Recombinant Debaryomyces hansenii Autophagy-related protein 27 (ATG27), partial

What is Debaryomyces hansenii and why is it important for studying autophagy-related proteins?

Debaryomyces hansenii is a non-conventional yeast species with remarkable halotolerant (salt-tolerant) characteristics that make it valuable for industrial biotechnology applications. It can thrive in high-salt environments, with studies demonstrating improved performance under abiotic stresses when 1M NaCl is present . This yeast exhibits distinct physical and physiological features compared to the model yeast Saccharomyces cerevisiae, with genetic heterogeneity and chromosome polymorphism that may explain contradictory research findings .

The study of autophagy-related proteins in D. hansenii is particularly interesting because:

• D. hansenii has been historically difficult to study due to its resistance to typical techniques used for other yeast species, including cell wall disruption methods

• It has shown the ability to adapt and grow in the presence of different antibiotics, complicating selection processes

• Recent advances in genetic tools have made D. hansenii more accessible for molecular studies

• Comparison of autophagy pathways between different yeast species can reveal evolutionary conservation and specialization of these critical cellular processes

Understanding autophagy in D. hansenii could provide insights into how this stress-resistant organism maintains cellular homeostasis under extreme conditions.

What is the known function of ATG27 in yeast autophagy pathways?

ATG27 is a transmembrane protein that plays crucial roles in autophagy-related pathways in yeast. Based on studies primarily in S. cerevisiae and other model yeasts:

The proper functioning of ATG27 is essential for maintaining normal autophagic flux, which becomes especially important under stress conditions when autophagy is upregulated.

How do I identify orthologs of ATG27 in Debaryomyces hansenii?

To identify ATG27 orthologs in D. hansenii, researchers should employ a systematic bioinformatics approach:

• Sequence-based identification: Use protein-protein BLASTP searches against Debaryomyces hansenii (taxid:4959) using well-characterized ATG27 sequences from model organisms like S. cerevisiae .

• Conserved domain analysis: Examine the presence of characteristic domains of ATG27, including transmembrane domains and potential sorting signals.

• Synteny analysis: Compare the genomic context of putative ATG27 genes in D. hansenii with known ATG27 genes in other yeasts.

• Experimental validation: Once potential orthologs are identified, confirm their identity through:

  • PCR amplification using primers designed based on the predicted sequence

  • Expression analysis under autophagy-inducing conditions

  • Functional complementation studies (expressing D. hansenii ATG27 in S. cerevisiae atg27Δ strains)

One approach successfully used for other proteins involved a methodology where "respective orthologues were identified using protein-protein BLASTP against Debaromyces hansenii (taxid:4959)" . This approach could be similarly applied for ATG27 identification.

What are the structural characteristics of ATG27 and how might they differ in D. hansenii?

ATG27 possesses several important structural features that influence its function:

• Transmembrane topology: In S. cerevisiae, ATG27 was initially mischaracterized as a type II transmembrane protein, but further analysis revealed it to be a type I transmembrane protein with an N-terminal signal sequence .

• Sorting motifs: ATG27 contains a C-terminal tyrosine sorting motif (YSAV) that is crucial for its trafficking through the trans-Golgi network and endosomal compartments .

• Domain organization: The protein likely contains domains for interaction with other autophagy-related proteins, including ATG9.

In D. hansenii, structural characteristics might differ due to evolutionary divergence. When comparing D. hansenii proteins to S. cerevisiae:

• D. hansenii proteins often show sequence divergence while maintaining functional conservation

• The halotolerant nature of D. hansenii might influence protein structure to function optimally in high-salt environments

• Domain architecture is generally conserved across species, but specific motifs may vary

Comparative structural analysis between ATG27 from D. hansenii and S. cerevisiae could reveal adaptations specific to each organism's environmental niche and autophagy requirements.

What genetic modification techniques are available for studying ATG27 in D. hansenii?

Recent advances have made genetic modification of D. hansenii more accessible. The following methods can be applied to study ATG27:

• PCR-based gene targeting: A highly efficient method using PCR amplification with 50 bp flanking regions homologous to the target site . This technique achieved:

  • Integration efficiency >75% through homologous recombination

  • Successful targeting in wild-type isolates without requiring auxotrophic markers

• Selectable marker systems:

  • Heterologous markers conferring Hygromycin B or G418 resistance

  • Codon-adapted markers to account for D. hansenii's alternative codon usage

  • Previously developed auxotrophic systems using DhHIS4 or DhARG1 markers

• Expression systems:

  • Safe harbor genomic integration sites for heterologous gene expression

  • Promoter options including TEF1 from Arxula adeninivorans and native D. hansenii promoters

• In vivo DNA assembly: Recent work has demonstrated successful in vivo assembly of up to three DNA fragments with 30-bp homologous overlapping overhangs .

The choice of method depends on the specific experimental goals. For ATG27 studies, PCR-based gene targeting with fluorescent protein tags would be particularly useful for localization studies.

How does salt concentration affect ATG27 function and autophagy in D. hansenii?

D. hansenii is renowned for its halotolerance, and salt concentration significantly affects its cellular processes, likely including ATG27 function and autophagy:

• Growth optimization: D. hansenii strains show improved performance under abiotic stresses when grown in media containing 1M NaCl , suggesting potential enhancement of cellular processes including autophagy.

• Synergistic effects: Studies have documented "a positive and summative effect on growth in pH 4 and high salt content" , indicating that D. hansenii's stress response mechanisms, potentially including autophagy, may be optimized under these conditions.

• Strain-specific responses: Different D. hansenii strains exhibit varying degrees of sodium-induced optimization, with "some strains exerting a more notable induction by the presence of salt than the standard strain (CBS767)" .

While no direct studies on ATG27 function under varying salt conditions are available in the search results, the general pattern suggests that:

• Autophagy pathways may be upregulated under high-salt conditions as part of stress adaptation

• Protein trafficking, including ATG27-mediated processes, likely shows adaptation to function optimally in high-salt environments

• The interplay between pH and salt concentration may influence ATG27 trafficking routes

A methodological approach to studying this would involve expressing tagged ATG27 in D. hansenii and monitoring its localization and function under varying salt concentrations.

What specific experimental challenges exist when working with recombinant ATG27 in D. hansenii?

Working with recombinant ATG27 in D. hansenii presents several unique challenges:

• Genetic manipulation difficulties:

  • Historically, D. hansenii has been "intractable to typical techniques used for other yeast species"

  • "The slow development of genetic tools for D. hansenii" has hindered progress

  • Genetic heterogeneity across strains may produce variable results

• Antibiotic resistance issues:

  • D. hansenii has "the ability to adapt and grow in the presence of different antibiotics" , complicating selection strategies

  • Careful selection of appropriate markers is essential (Hygromycin B and G418 have proven effective )

• Protein expression optimization:

  • Codon optimization is necessary due to D. hansenii's alternative genetic code

  • The TEF1 promoter (from Arxula adeninivorans) has shown highest efficiency for heterologous protein expression

• Strain selection concerns:

  • Significant strain-specific differences exist within D. hansenii

  • For optimal experimental consistency, researchers should characterize multiple strains

• Environmental conditions:

  • Salt concentration dramatically affects D. hansenii physiology and should be carefully controlled

  • Growth rates are typically slower than S. cerevisiae, requiring longer experimental timeframes

A systematic approach to overcome these challenges would involve testing multiple expression systems, strains, and growth conditions to optimize recombinant ATG27 expression and function.

How can fluorescent protein tagging be optimized for visualizing ATG27 in D. hansenii?

Optimizing fluorescent protein tagging for ATG27 visualization in D. hansenii requires considerations specific to this yeast species:

• Choice of fluorescent protein:

  • YFP has been successfully expressed and secreted in D. hansenii

  • eYFP and eCFP have been used for protein visualization in D. hansenii

  • For colocalization studies, consider the mCherry-GFP combination, which has been successfully used in yeast autophagy studies

• Fusion strategy:

  • C-terminal tagging: ATG27-GFP fusions have been successfully used in S. cerevisiae

  • Consider both N- and C-terminal tagging options to determine which preserves function

  • Use flexible linkers (e.g., GGGGS) to minimize interference with protein function

• Expression optimization:

  • Promoter selection: The TEF1 promoter from A. adeninivorans has shown highest expression levels

  • Terminator efficiency: The CYC1 terminator from S. cerevisiae has performed well in D. hansenii

  • For secreted constructs, the α-mating factor signal peptide from S. cerevisiae has worked effectively

• Integration method:

  • PCR-based gene targeting with 50 bp homologous flanking regions has shown >75% efficiency

  • Consider genomic safe harbor sites for consistent expression levels

• Imaging considerations:

  • D. hansenii's smaller cell size may require higher-resolution imaging techniques

  • Differential Interference Contrast (DIC) has been successfully used for initial imaging experiments with D. hansenii

A systematic comparison of different tagging strategies would be advisable to determine optimal visualization conditions for ATG27 in D. hansenii.

What is the role of ATG27 in the trafficking of ATG9 in yeasts and how might this function in D. hansenii?

ATG27 plays a crucial role in ATG9 trafficking, which is essential for autophagosome formation:

In D. hansenii, these functions might be conserved but adapted to its unique physiology:

• The salt-tolerant nature of D. hansenii might require specialized membrane trafficking

• Higher salt concentrations could affect membrane dynamics and thus ATG9/ATG27 trafficking

• The potential synergy between salt tolerance mechanisms and autophagy pathways could result in unique adaptations

A comparative study between ATG27-mediated ATG9 trafficking in S. cerevisiae and D. hansenii would provide valuable insights into the evolution of this pathway.

How can the ATG27 tyrosine sorting motif be studied in D. hansenii?

The tyrosine sorting motif (YSAV) of ATG27 is crucial for its trafficking and function. To study this motif in D. hansenii:

• Mutational analysis approach:

  • Create site-directed mutations of the YSAV motif (complete deletion or point mutations)

  • Utilize the PCR-based gene targeting method with >75% efficiency in D. hansenii

  • Compare YSAV mutants with wild-type ATG27 for localization and function

• Localization studies:

  • Generate fluorescently tagged wild-type and mutant ATG27 proteins

  • Monitor localization to key compartments:

    • PAS (using RFP-APE1 as marker)

    • Vacuolar membrane (dependent on AP-3 pathway)

    • TGN/endosomal compartments

  • Quantify colocalization percentages with appropriate markers

• Functional assays:

  • Monitor ATG9 localization in cells expressing wild-type versus YSAV-mutant ATG27

  • Assess autophagy function using standard assays adapted for D. hansenii

  • Examine vacuolar delivery of ATG9 as a measure of missorting

• Comparative analysis:

  • Compare results with known effects in S. cerevisiae where "deletion of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments"

  • Investigate whether PAS localization remains normal as observed in S. cerevisiae

• Environmental influence assessment:

  • Test how salt concentration affects sorting motif function

  • Examine whether pH and salt synergistically affect ATG27 trafficking

Such studies would provide valuable insights into the conservation and potential adaptation of this important sorting mechanism in D. hansenii.

What methods are available for monitoring autophagy flux in D. hansenii?

Monitoring autophagy flux in D. hansenii requires adaptation of established techniques to this non-conventional yeast:

• Western blot analysis of autophagy markers:

  • Track processing of APE1 (aminopeptidase I) as demonstrated in the pulse-chase analysis of prAPE1 processing in S. cerevisiae

  • Monitor ATG8 lipidation (ATG8-PE) as an indicator of autophagosome formation

  • Use appropriate antibodies raised against orthologous proteins (approach validated for other proteins in D. hansenii)

• Fluorescent protein-based assays:

  • GFP-ATG8 processing assay: Monitor free GFP release by western blot

  • mCherry-GFP-ATG8 dual reporter: Track changes in red/green signal ratio

  • Use the TEF1 promoter from A. adeninivorans for optimal expression of reporter constructs

• Microscopy-based approaches:

  • Differential Interference Contrast (DIC) has been successful for initial imaging of D. hansenii

  • Track autophagosome formation using fluorescently tagged autophagy proteins

  • Monitor colocalization of ATG markers with the PAS (pre-autophagosomal structure)

• Electron microscopy techniques:

  • Transmission electron microscopy to visualize autophagic structures

  • Immunogold labeling of ATG proteins to track localization

  • May require optimization of fixation protocols for D. hansenii's cell wall

• Biochemical assays:

  • Measure activity of vacuolar enzymes released during autophagy

  • Track degradation of long-lived proteins as a measure of bulk autophagy

Researchers should validate these methods specifically for D. hansenii, as physiological differences from model yeasts may influence assay performance.

How do D. hansenii ATG proteins compare to those in S. cerevisiae and other yeasts?

Comparative analysis of ATG proteins across yeast species reveals important evolutionary patterns:

• Conservation and divergence patterns:

  • Core autophagy machinery is generally conserved across yeast species

  • Some species possess specialized ATG proteins, such as ATG35 which is "a micropexophagy-specific protein"

  • Certain ATG proteins show variable distribution: "Candida albicans, Debaryomyces hansenii, P. pastoris and Hansenula polymorpha have both Atg28 and Atg35, but S. cerevisiae, Candida glabrata [do not]"

• Functional conservation despite sequence divergence:

  • Orthologous ATG proteins can be identified using protein-protein BLASTP

  • Cross-species antibody recognition is possible: antibodies raised against S. cerevisiae proteins have successfully recognized D. hansenii orthologs

• Structural adaptations:

  • Membrane-associated ATG proteins may show adaptations related to D. hansenii's halotolerance

  • Sorting signals and trafficking motifs may be modified to function in different cellular environments

• Expression patterns:

  • Environmental stress responses differ between yeasts, potentially affecting ATG gene regulation

  • Salt stress may differentially regulate autophagy in halotolerant versus non-halotolerant yeasts

• Species-specific autophagy processes:

  • Selective autophagy pathways may show greater divergence than core machinery

  • The interplay between autophagy and halotolerance mechanisms represents an interesting area for exploration in D. hansenii

A comprehensive comparison would require systematic analysis of ATG protein sequences, structures, and functions across multiple yeast species, with particular attention to adaptations related to each species' ecological niche.

What are the best growth conditions for studying ATG27 function in D. hansenii?

Optimizing growth conditions is crucial for studying ATG27 function in D. hansenii, considering its unique physiological characteristics:

• Salt concentration optimization:

  • Include 1M NaCl in media as D. hansenii strains show "improved performance under abiotic stresses" in this condition

  • Consider creating a salt concentration gradient to determine optimal conditions for your specific strain

• pH considerations:

  • Test both neutral (pH 6) and acidic (pH 4) conditions

  • A "positive and summative effect on growth" has been observed "when pH 4 and high sodium was used"

• Carbon source selection:

  • D. hansenii can utilize various carbon sources including volatile fatty acids (VFAs)

  • Different carbon sources may affect autophagy induction differently

• Autophagy induction methods:

  • Rapamycin treatment has been used to induce autophagy in D. hansenii studies

  • Nutrient starvation (nitrogen or carbon) is another standard induction method

  • Optimize concentration and exposure time for consistent results

• Growth monitoring approach:

  • Use scattered light signal as an indicator for biomass formation in micro-fermentations

  • For comparative analysis, focus on "maximum specific growth rates restricted to the exponential growth phase"

The table below summarizes growth parameters observed in different D. hansenii strains under varying conditions:

Strain-specific optimization is recommended, as significant intraspecies variation has been documented in D. hansenii.

How can protein-protein interactions involving ATG27 be studied in D. hansenii?

Investigating protein-protein interactions involving ATG27 in D. hansenii requires specialized approaches:

• Yeast two-hybrid (Y2H) systems:

  • Y2H library screening has been successfully used to identify interaction partners of autophagy proteins in yeasts

  • Adapt existing Y2H systems for D. hansenii by using appropriate selectable markers

  • Consider developing a D. hansenii-specific Y2H library

• Co-immunoprecipitation approaches:

  • Express epitope-tagged versions of ATG27 (e.g., HA, FLAG, or Myc tags)

  • Use crosslinking agents to capture transient interactions

  • Optimize lysis conditions for D. hansenii's robust cell wall

  • Identify interaction partners through mass spectrometry

• Fluorescence-based interaction detection:

  • Bimolecular Fluorescence Complementation (BiFC)

  • Fluorescence Resonance Energy Transfer (FRET)

  • Split-GFP or split-luciferase complementation assays

  • Utilize the optimized fluorescent protein expression systems for D. hansenii

• Proximity labeling techniques:

  • BioID or TurboID fusion proteins to identify proximal proteins

  • APEX2-based proximity labeling

  • May require codon optimization for expression in D. hansenii

• Colocalization studies:

  • Dual-color fluorescence microscopy with differentially tagged proteins

  • Quantitative colocalization analysis under various conditions

  • Examples from other studies: "33–43% Atg9 puncta exhibited Atg27 fluorescence" in colocalization experiments

These methods should be optimized for D. hansenii's specific characteristics, particularly its cell wall properties and growth conditions. The high-efficiency PCR-based gene targeting method (>75% efficiency) can be used to generate the necessary tagged protein constructs for these interaction studies.

What is the current understanding of how ATG27 regulates selective autophagy in yeasts?

The role of ATG27 in selective autophagy pathways is complex and continues to evolve:

• ATG27 in different autophagy pathways:

  • Initially thought to be specific to the Cvt pathway, ATG27 is now known to be "required for efficient bulk autophagy and pexophagy"

  • ATG27 function affects multiple selective autophagy pathways including pexophagy (peroxisome degradation)

• Selective autophagy mechanisms:

  • Selective autophagy requires specific receptors to recognize cargo for degradation

  • In ribosome-selective autophagy, "Rsa1p functions as the ribosome-specific receptor"

  • ATG27 may facilitate the trafficking of various selective autophagy receptors

• ATG27 in cargo recognition and trafficking:

  • ATG27 helps form "Atg9 transport vesicles destined for the site of autophagosome formation"

  • It plays a role in retaining "Atg9 in endosomal reservoirs that can be mobilized during autophagy"

  • This reservoir function may be particularly important for rapid upregulation of selective autophagy

• Regulation by post-translational modifications:

  • Phosphorylation and other modifications likely regulate ATG27 function

  • The tyrosine sorting motif (YSAV) suggests potential regulation by tyrosine kinases/phosphatases

• Environmental regulation:

  • In D. hansenii, high salt and specific pH conditions alter cellular processes

  • These conditions likely affect selective autophagy pathways differently than bulk autophagy

  • The halotolerant nature of D. hansenii may involve specialized autophagy regulation mechanisms

The micropexophagy-specific protein ATG35 represents an example of specialized factors involved in selective autophagy that might interact with the core machinery including ATG27. Interestingly, "both deletion and overexpression of the ATG35 gene specifically inhibited MIPA formation, but not pexophagosome formation or macropexophagy" , highlighting the complexity of selective autophagy regulation.