: Unlike in some regulatory systems, S. cerevisiae CAF20 was found not to be a target of the TOR pathway , but may participate in other stress response mechanisms particularly relevant to D. hansenii's extreme environments.

• Lipid metabolism: Given D. hansenii's oleaginous nature , CAF20 may regulate genes involved in lipid biosynthesis and accumulation.

How can CRISPR-Cas9 be adapted for CAF20 modification in D. hansenii?

While the search results don't directly address CRISPR-Cas9 use in D. hansenii, the system could be adapted based on principles from the PCR-based gene targeting approach:

• Design considerations:

  • Optimize codon usage of Cas9 for D. hansenii expression

  • Select promoters that function efficiently in D. hansenii

  • Design gRNAs targeting CAF20 with high specificity

• Delivery method:

  • Integrate the system using the high-efficiency homologous recombination approach with 50 bp homology flanks

  • Use established selectable markers (HygR or KanR)

• Verification strategies:

  • PCR-based genotyping

  • Sequencing of edited regions

  • Western blot for protein expression/deletion confirmation

How might CAF20 manipulation enhance biotechnological applications of D. hansenii?

D. hansenii has biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe . CAF20 manipulation could enhance these properties:

• Improved stress tolerance: Modifying CAF20 to optimize translational response during stress could enhance D. hansenii's performance in challenging industrial conditions.

• Enhanced heterologous protein expression: Creating CAF20 mutants that selectively reduce its repression of specific mRNAs could improve production of recombinant proteins.

• Metabolic engineering: Targeted modification of CAF20 to alter translation of key metabolic enzymes could enhance lipid production or other valuable metabolites.

• Bioprocess optimization: Engineering CAF20 variants responsive to specific industrial conditions could create adaptable strains that optimize performance based on environmental parameters.

What comparative insights can be gained by studying CAF20 across different yeast species?

Comparative analysis between D. hansenii CAF20 and homologs in other yeasts may reveal:

• Evolutionary adaptations: Sequence and functional differences that correlate with each species' ecological niche.

• Conserved mechanisms: Core functions preserved across diverse yeast lineages representing fundamental aspects of translational control.

• Species-specific targets: Differences in mRNA targeting that reflect specialized metabolic or stress response pathways.

• Structural variations: Modifications to protein interaction domains that may confer different regulatory properties.

This comparative approach could be particularly valuable given D. hansenii's extreme halotolerance compared to conventional model yeasts like S. cerevisiae.