Recombinant Ashbya gossypii Autophagy-related protein 23 (ATG23)

What is the structural organization of ATG23 in Ashbya gossypii and how does it relate to its function?

ATG23 in A. gossypii exists primarily as a homodimer, with dimerization facilitated by a putative amphipathic helix. Small-angle X-ray scattering (SAXS) analysis reveals that ATG23 possesses an extended rod-like structure spanning approximately 320 Å .

The putative amphipathic helix contains conserved hydrophobic residues (notably L171, I182, L189) that are essential for dimerization. When these residues are mutated to alanines (creating the Atg23[LIL] mutant), dimerization is completely disrupted, as demonstrated by coimmunoprecipitation (coIP) assays .

This dimerization is critical for ATG23's function in membrane tethering and autophagy. Research shows that mutation of the hydrophobic face of the putative amphipathic helix completely prevents dimer formation, leading to severely impaired subcellular localization, vesicle tethering, ATG9 binding, and ultimately reduced autophagic efficiency .

Recommended methodological approaches:

How does ATG23 contribute to membrane dynamics during autophagy in A. gossypii?

ATG23 plays a crucial role in membrane dynamics by binding directly to membranes through electrostatic interactions and facilitating vesicle tethering. Significantly, this tethering function occurs in an ATG9-independent manner .

Research demonstrates that ATG23 can bind to membranes and tether vesicles on its own, which is essential for autophagosome formation. The dimerization of ATG23 is critical for this function - when dimerization is disrupted through mutations in the amphipathic helix, membrane tethering ability is severely compromised .

In the absence of ATG23, cargo molecules like prApe1 (aminopeptidase I) are correctly recruited to the pre-autophagosomal structure (PAS), but this structure is unable to give rise to Cvt vesicles . This indicates that ATG23 is not involved in cargo selection but rather in the membrane dynamics required for vesicle formation.

Experimental approaches to study ATG23's membrane interactions:

• Liposome binding assays with purified recombinant ATG23

• Fluorescence-based membrane binding assays using environment-sensitive dyes

• Membrane tethering assays comparing wild-type ATG23 with dimerization-deficient mutants

• FRET-based measurements to monitor ATG23 oligomerization upon membrane binding

What are the phenotypic consequences of ATG23 deletion in Ashbya gossypii and related yeasts?

ATG23 deletion results in distinctive phenotypes related to autophagy pathways. Based on studies in related yeast species, atg23Δ mutants exhibit:

• Defective Cvt pathway: ATG23 deletion prevents the cytoplasm-to-vacuole targeting pathway from functioning properly during nutrient-rich conditions .

• Impaired but not abolished autophagy: Interestingly, atg23Δ cells retain partial autophagic capacity during starvation conditions. Quantitative measurements show that atg23Δ cells induce approximately 40% of the Pho8Δ60 activity (a marker for autophagy) compared to wild-type cells during nitrogen starvation .

• Intermediate starvation resistance: While autophagy-defective mutants (e.g., atg9Δ) die rapidly upon nitrogen removal (becoming inviable after 3-4 days), atg23Δ mutants remain viable during starvation conditions for several days, only beginning to die after approximately 6-8 days .

• Reduced autophagosome formation: Electron microscopy reveals that atg23Δ mutants accumulate significantly fewer autophagic bodies compared to wild-type:

This 70% reduction in autophagic bodies correlates well with the biochemical data demonstrating decreased autophagy efficiency .

• Normal pexophagy: Despite its role in the Cvt pathway and general autophagy, ATG23 is not required for pexophagy (selective autophagy of peroxisomes). Deletion mutants can degrade the peroxisomal marker protein Fox3 at rates comparable to wild-type cells .

These phenotypes collectively suggest that ATG23 has a specific role in certain autophagy-related pathways rather than being essential for all autophagic processes.

How do ATG23 and ATG9 interact to regulate autophagy?

ATG23 and ATG9 have a complex functional relationship that is critical for autophagy. Key aspects of this interaction include:

• Physical interaction: ATG9 transiently interacts with ATG23, as demonstrated by coimmunoprecipitation studies .

• Membrane recruitment: ATG23 is a peripheral membrane protein that requires the presence of ATG9 to be specifically targeted to lipid bilayers . In the absence of ATG9, ATG23's membrane association is severely compromised.

• Similar localization patterns: Both proteins localize to the pre-autophagosomal structure (PAS) and other cytosolic punctate compartments .

• Functional relationship: While ATG23 requires ATG9 for membrane recruitment, ATG23 can tether vesicles independently of ATG9 once it reaches the membrane . This suggests a sequential relationship where ATG9 first recruits ATG23 to membranes, after which ATG23 can perform membrane tethering functions semi-autonomously.

• Differential essentiality: ATG9 deletion results in complete autophagy defects, while ATG23 deletion allows partial autophagy , indicating that ATG9 has additional functions beyond ATG23 recruitment.

Methodological approaches to study ATG23-ATG9 interactions:

• Coimmunoprecipitation assays with tagged proteins

• Fluorescence microscopy for colocalization studies

• Bimolecular fluorescence complementation (BiFC) to visualize direct interactions

• Membrane fractionation studies comparing wild-type, atg9Δ, and atg23Δ strains

What methods are optimal for expressing and purifying recombinant ATG23 from A. gossypii?

The expression and purification of recombinant ATG23 from A. gossypii require specialized approaches tailored to this filamentous fungus. Based on successful strategies for other proteins in A. gossypii, the following methodological workflow is recommended:

Expression system selection:

• Use integrative expression cassettes rather than episomal vectors, as "plasmids are not fully stable in the multinucleated syncytium of A. gossypii" .

• Target genomic integration using recombinogenic flanks, which is "the main mechanism for DNA integration in A. gossypii" .

• Utilize strong promoters such as the GPD1 (TDH3) promoter or the constitutive TEF promoter (translation elongation factor 1α) for high-level expression .

Marker selection:

• The loxP-kanMX-loxP marker system conferring G418 resistance is effective in A. gossypii .

• Marker recycling using Cre recombinase allows multiple genetic modifications .

Expression optimization:

• Culture in defined minimal medium (DMM) or rich medium (AFM) with sucrose as the primary carbon source .

• Monitor growth phases carefully, as protein expression may vary between the trophic phase and the riboflavin-productive phase .

Purification strategy:

• Affinity tags such as His6-tag or MBP-tag can facilitate purification.

• Include protease inhibitors during extraction to prevent degradation.

• Consider using a dual tag approach for increased purity.

Quality control:

• Verify protein integrity by SDS-PAGE and Western blotting.

• Confirm proper folding through circular dichroism or limited proteolysis assays.

• Assess oligomerization state by size exclusion chromatography or native PAGE.

How can researchers investigate the dimerization-dependent membrane tethering function of ATG23?

The dimerization-dependent membrane tethering function of ATG23 can be investigated through a combination of structural, biochemical, and microscopy approaches:

Structure-based investigations:

• Mapping the dimerization interface: Use site-directed mutagenesis to target residues in the putative amphipathic helix, particularly focusing on the hydrophobic face (residues L171, I182, L189) .

• Structural analysis: Employ small-angle X-ray scattering (SAXS) to examine how mutations affect the extended rod-like structure (approximately 320 Å) of ATG23 .

Biochemical approaches:

• In vitro vesicle tethering assays: Compare the ability of wild-type ATG23 and dimerization-deficient mutants to tether liposomes.

• Oligomerization analysis: Use native mass spectrometry to determine if ATG23 forms higher-order oligomers beyond dimers when bound to membranes. Research shows that "Native mass spectrometry confirmed the formation of ATG23 oligomers" .

• FRET-based stopped-flow measurements: These can reveal the kinetics of ATG23 oligomerization upon membrane binding. Studies show that "ATG23 rapidly oligomerizes upon membrane binding while it is mainly monomeric in solution" .

• Membrane-binding analysis: Use fluorescence-based techniques with environment-sensitive dyes to monitor how dimerization affects membrane interaction.

Cell biological approaches:

• Localization studies: Compare subcellular localization of wild-type ATG23 with dimerization-deficient mutants using fluorescence microscopy.

• Functional complementation: Test whether expression of dimerization-deficient ATG23 mutants can rescue autophagy defects in atg23Δ cells.

• Electron microscopy: Examine ultrastructural changes in autophagosome formation in cells expressing dimerization-deficient ATG23 mutants.

Key experimental controls:

• Include the ATG23[LIL] mutant (L171A, I182A, L189A) as a negative control for dimerization .

• Use purified monomeric proteins as controls in tethering assays.

• Include conditions that promote or inhibit membrane curvature to test whether this affects ATG23's tethering function.

What is the relationship between ATG23 function and riboflavin production in A. gossypii?

A. gossypii is industrially important for its natural ability to overproduce riboflavin (vitamin B2) . The relationship between ATG23 function and riboflavin production represents an intriguing area for exploration, particularly given the role of autophagy in nutrient recycling and metabolic regulation.

While direct evidence linking ATG23 to riboflavin biosynthesis is limited, several connections can be hypothesized and investigated:

Metabolic connections:

• Nutrient recycling: Autophagy recycles cellular components during nutrient limitation, potentially providing precursors for riboflavin biosynthesis. ATG23's role in efficient autophagy may influence this recycling process .

• Purine metabolism: Riboflavin biosynthesis in A. gossypii involves purine metabolism . Since autophagy affects nucleotide pools, ATG23-dependent autophagy may indirectly influence purine availability for riboflavin production.

• Glycine pathway: Research shows that "riboflavin production in A. gossypii is limited by glycine, an early precursor required for purine synthesis" . Autophagy may affect amino acid pools, including glycine.

Growth phase regulation:

A. gossypii produces riboflavin primarily during the "productive phase" after active growth has ceased:

"In terms of riboflavin production, two stages can be differentiated during A. gossypii culture: a trophic phase when riboflavin production is minimal and the growth rate increases, and a productive phase when the growth rate decreases and riboflavin is overproduced" .

Since autophagy is typically induced during nutrient limitation and growth cessation, ATG23-dependent autophagy may be particularly important during the transition from trophic to productive phase.

Experimental approaches:

• Compare riboflavin production in wild-type and atg23Δ strains throughout growth phases.

• Analyze metabolic profiles of wild-type and atg23Δ strains, focusing on purine metabolites and glycine.

• Investigate ATG23 expression and localization during the transition from trophic to productive phase.

• Test whether autophagy inducers or inhibitors affect riboflavin production.

How does the function of A. gossypii ATG23 compare with its homologs in other fungi?

A comparative analysis of ATG23 across fungal species reveals both conserved and divergent features that provide insights into its evolutionary significance and specialized functions:

Functional conservation:

• Role in selective autophagy: ATG23 is required for the cytoplasm-to-vacuole targeting (Cvt) pathway across fungal species that possess this pathway . This suggests conservation of its core function in selective autophagy.

• Interaction with ATG9: The interaction between ATG23 and ATG9 appears to be conserved across fungi, with ATG9 recruiting ATG23 to membranes .

• Partial requirement for general autophagy: In both S. cerevisiae and other studied fungi, ATG23 deletion reduces but does not completely abolish starvation-induced autophagy , indicating a conserved auxiliary role.

Structural conservation:

Sequence analysis suggests conservation of key structural features:

• Amphipathic helix: The putative amphipathic helix responsible for dimerization appears to be conserved across fungal ATG23 homologs .

• Dimerization mechanism: The hydrophobic residues (L171, I182, L189) critical for dimerization are likely conserved in ATG23 homologs from related fungi .

Species-specific differences:

• Dispensability for pexophagy: In S. cerevisiae, ATG23 is not required for pexophagy , but this may differ in methylotrophic yeasts like Pichia pastoris where peroxisome metabolism is more central.

• Integration with other cellular processes: The relationship between ATG23 and species-specific processes (such as riboflavin overproduction in A. gossypii) may create unique functional contexts for ATG23 in different fungi.

Methodological approaches for comparative studies:

• Complementation experiments: Test whether ATG23 from one fungal species can restore function in an atg23Δ mutant of another species.

• Domain swapping: Create chimeric ATG23 proteins with domains from different fungal species to identify functionally important regions.

• Comparative localization studies: Compare the subcellular localization and dynamics of ATG23 across fungal species.

• Evolution rate analysis: Examine the evolutionary rate of different ATG23 domains to identify regions under selective pressure.

What techniques can effectively visualize ATG23 trafficking and dynamics in A. gossypii?

Visualizing ATG23 trafficking and dynamics in A. gossypii presents unique challenges due to its filamentous morphology and multinucleate nature. The following advanced imaging approaches are recommended:

Fluorescent protein tagging strategies:

• Genomic integration of fluorescent tags: Create C-terminal or N-terminal fusions of ATG23 with fluorescent proteins (GFP, mCherry, etc.) at the endogenous locus to maintain native expression levels .

• Dual-color imaging: Co-express ATG23 fused to one fluorophore along with markers for specific subcellular compartments (PAS, vacuole, endosomes) fused to spectrally distinct fluorophores.

• Functional verification: Confirm that tagged versions of ATG23 complement the phenotypes of atg23Δ mutants to ensure the tags don't disrupt function.

Advanced microscopy techniques:

• 4D imaging (x,y,z,t): Capture the dynamic behavior of ATG23 in three spatial dimensions over time, which is particularly important for the complex hyphal architecture of A. gossypii.

• Photoactivatable/photoconvertible tags: Use photoactivatable GFP or photoconvertible proteins like Dendra2 fused to ATG23 to track specific populations of the protein over time.

• Super-resolution microscopy: Techniques such as structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) can resolve ATG23 localization beyond the diffraction limit.

• FRAP (Fluorescence Recovery After Photobleaching): Apply this technique to measure the mobility and exchange rates of ATG23 at specific subcellular locations.

• Correlative light and electron microscopy (CLEM): Combine fluorescence imaging of ATG23 with electron microscopy to correlate its localization with ultrastructural features.

Experimental considerations specific to A. gossypii:

• Growth conditions: Culture A. gossypii in microfluidic devices that allow continuous perfusion with fresh media and rapid media exchanges to monitor ATG23 dynamics during nutrient shifts.

• Stage of development: Consider the developmental stage of A. gossypii hyphae, as protein localization may differ between germlings, young hyphae, and mature mycelium.

• Hyphal tip focus: Pay special attention to hyphal tips, which are active growth zones with distinctive membrane dynamics that may involve ATG23.

• Environmental triggers: Monitor ATG23 localization during transitions that trigger autophagy, such as nitrogen starvation or rapamycin treatment.

How can researchers analyze the effect of ATG23 on autophagic flux in A. gossypii?

Measuring autophagic flux in A. gossypii requires specialized approaches that account for its filamentous growth and unique cellular organization. The following comprehensive methodological framework is recommended:

Biochemical assays:

• GFP-ATG8 processing assay: Express GFP-ATG8 in wild-type and atg23Δ strains and monitor the appearance of free GFP by Western blot after autophagy induction . When GFP-ATG8 is delivered to the vacuole through autophagy, the ATG8 portion is degraded while GFP remains relatively stable, resulting in detectable free GFP.

• Aminopeptidase I (Ape1) maturation assay: Monitor the conversion of prApe1 to mature Ape1 by Western blot in wild-type and atg23Δ strains under different nutrient conditions . This assay specifically measures the Cvt pathway under nutrient-rich conditions and autophagy under starvation.

• Pho8Δ60 activity assay: This quantitative enzymatic assay measures the delivery of the cytosolic Pho8Δ60 reporter to the vacuole through non-selective autophagy . The assay has been successfully used to show that "atg23Δ cells induced ∼40% of the amount of Pho8Δ60 activity compared with wild type" .

Microscopy-based methods:

• Autophagosome quantification: Use fluorescently-tagged markers such as ATG8 to visualize and count autophagosomes in living cells. Compare the number, size, and dynamics of autophagosomes between wild-type and atg23Δ strains.

• Electron microscopy: This technique provides ultrastructural evidence of autophagy. Quantify autophagic bodies in the vacuole using strains lacking vacuolar proteases (e.g., pep4Δ) to prevent their degradation . Research has shown a "70% reduction in the number of autophagic bodies accumulating in atg23Δ cells compared with wild type" .

• Cargo-specific autophagy: For selective forms of autophagy, express specific cargo markers (e.g., peroxisomal RFP-SKL for pexophagy) and monitor their delivery to the vacuole.

Pharmacological approaches:

• Autophagy modulation: Use rapamycin to induce autophagy or 3-methyladenine to inhibit it, then compare responses between wild-type and atg23Δ strains.

• Vacuolar protease inhibitors: Apply PMSF or E-64d to prevent degradation of autophagic bodies, allowing their accumulation and quantification.

• Concanamycin A: This V-ATPase inhibitor raises vacuolar pH and inhibits vacuolar hydrolases, providing an alternative method to visualize accumulated autophagic bodies.

Experimental design considerations:

• Growth conditions: Standardize culture conditions and growth phases for consistent comparisons.

• Starvation protocols: Implement controlled nitrogen starvation protocols to induce autophagy reliably.

• Time-course analysis: Measure autophagic flux at multiple time points after autophagy induction to capture the kinetics of the process.

• Multiple assays: Combine biochemical and microscopy-based approaches for comprehensive assessment of autophagic flux.

What role does ATG23 play in the transition between growth phases in A. gossypii?

A. gossypii exhibits distinct growth phases that culminate in riboflavin overproduction. Understanding ATG23's role in these transitions could provide insights into both autophagy regulation and industrial applications:

Growth phases in A. gossypii:

A. gossypii culture can be divided into two distinct phases:

• Trophic phase: Characterized by minimal riboflavin production and increasing growth rate .

• Productive phase: Characterized by decreasing growth rate and riboflavin overproduction .

The transition between these phases involves "many physiological and morphological changes" , but the mechanisms triggering this transition are not fully understood.

Potential roles for ATG23 in phase transitions:

• Nutrient sensing and response: ATG23-dependent autophagy may help cells adapt to changing nutrient availability during the transition from trophic to productive phase.

• Metabolic remodeling: Selective autophagy mediated by ATG23 might contribute to the metabolic shift required for riboflavin overproduction.

• Cellular development: A. gossypii transitions from spore germination to filamentous growth, with ATG23 potentially playing a role in these developmental processes, similar to how BAS1 has been shown to affect "a delay in the germination of the spores and an abnormally prolonged trophic phase" .

Experimental approaches to investigate this relationship:

• Growth phase-specific expression analysis: Compare ATG23 expression levels between trophic and productive phases using RT-qPCR or RNA-seq.

• Phase transition analysis: Determine whether atg23Δ mutants show altered timing or efficiency of the transition from trophic to productive phase.

• Conditional expression: Use regulatable promoters to control ATG23 expression at different growth phases and assess the impact on phase transitions.

• Metabolomic analysis: Compare metabolite profiles between wild-type and atg23Δ strains during phase transitions, focusing on riboflavin precursors and related metabolites.

• Interaction with known regulators: Investigate potential interactions between ATG23 and established regulators of the trophic-to-productive phase transition, such as BAS1, which has been shown to be "involved in the regulation of different physiological processes, such as purine and glycine biosynthesis, riboflavin overproduction, and growth" .

How do the dual functions of ATG23 in selective and non-selective autophagy differ mechanistically?

ATG23 has distinct roles in selective autophagy (particularly the Cvt pathway) and non-selective, starvation-induced autophagy. Understanding the mechanistic differences between these functions provides insights into how a single protein can contribute to multiple autophagy-related pathways:

Comparative impact of ATG23 deletion:

• Selective autophagy (Cvt pathway): ATG23 deletion completely blocks the Cvt pathway under nutrient-rich conditions, preventing the maturation of prApe1 .

• Non-selective autophagy: ATG23 deletion reduces but does not eliminate starvation-induced autophagy, with atg23Δ cells retaining approximately 40% of wild-type autophagic activity .

• Pexophagy: Interestingly, ATG23 is dispensable for pexophagy, with atg23Δ mutants degrading the peroxisomal marker Fox3 at rates comparable to wild-type .

Mechanistic distinctions:

• PAS organization: The pre-autophagosomal structure (PAS) organization differs between selective and non-selective autophagy:

  • The Cvt-specific PAS depends on the cargo-receptor-adaptor complex of Ape1, Atg19, and Atg11 .

  • The autophagy-specific PAS depends on Atg17 complexes: Atg1-Atg13-Atg17 and Atg17-Atg29-Atg31 .

  ATG23 may interact differently with these distinct PAS organizations.

• Vesicle size regulation: Cvt vesicles (150 nm diameter) are considerably smaller than autophagosomes (300-900 nm diameter) . ATG23 may contribute to this size regulation, potentially explaining why its absence completely blocks Cvt vesicle formation but only partially impairs autophagosome formation.

• Cargo-specific interactions: In selective autophagy, ATG23 may engage with cargo-specific receptors that are absent in non-selective autophagy.

• Threshold requirements: Non-selective autophagy may have redundant mechanisms that can partially compensate for ATG23 loss, whereas the Cvt pathway may have an absolute requirement for ATG23.

Experimental approaches to distinguish these mechanisms:

• Separation-of-function mutations: Identify and characterize ATG23 mutations that specifically affect either selective or non-selective autophagy.

• Interaction profiling: Compare ATG23 protein interaction networks under nutrient-rich (Cvt-inducing) versus starvation (autophagy-inducing) conditions.

• Structure-function analysis: Map domains of ATG23 required for different functions through systematic deletion and mutation analysis.

• Cargo specificity: Investigate whether ATG23 directly or indirectly interacts with specific Cvt cargo proteins such as prApe1.

How can high-throughput approaches be applied to study ATG23 interactions and functions?

Modern high-throughput technologies offer powerful approaches to comprehensively characterize ATG23's interactome, functional networks, and regulatory mechanisms:

Interactome mapping:

• Proximity-based labeling: Express ATG23 fused to enzymes like BioID or APEX2 to biotinylate proteins in close proximity, followed by streptavidin pulldown and mass spectrometry.

• Systematic yeast two-hybrid screens: Screen ATG23 against an A. gossypii genomic library to identify direct protein-protein interactions.

• Affinity purification-mass spectrometry (AP-MS): Use epitope-tagged ATG23 for immunoprecipitation followed by mass spectrometry to identify protein complexes.

• Cross-linking mass spectrometry (XL-MS): Apply protein cross-linkers followed by mass spectrometry to capture transient interactions and determine interaction interfaces. Research shows that "the amine-specific cross-linker Bis(sulfosuccinimidyl)suberate (BS3), which is used for protein-protein cross-linking can also be applied for cross-linking proteins and phosphatidylethanolamine (PE)" , which could be valuable for studying ATG23-membrane interactions.

Functional genomics:

• Genome-wide synthetic genetic array (SGA): Cross atg23Δ with a comprehensive deletion library to identify genetic interactions through fitness analysis.

• CRISPR screens: Conduct genome-wide CRISPR screens in ATG23 wild-type and knockout backgrounds to identify genes that genetically interact with ATG23.

• Pooled barcoded mutant screens: Create a library of ATG23 variants with different mutations, each linked to a unique barcode, and screen for functionality under different conditions.

Systems-level analyses:

• Integrative multi-omics: Combine transcriptomics, proteomics, and metabolomics data from wild-type and atg23Δ strains to build comprehensive regulatory networks.

• Computational modeling: Develop mathematical models of autophagy that incorporate ATG23 functions and predict systemic responses to perturbations.

• Network analysis: Apply graph theory and network algorithms to identify key nodes and pathways connecting ATG23 to other cellular processes.

High-content microscopy:

• Automated image analysis: Develop image analysis pipelines to quantify ATG23 localization, autophagosome formation, and cargo transport in large image datasets.

• Multiplexed imaging: Use spectral unmixing or sequential labeling approaches to simultaneously visualize multiple proteins involved in autophagy.

• Machine learning classification: Train algorithms to recognize and classify autophagy-related phenotypes in microscopy images of cells with wild-type or mutant ATG23.

What post-translational modifications regulate ATG23 function and how can they be studied?

Post-translational modifications (PTMs) often serve as key regulatory mechanisms for autophagy proteins. Although specific PTMs of ATG23 have not been extensively characterized, several approaches can be employed to identify and study potential modifications:

Potential PTMs affecting ATG23:

• Phosphorylation: Many autophagy proteins are regulated by phosphorylation in response to nutrient signaling through kinases like Tor1/mTOR, AMPK, and PKA.

• Ubiquitination: Ubiquitin modifications can affect protein stability, localization, or interactions.

• Acetylation: This modification can influence protein-protein or protein-membrane interactions.

• Lipidation: Similar to ATG8/LC3 lipidation, membrane-associated proteins can be modified with lipids.

Methodological approaches for PTM identification:

• Mass spectrometry-based PTM mapping:

  • Enrich for phosphopeptides using titanium dioxide or immobilized metal affinity chromatography

  • Use specific antibodies to enrich for ubiquitinated or acetylated peptides

  • Apply multiple proteolytic enzymes to improve sequence coverage

  • Employ both bottom-up and top-down proteomics approaches

• Site-specific mutational analysis:

  • Mutate predicted modification sites (e.g., S/T to A for phosphorylation sites)

  • Create phosphomimetic mutations (S/T to D/E)

  • Assess the impact of mutations on ATG23 function, localization, and interactions

• PTM-specific antibodies:

  • Develop antibodies recognizing specific modified forms of ATG23

  • Use these antibodies for Western blotting and immunofluorescence microscopy

• Pharmacological approaches:

  • Apply kinase inhibitors or phosphatase inhibitors to modulate phosphorylation status

  • Use deacetylase inhibitors to enhance acetylation signals

  • Employ proteasome inhibitors to stabilize ubiquitinated forms

Functional analysis of PTMs:

• Condition-dependent modification: Compare PTM profiles across different conditions:

  • Nutrient-rich versus starvation conditions

  • Trophic versus productive phase

  • Various stress conditions

• Modification-specific interactome: Identify proteins that specifically interact with modified versus unmodified ATG23.

• Dynamic regulation: Use pulse-chase approaches to determine the kinetics of PTM acquisition and removal.

• Structure-function relationships: Determine how modifications affect protein structure, dimerization, membrane binding, and tethering activity.

How can computational approaches enhance our understanding of ATG23 function?

Computational biology offers powerful tools to predict, model, and analyze ATG23 function across multiple scales:

Structural bioinformatics:

• Protein structure prediction: Apply AlphaFold2 or RoseTTAFold to predict the three-dimensional structure of ATG23, with particular focus on the putative amphipathic helix identified as critical for dimerization .

• Molecular dynamics simulations: Simulate ATG23 interaction with membranes to understand how it contributes to membrane tethering.

• Protein-protein docking: Model the interaction between ATG23 and known binding partners like ATG9.

• Mutation effect prediction: Use computational tools to predict the impact of mutations on protein stability, function, and interactions.

Comparative genomics:

• Evolutionary analysis: Compare ATG23 sequences across fungal species to identify conserved regions that may be functionally important.

• Coevolution analysis: Identify residues that coevolve, potentially indicating functional interactions or structural constraints.

• Synteny analysis: Examine the genomic context of ATG23 across species to identify potential functional relationships with neighboring genes.

Systems biology:

• Network inference: Build gene regulatory networks from transcriptomic data to understand how ATG23 is regulated and how it affects downstream processes.

• Flux balance analysis: Model metabolic networks to predict how ATG23-dependent autophagy affects cellular metabolism, particularly in relation to riboflavin production.

• Agent-based modeling: Create computational models of autophagosome formation incorporating ATG23 function based on experimental data.

Machine learning applications:

• Feature extraction from microscopy data: Apply computer vision algorithms to extract quantitative features from images of ATG23 localization and autophagosome formation.

• Predictive modeling: Train models to predict autophagy efficiency based on ATG23 variants or experimental conditions.

• Literature mining: Use natural language processing to systematically extract information about ATG23 and related proteins from scientific literature.

Resources and databases:

• Ashbya Genome Database (http://agd.unibas.ch/): A comprehensive resource for A. gossypii genomics .

• ScanProsite tool (http://www.expasy.org/tools/scanprosite/): Useful for identifying functional motifs in ATG23 .

• AutophagyDB: A database dedicated to autophagy-related proteins and their interactions.

• Saccharomyces Genome Database (SGD): Contains valuable information on S. cerevisiae homologs that can inform A. gossypii research.