Recombinant Debaryomyces hansenii Autophagy-related protein 23 (ATG23), partial

What is the structure and function of ATG23 in D. hansenii?

ATG23 in D. hansenii is a peripheral membrane protein involved in autophagy, similar to its well-characterized homolog in Saccharomyces cerevisiae. The protein exists primarily as a homodimer, with dimerization facilitated by a putative amphipathic helix. Structural analysis using small-angle X-ray scattering reveals an extended rod-like structure spanning approximately 320 Å .

Functionally, ATG23 is essential for the cytoplasm-to-vacuole targeting (Cvt) pathway and efficient non-selective autophagy, though it is not required for pexophagy. Its primary roles include:

• Membrane tethering through direct interaction with lipid bilayers

• Interaction with Atg9, facilitating vesicle formation

• Localization to the pre-autophagosomal structure (PAS) and other cytosolic punctate compartments

How does D. hansenii ATG23 differ from its homologs in other yeast species?

While the fundamental function of ATG23 is conserved across yeast species, D. hansenii's extreme halotolerance suggests possible adaptations in its autophagy machinery. In contrast to S. cerevisiae, which has stable "capped" Atg13 not requiring Atg101 for stabilization, D. hansenii possesses different autophagy complex components. D. hansenii, along with Candida albicans and Hansenula polymorpha, has Atg28 but lacks Atg29 and Atg31, which are present in S. cerevisiae .

In S. cerevisiae, Atg23 is required for the Cvt pathway and contributes to non-selective autophagy, but is dispensable for pexophagy. Research comparing atg23Δ mutants in S. cerevisiae showed that these cells remain viable during starvation for several days before beginning to die after approximately 6-8 days, displaying an intermediate starvation-resistance phenotype . Similar comparative studies with D. hansenii would be valuable to understand species-specific functions.

What experimental systems are available for studying D. hansenii ATG23?

Several experimental systems have been developed for studying D. hansenii ATG23:

• CRISPR/Cas9 genome editing: Recent development of efficient single- and dual-guide CRISPR systems allows for markerless genome editing of D. hansenii with high efficiency (up to 95%). The single-guide system permits mutation of genes or regulatory elements, while the dual-guide system enables efficient deletion of genomic loci .

• PCR-based gene targeting: An efficient method using PCR-based amplification that extends a heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%) .

• In vivo DNA assembly: Recently demonstrated feasibility of performing in vivo DNA assembly in D. hansenii. Up to three different DNA fragments containing 30-bp homologous overlapping overhangs can be co-transformed into the yeast and fused in the correct order in a single step .

What are the optimal conditions for expressing recombinant D. hansenii ATG23?

For optimal expression of recombinant D. hansenii ATG23, several factors should be considered:

Expression System Selection:

• For homologous expression, the TEF1 promoter (from Arxula adeninivorans) combined with the CYC1 terminator has shown the highest production of recombinant proteins in D. hansenii .

Culture Conditions:

• Temperature: Optimal growth at 25°C

• Media: YPD (1% yeast extract, 2% peptone, 2% dextrose)

• Salt concentration: D. hansenii thrives in high-salt conditions (up to 6% NaCl), which can be utilized to enhance recombinant protein expression while inhibiting other microorganisms

Purification Strategy:
For membrane-associated proteins like ATG23:

• Cell disruption in buffer containing protease inhibitors

• Differential centrifugation to separate membrane fractions

• Solubilization with mild detergents

• Affinity chromatography using tagged recombinant protein

How can the dimerization of D. hansenii ATG23 be assessed experimentally?

The dimerization of D. hansenii ATG23 can be assessed using several complementary approaches:

Coimmunoprecipitation (CoIP) Assay:

• Express differentially tagged versions of ATG23 (e.g., ATG23-MYC and ATG23-PA)

• Perform immunoprecipitation with one tag

• Detect the presence of the other tag in the precipitate by Western blotting

This approach has been successfully used to demonstrate ATG23 dimerization in other yeast species .

Native PAGE Analysis:
Compare the migration patterns of wild-type ATG23 and mutant versions with disrupted dimerization motifs. Wild-type dimeric ATG23 migrates slower than monomeric mutants .

What approaches can be used to study D. hansenii ATG23 interactions with Atg9?

To study the interactions between D. hansenii ATG23 and Atg9, the following approaches are recommended:

Yeast Two-Hybrid (Y2H) Analysis:

• Clone ATG23 and ATG9 into appropriate Y2H vectors

• Transform into a suitable yeast reporter strain

• Assess interaction by monitoring reporter gene expression

• Map interaction domains using truncated constructs

Bimolecular Fluorescence Complementation (BiFC):

• Fuse ATG23 and ATG9 to complementary fragments of a fluorescent protein

• Express in D. hansenii

• Visualize interaction by fluorescence microscopy when the fragments come together

Pull-Down Assays:

• Express recombinant ATG23 with an affinity tag

• Immobilize on appropriate resin

• Incubate with D. hansenii cell lysate

• Analyze bound proteins by SDS-PAGE and mass spectrometry

Co-localization Studies:

• Generate strains expressing fluorescently tagged ATG23 and ATG9

• Visualize by fluorescence microscopy

• Quantify co-localization under different conditions (normal growth, starvation, stress)

How does salt stress influence ATG23 function in the halotolerant D. hansenii?

D. hansenii exhibits remarkable halotolerance, and salt stress significantly impacts its cellular processes, including autophagy. The influence of salt stress on ATG23 function involves several regulatory mechanisms:

Transcriptional Regulation:
Salt stress in D. hansenii activates the DhHog1 MAP kinase pathway, which plays a critical role in regulating stress responses. Analysis of a Dhhog1Δ mutant has shown that DhHog1 is involved in regulating stress response under both H₂O₂ and NaCl conditions . Since ATG23 is part of the autophagy machinery, its expression may be similarly regulated under salt stress.

Post-translational Modifications:
Under salt stress, increased phosphorylation of various proteins has been observed in D. hansenii. Two ATPase-coupled cation transmembrane transporters, DEHA2G09108p and DEHA2C02552p, show the highest significance among all proteins upregulated in saline treatments . These changes in the cellular protein phosphorylation landscape likely affect ATG23 function, either directly or indirectly.

Membrane Dynamics:
Salt stress induces changes in membrane composition and fluidity in D. hansenii, which may influence ATG23's membrane interactions and its ability to facilitate vesicle formation. The upregulation of proteins involved in O-glycosylation, which is essential for cell wall rigidity, indicates significant membrane remodeling under salt stress .

Energy Metabolism:
Salt stress requires enhanced provision of ATP for transporter functioning, increasing cellular energy demand . This energy redistribution likely impacts energy-dependent processes like autophagy, potentially affecting ATG23-dependent pathways.

What is the role of ATG23 in D. hansenii's response to oxidative stress?

D. hansenii shows remarkable tolerance to oxidative stress, with high catalase activity from DhCTA and DhCTT genes playing a significant role. The relationship between ATG23 and oxidative stress response involves several interacting pathways:

Oxidative Stress Signaling:
D. hansenii responds to H₂O₂-induced oxidative stress through the DhHog1 MAP kinase pathway, which regulates catalase expression . Experimental evidence indicates that exposure to H₂O₂ shock reduces cell viability while transiently increasing catalase expression and activity. Chromatin organization analysis reveals low nucleosome occupancy in promoter regions of catalase genes, correlating with active gene expression .

Autophagy Induction:
Oxidative stress induces autophagy as a protective mechanism. In D. hansenii, this likely involves ATG23-dependent pathways. Research in S. cerevisiae has shown that autophagy proteins, including proteasome components, contribute to extremophilic properties . In D. hansenii, mutation of proteasome subunits results in sensitivity to geno- and proteotoxic stresses as well as high salinity and osmolarity .

Comparative Stress Response Data:
The table below shows the relative contributions (%) of various organisms to ethanol production under stress conditions, indicating how D. hansenii participates in metabolism under stress:

This data suggests that D. hansenii's metabolic contribution doubles under stress conditions, indicating significant metabolic adaptation in which ATG23-mediated autophagy may play a role .

What potential applications exist for manipulating ATG23 function in D. hansenii biotechnology?

Manipulating ATG23 function in D. hansenii offers several promising biotechnological applications:

Enhanced Stress Tolerance:
Modifying ATG23 expression could potentially enhance D. hansenii's already impressive stress tolerance. This could produce strains with improved ability to function in industrial fermentation processes under harsh conditions. Since ATG23 is involved in autophagy but not essential for survival, its careful manipulation could fine-tune cellular responses without compromising viability.

Improved Recombinant Protein Production:
D. hansenii has been established as a promising cell factory for recombinant protein production, particularly in salt-rich industrial side-streams . Enhancing ATG23-dependent autophagy pathways could help manage cellular stress during high-level protein expression, potentially increasing yields of target proteins.

Anti-Candida Applications:
D. hansenii produces killer toxins effective against pathogenic Candida species. In studies examining 42 strains of D. hansenii isolated from cheese, many exhibited killer activity against C. albicans and C. tropicalis . Manipulating ATG23 to enhance cellular fitness under production conditions could potentially increase killer toxin yield.

Optimization Strategy:
For optimizing ATG23 function, a combined genomic and transcriptomic approach is recommended:

• CRISPR/Cas9-mediated precise modification of ATG23 and interacting partners

• Screening of modified strains under relevant industrial conditions

• Multi-omics analysis of high-performing strains to understand mechanism of enhancement

What are common challenges in purifying recombinant D. hansenii ATG23, and how can they be addressed?

Challenge: Protein Solubility Issues
ATG23 is a peripheral membrane protein that may have solubility issues during purification.

Solution:

• Use mild detergents like CHAPS or n-dodecyl-β-D-maltoside (DDM) in purification buffers

• Test different salt concentrations (100-500 mM NaCl) to enhance solubility

• Add 10% glycerol to stabilize the protein

• Consider fusion tags that enhance solubility (e.g., MBP or SUMO)

Challenge: Maintaining Proper Folding
Since ATG23 forms dimers through a specific amphipathic helix, maintaining proper folding is crucial.

Solution:

• Avoid harsh denaturation conditions during purification

• Use native purification methods when possible

• Include reducing agents like DTT or β-mercaptoethanol to maintain proper disulfide bonds

• Purify at 4°C to minimize protein degradation and misfolding

Challenge: Low Expression Levels
Recombinant expression of heterologous proteins can be challenging in D. hansenii.

Solution:

• Optimize codon usage for D. hansenii

• Test different promoters; the TEF1 promoter from Arxula adeninivorans has shown good results

• Growth in media with 6% NaCl can enhance expression while inhibiting contaminating microorganisms

• Consider using protease-deficient strains to minimize degradation

Challenge: Protein Degradation
ATG23 may be susceptible to proteolytic degradation during purification.

Solution:

• Include a comprehensive protease inhibitor cocktail in all buffers

• Minimize time between cell lysis and purification

• Keep samples cold throughout the purification process

• Consider adding 1-2 mM EDTA to inhibit metalloproteases

How can researchers differentiate between functional effects of ATG23 and other autophagy-related proteins in D. hansenii?

Differentiating the specific roles of ATG23 from other autophagy proteins requires strategic experimental approaches:

Genetic Approaches:

• Precise gene deletions and mutations: Use CRISPR/Cas9-mediated genome editing to create clean deletions or point mutations in ATG23 and other autophagy genes . The single-guide CRISPR system allows high-efficiency (up to 95%) mutation of genes in D. hansenii.

• Complementation studies: Express wild-type or mutant ATG23 in atg23Δ strains to verify phenotypes are specifically due to ATG23 function.

• Double mutant analysis: Create double knockouts (e.g., atg23Δ atg9Δ) to study genetic interactions and pathway relationships.

Biochemical Approaches:

• Protein-protein interaction mapping: Use immunoprecipitation followed by mass spectrometry to identify ATG23-specific interacting partners versus those common to multiple ATG proteins.

• Membrane tethering assays: Since ATG23 has vesicle tethering activity, perform in vitro vesicle tethering assays with purified ATG23 and compare with other autophagy proteins.

Cellular and Microscopy Approaches:

• Differential localization: Use fluorescently tagged proteins to track the localization of ATG23 versus other autophagy proteins under different conditions.

• Selective autophagy assays: ATG23 is required for the Cvt pathway but dispensable for pexophagy . This differential requirement can be used to distinguish ATG23 functions.

Pathway-Specific Markers:
Monitor specific autophagy processes using established markers:

• Cvt pathway: Track prApe1 processing (ATG23-dependent)

• Pexophagy: Monitor degradation of the peroxisomal thiolase Fox3 (ATG23-independent)

• General autophagy: Use the Pho8Δ60 alkaline phosphatase assay (partially ATG23-dependent)

What considerations should be taken into account when designing ATG23 mutants in D. hansenii?

When designing ATG23 mutants in D. hansenii, several key considerations should be addressed:

Functional Domains:

Technical Considerations:

• Codon optimization: D. hansenii has a distinct codon usage bias. When introducing mutations, maintain codon optimization for efficient expression.

• Selection markers: Choose appropriate selection markers compatible with your D. hansenii strain. Recent developments include completely heterologous selection markers that allow for efficient targeted genome modification .

• Safe landing sites: For heterologous expression of mutant variants, consider using identified safe landing sites in the D. hansenii genome .

Experimental Design:

• Control mutations: Include conservative mutations (same amino acid charge/size) as controls.

• Comprehensive mutation series: Design a series of mutations ranging from subtle to severe to capture the full spectrum of functional impacts.

• Tagged versions: Include versions with C-terminal or N-terminal tags for detection, but verify these don't interfere with function.

Phenotypic Validation:
Ensure mutants are evaluated across multiple assays to fully characterize functional impacts:

• Protein-protein interactions (particularly with ATG9)

• Membrane binding and vesicle tethering capabilities

• Subcellular localization patterns

• Autophagy flux measurements

• Stress response phenotypes (especially salt and oxidative stress)

How might the unique properties of D. hansenii ATG23 be exploited for therapeutic applications?

D. hansenii ATG23's role in autophagy, combined with the yeast's unique properties, suggests several potential therapeutic applications:

Anti-Candida Therapeutics:
D. hansenii produces killer toxins effective against pathogenic Candida species. Studies have isolated 42 strains of D. hansenii from cheese with killer activity against C. albicans and C. tropicalis . By understanding how ATG23-mediated autophagy affects killer toxin production, enhanced therapeutic strains could be developed. Recent studies show killer toxin production optimized in YMB medium containing NaCl (6%) and DMSO (1000 ppm) at pH 4.0 and 20°C .

Inflammatory Bowel Disease Interventions:
Research has revealed that D. hansenii is enriched in inflamed tissue from Crohn's disease patients and contributes to delayed wound healing in mice . By targeting ATG23 to modulate D. hansenii's inflammatory properties, novel therapeutic approaches could be developed. D. hansenii stimulates upregulation of Ccl5 in macrophages, interfering with healing of wounded intestine; this suggests Ccl5 as a potential therapeutic target .

Probiotic Applications:
Studies have shown that D. hansenii combined with Qiweibaizhu powder extract (QCD) has synergistic effects in recovering gut microbiota after antibiotic-associated diarrhea . The QCD treatment increased species richness and diversity of gut microbiota, though not to original levels. By engineering ATG23 to enhance D. hansenii's stress resistance and survival in the gastrointestinal tract, more effective probiotic formulations could be developed.

Drug Delivery Systems:
ATG23's membrane-binding and vesicle-tethering properties could potentially be exploited to develop novel drug delivery systems. As a peripheral membrane protein capable of vesicle tethering through direct membrane interaction , ATG23 or its derivatives might serve as components in engineered vesicles for targeted drug delivery.

What insights might comparative studies of ATG23 across different yeast species provide about autophagy evolution?

Comparative studies of ATG23 across yeast species could yield valuable insights into autophagy evolution:

Structural Conservation and Divergence:
ATG23 maintains core functions across yeast species but shows adaptations in different environmental niches. In S. cerevisiae, Atg23 is essential for the Cvt pathway and contributes to efficient autophagy . Examining structural differences in ATG23 from D. hansenii could reveal adaptations related to extreme salt tolerance and stress resistance.

Autophagy Complex Composition:
Different yeasts have evolved distinct autophagy complex components. While S. cerevisiae has Atg29 and Atg31, D. hansenii, along with Candida albicans and Hansenula polymorpha, possesses Atg28 but lacks these components . This suggests divergent evolutionary paths in autophagy machinery assembly.

Functional Specialization:
ATG23's role varies somewhat across species. In S. cerevisiae, atg23Δ mutants show an intermediate starvation-resistance phenotype, remaining viable for 6-8 days before dying . Comparing the precise functions of ATG23 across species could reveal how autophagy has been adapted for different ecological niches.

Interaction Networks:
The protein-protein interaction networks of ATG23 likely differ between yeast species. In S. cerevisiae, Atg23 interacts with Atg9 and affects its trafficking . Comparative studies could map how these interaction networks have evolved and identify species-specific partners that reflect unique autophagy adaptations.

How can advanced imaging techniques enhance our understanding of D. hansenii ATG23 dynamics in vivo?

Advanced imaging techniques offer powerful approaches to study D. hansenii ATG23 dynamics:

Super-Resolution Microscopy:
Techniques like Stimulated Emission Depletion (STED) or Stochastic Optical Reconstruction Microscopy (STORM) can visualize ATG23 localization with nanometer precision, revealing details of its distribution at the pre-autophagosomal structure (PAS) and other punctate compartments that conventional microscopy cannot resolve.

Live-Cell Time-Lapse Imaging:
Using fluorescently tagged ATG23 constructs with the recently developed gene editing tools for D. hansenii , researchers can track ATG23 dynamics in real-time during autophagy induction. This approach can reveal:

• The kinetics of ATG23 recruitment to the PAS

• The dynamics of ATG23-positive vesicles

• The interaction timing with ATG9 and other autophagy proteins

Förster Resonance Energy Transfer (FRET):
FRET microscopy can detect protein-protein interactions in living cells with high sensitivity. By tagging ATG23 and interaction partners (e.g., ATG9) with appropriate fluorophore pairs, researchers can:

• Map interaction domains in vivo

• Determine the spatial and temporal dynamics of these interactions

• Assess how environmental factors (salt, oxidative stress) affect these interactions

Correlative Light and Electron Microscopy (CLEM):
CLEM combines the specificity of fluorescence microscopy with the ultrastructural detail of electron microscopy. This approach allows researchers to:

• Precisely locate ATG23-positive structures at the ultrastructural level

• Examine membrane tethering events mediated by ATG23

• Visualize the formation of autophagosomes in relation to ATG23 localization

Lattice Light-Sheet Microscopy:
This technique enables long-term 3D imaging with minimal phototoxicity, allowing researchers to:

• Track ATG23 through complete autophagy cycles

• Observe rare or transient events in ATG23 dynamics

• Quantify the movement patterns of ATG23-positive structures in 3D space