Recombinant Ashbya gossypii Ubiquitin-like modifier-activating enzyme ATG7 (ATG7), partial

What is ATG7 and what is its role in Ashbya gossypii?

ATG7 (Ubiquitin-like modifier-activating enzyme ATG7) functions as an E1-like activating enzyme involved in two ubiquitin-like systems required for cytoplasm to vacuole transport (Cvt) and autophagy . In cellular systems, ATG7 activates ATG12 for its conjugation with ATG5 as well as the ATG8 family proteins for their conjugation with phosphatidylethanolamine . Both systems are essential for the association of ATG8 to Cvt vesicles and autophagosome membranes .

While specific research on A. gossypii ATG7 is limited in the provided context, comparative studies in other organisms show that ATG7 plays crucial roles in stress response mechanisms. Studies in Drosophila demonstrate that ATG7 is required for starvation-induced autophagy, with ATG7 mutants showing severe impairment in autophagy induction in response to starvation . The autophagy process is likely important in A. gossypii for maintaining cellular health during various stress conditions, particularly given its filamentous growth pattern and industrial applications.

How does ATG7 in A. gossypii compare to ATG7 in other organisms?

ATG7 is highly conserved across eukaryotes, but with functional specializations depending on the organism. While direct comparative studies of A. gossypii ATG7 with other organisms aren't explicitly detailed in the provided sources, we can draw inferences from related research.

Studies in Drosophila show that ATG7 mutants exhibit severe defects in autophagy but remain viable, suggesting that while autophagy is crucial for stress survival and continuous cellular renewal, it may not be essential for all developmental processes . In mammals, ATG7 has additional roles in mitophagy and modulating p53/TP53 activity to regulate cell cycle and survival during metabolic stress .

A. gossypii is phylogenetically close to Saccharomyces cerevisiae while exhibiting filamentous growth, making it an interesting model to study ATG7 function in the context of different growth morphologies . Its genome sequence completion allows for comparative genomic analyses with yeast ATG7, potentially revealing insights into how this protein may have specialized functions in filamentous versus unicellular growth patterns.

What is known about the structure and functional domains of A. gossypii ATG7?

While the provided search results don't contain specific structural information about A. gossypii ATG7, we can infer its structure based on the conserved nature of this protein. ATG7 contains an E1-like activating enzyme domain responsible for the ATP-dependent activation of ATG12 and ATG8 family proteins during the initial steps of the two conjugation systems essential for autophagosome formation .

The protein likely contains binding sites for ATG12 and ATG8 as well as ATP-binding domains characteristic of E1 enzymes. Specific structural features that might distinguish A. gossypii ATG7 from its counterparts in other organisms would require structural studies using techniques such as X-ray crystallography or cryo-electron microscopy, which appear to be currently lacking in the literature for this specific protein.

For researchers interested in structural analyses, expressing and purifying recombinant A. gossypii ATG7 using systems like those offered by CUSABIO (partial recombinant protein available) could provide material for such studies .

How does ATG7 expression affect protein secretion stress responses in A. gossypii?

A. gossypii has been explored as a host system for recombinant protein production, making the relationship between autophagy and secretion stress particularly relevant . Studies examining DTT-induced secretion stress in A. gossypii reveal important insights that may relate to ATG7 function, though direct examination of ATG7's role isn't detailed in the provided sources.

When A. gossypii cells were treated with 10 mM DTT (a known inducer of secretion stress), they showed a substantial and immediate reduction in specific growth rate . This suggests that secretion stress response mechanisms, which may involve autophagy pathways regulated by ATG7, significantly impact cellular growth. While the specific transcriptional changes related to ATG7 weren't reported, the study identified 21 differentially expressed genes during recombinant protein secretion, with GO enrichment analyses suggesting translation down-regulation and ion and amino acid transmembrane transport up-regulation .

Researchers investigating ATG7's role in secretion stress might design experiments comparing wild-type and ATG7-deficient A. gossypii strains under protein secretion stress conditions, monitoring not only growth parameters but also specific stress markers and transcriptional responses.

What role might ATG7-dependent autophagy play in riboflavin production by A. gossypii?

A. gossypii is industrially important for riboflavin (vitamin B2) production . While direct studies on ATG7's role in riboflavin production aren't detailed in the provided sources, we can propose potential connections based on known metabolic pathways.

Riboflavin production in A. gossypii involves complex metabolic networks, with carbon flux studies showing intricate connections between central carbon metabolism, amino acid synthesis, and vitamin production . Research using 13C tracer studies revealed that during the riboflavin production phase, there are specific metabolic bottlenecks, particularly in the carbon-one metabolism which was transiently limiting but could be overcome by supplementation with formate and serine .

ATG7-dependent autophagy might influence riboflavin production through several potential mechanisms:

• Nutrient recycling during autophagy could provide precursors for riboflavin biosynthesis

• Autophagy might help cells adapt to stress conditions during fermentation

• Removal of damaged organelles through selective autophagy could optimize cellular metabolism

Researchers could investigate these hypotheses by comparing riboflavin production in wild-type and ATG7-deficient strains under various nutrient and stress conditions, potentially identifying new strategies to enhance industrial production.

How does ATG7 contribute to stress resistance and longevity in A. gossypii?

Studies in Drosophila demonstrate that ATG7 plays a crucial role in stress resistance and longevity, with ATG7 mutants showing increased sensitivity to nutrient and oxidative stress as well as reduced lifespan . When subjected to complete starvation or a sugar-only diet, ATG7 mutant flies showed significantly reduced survival compared to wild-type flies .

Furthermore, aged ATG7 mutant flies exhibited accumulation of ubiquitinated proteins and progressive neurodegeneration, suggesting that autophagy regulated by ATG7 is essential for preventing the accumulation of toxic protein aggregates . The number of ubiquitin-positive inclusion bodies increased over time in neurons of ATG7 mutant flies, correlating with reduced longevity .

While these specific findings are from Drosophila, they suggest that ATG7-dependent autophagy likely plays similar roles in A. gossypii. Researchers investigating stress resistance in A. gossypii might design experiments comparing wild-type and ATG7-deficient strains under various stress conditions, including:

• Nutrient limitation

• Oxidative stress (e.g., hydrogen peroxide exposure)

• Heat shock

• ER stress (e.g., DTT treatment)

Measuring cellular viability, growth rates, and accumulation of protein aggregates would provide insights into ATG7's role in stress protection in this filamentous fungus.

What are the best methods for expressing and purifying recombinant A. gossypii ATG7?

While the search results don't provide specific protocols for ATG7 expression and purification, information from CUSABIO indicates that recombinant A. gossypii ATG7 (partial) can be produced in various expression systems including E. coli, baculovirus, mammalian cells, and through in vivo biotinylation .

For researchers seeking to express and purify this protein, the following methodological approach is recommended:

• Expression system selection:

  • E. coli systems are suitable for high-yield production but may lack post-translational modifications

  • Baculovirus expression in insect cells provides eukaryotic processing capabilities

  • Mammalian expression systems offer full eukaryotic processing but with lower yields

• Construct design considerations:

  • Include appropriate purification tags (His-tag, GST-tag, etc.)

  • Consider codon optimization for the chosen expression system

  • For partial ATG7 expression, identify functional domains of interest

• Purification strategy:

  • Affinity chromatography based on the chosen tag

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography for final polishing and buffer exchange

• Quality control:

  • SDS-PAGE for purity assessment

  • Western blot using specific antibodies (available from suppliers like CUSABIO)

  • Activity assays to confirm functional protein production

How can researchers effectively measure ATG7-dependent autophagy in A. gossypii?

Based on methodologies described in the Drosophila studies, several approaches can be adapted to measure ATG7-dependent autophagy in A. gossypii :

• Fluorescent reporters:

  • Express GFP-Atg8a fusion proteins to visualize autophagosome formation

  • Monitor localization changes from diffuse cytoplasmic to punctate structures upon autophagy induction

  • Quantify the number of GFP-positive puncta per cell under different conditions

• Lysotracker staining:

  • Use Lysotracker Red to stain acidic organelles including autolysosomes

  • Visualize using fluorescence microscopy

  • Quantify the number and size of Lysotracker-positive vesicles

• Transmission electron microscopy (EM):

  • Directly visualize autophagosomes and autolysosomes at the ultrastructural level

  • Perform morphometric analysis to quantify the volume ratio occupied by autophagic structures

  • In Drosophila studies, this approach revealed that autophagosomes occupied 0.53% and autolysosomes 1.79% of the cytoplasm in controls, compared to 0.08% and 0.04% respectively in ATG7 mutants

• Biochemical assays:

  • Monitor conversion of LC3-I to LC3-II (ATG8 homolog) by western blot

  • Assess degradation of autophagic substrates

  • Measure activity of ATG7 through its ability to activate ATG8 in vitro

When designing experiments, researchers should include appropriate controls such as starvation-induced autophagy (positive control) and autophagy inhibitors like 3-methyladenine (negative control).

What genetic modification techniques are most effective for studying ATG7 function in A. gossypii?

A. gossypii is noted for its ease of genetic manipulation, making it well-suited for functional studies of genes like ATG7 . While specific techniques for ATG7 modification aren't detailed in the provided sources, we can recommend approaches based on established methods for this organism:

• Gene deletion/knockout:

  • Homologous recombination-based approaches can be used to replace the ATG7 gene with selection markers

  • PCR-based targeting with long flanking homology regions increases efficiency

  • Verification can be done using PCR, Southern blotting, and functional assays

• Gene replacement with mutant variants:

  • Introduction of point mutations to study specific domains

  • Creation of temperature-sensitive alleles

  • Fluorescent protein tagging for localization studies

• Conditional expression systems:

  • Inducible promoters to control ATG7 expression levels

  • Allows study of essential gene functions that might be lethal when completely deleted

• CRISPR-Cas9 genome editing:

  • Increasing evidence suggests CRISPR systems can be applied to filamentous fungi

  • Offers precision editing capabilities for introducing specific mutations

  • Can be used for multiplexed gene targeting

When designing genetic modification experiments, researchers should consider:

• The haploid nature of A. gossypii facilitates genetic studies as phenotypes are immediately visible

• Transformation efficiencies vary based on strain backgrounds

• Verification of modifications at both the genomic and protein expression levels is essential