Recombinant Ashbya gossypii Autophagy-related protein 27 (ATG27), partial

What is the role of ATG27 in Ashbya gossypii autophagy pathways?

ATG27 in A. gossypii, similar to its orthologs in related fungi, functions as a membrane protein involved in the autophagy pathway. Based on studies in related organisms, ATG27 likely participates in the cycling of ATG9, a transmembrane protein essential for autophagosome formation . The protein is believed to shuttle between the pre-autophagosomal structure (PAS) and peripheral compartments, facilitating membrane recruitment during autophagosome biogenesis . In yeast models, ATG27 interacts with retromer subunits (such as Vps17 and Vps5), suggesting its involvement in membrane trafficking pathways that support autophagy . These interactions are particularly important for maintaining proper ATG9 cycling, which when disrupted can lead to partial defects across multiple autophagy-related pathways.

How does the genome structure of A. gossypii influence ATG27 expression?

A. gossypii possesses a relatively compact genome with high gene order conservation compared to Saccharomyces cerevisiae (91% synteny), despite having a lower level of genomic complexity . This genomic architecture impacts the expression patterns of genes including ATG27. Based on genome-wide annotation studies, the expression of ATG27 is likely influenced by the organism's filamentous growth pattern, which differs from budding yeasts like S. cerevisiae . The genome contains approximately 4,776 annotated genes, with only about 5% having no homologs in S. cerevisiae, suggesting that A. gossypii ATG27 likely shares significant sequence and functional similarity with its S. cerevisiae counterpart . This genomic context is crucial for understanding how ATG27 is regulated within the specific metabolic framework of A. gossypii.

What expression systems are optimal for producing recombinant ATG27 in A. gossypii?

For optimal expression of recombinant ATG27 in A. gossypii, researchers should consider several promoter options identified through recent metabolic engineering studies. Strong constitutive promoters like P_CCW12 have demonstrated high efficiency in A. gossypii expression systems and would be suitable for constitutive expression of ATG27 . For controlled expression, carbon source-regulatable promoters provide valuable tools for experimental manipulation of ATG27 levels . The Dual Luciferase Reporter (DLR) Assay has been adapted for promoter analysis in A. gossypii, allowing for quantitative assessment of promoter strength through integrative cassettes . This system enables researchers to optimize expression conditions for recombinant ATG27 based on experimental requirements.

What techniques are available for detecting and quantifying ATG27 expression in A. gossypii?

Several techniques can be employed to detect and quantify ATG27 expression in A. gossypii. For visualization and localization studies, fluorescent tagging with GFP fusion proteins expressed under native promoters provides valuable insights into protein distribution and dynamics, similar to approaches used for other autophagy-related proteins in fungi . Western blot analysis with specific antibodies against ATG27 or epitope tags can quantify protein levels, while RT-qPCR enables mRNA expression analysis. For functional studies, the GFP-ATG8 cleavage assay serves as an effective method to assess autophagy pathway functionality in strains with modified ATG27 expression . This assay measures the processing of GFP-ATG8 and accumulation of free GFP as indicators of autophagy flux. Additionally, nitrogen starvation viability assays can assess long-term functional consequences of ATG27 manipulation .

How does ATG27 interact with the PI3K complex in A. gossypii autophagy pathways?

The interaction between ATG27 and the PI3K complex in A. gossypii likely parallels mechanisms observed in related fungi, where the complex plays a critical role in autophagy regulation. Based on studies in similar systems, ATG27 function depends on the PI3K complex components, particularly Vps34, Vps15, and Atg6/Vps30 . The PI3K complex generates phosphatidylinositol 3-phosphate (PtdIns3P), which serves as a crucial lipid signaling molecule for membrane trafficking in autophagy pathways .

In experimental systems like P. pastoris, disruption of PI3K complex components (atg6Δ, vps15Δ, and vps34Δ) severely impairs autophagy, as evidenced by defects in GFP-Atg8 processing and dramatically reduced cell viability during nitrogen starvation . The relationship between ATG27 and the PI3K complex in A. gossypii likely involves:

• PtdIns3P-dependent recruitment of ATG27 to specific membrane compartments

• Coordination with other PtdIns3P-binding proteins like Atg24 and Vps17

• Facilitation of membrane trafficking events essential for ATG9 cycling

For researchers investigating these interactions, experimental approaches should include colocalization studies with fluorescently tagged proteins, co-immunoprecipitation assays, and functional analyses in strains with mutations in PI3K complex components.

What are the methodological challenges in creating ATG27 knockout strains in A. gossypii?

Creating ATG27 knockout strains in A. gossypii presents several methodological challenges specific to this organism's biology. A. gossypii is a multinucleate filamentous fungus, which complicates genetic manipulation compared to unicellular yeasts . Researchers must consider:

• Homologous recombination efficiency: While A. gossypii can undergo homologous recombination, optimizing transformation parameters for gene replacement is critical. The PCR-based gene targeting approach with long flanking homology regions (45-60 bp) may yield transformants, but screening multiple candidates is necessary to identify complete knockouts.

• Nuclear segregation: A. gossypii's multinucleate nature necessitates several rounds of selection to achieve homokaryotic transformants where all nuclei contain the knockout allele. Incomplete nuclear segregation can result in heterokaryotic mycelia that complicate phenotypic analysis.

• Verification strategies: Multiple verification methods are essential, including:

  • PCR verification of both insertion site junctions

  • Southern blot analysis

  • RT-PCR confirmation of transcript absence

  • Western blot verification of protein absence

• Functional complementation: Expressing wild-type ATG27 from an ectopic locus on a plasmid is crucial to confirm that observed phenotypes result specifically from ATG27 deletion rather than off-target effects.

• Selection marker considerations: Careful selection marker choice that functions efficiently in A. gossypii while minimizing impact on cellular metabolism is essential for reliable phenotypic characterization.

What role does ATG27 play in the regulation of the Cvt and pexophagy pathways in A. gossypii?

ATG27 likely plays significant roles in the cytoplasm-to-vacuole targeting (Cvt) and pexophagy pathways in A. gossypii, similar to its functions in related fungi. In P. pastoris, autophagy proteins participate in multiple membrane trafficking pathways, including both general autophagy and selective autophagy processes like Cvt and pexophagy .

Based on comparative studies, ATG27's functions in these pathways may include:

• Cvt pathway regulation: ATG27 likely facilitates the specific targeting of hydrolases like aminopeptidase I (Ape1) to the vacuole via the Cvt pathway. Its membrane trafficking function would support the formation of Cvt vesicles by coordinating with other autophagy-related proteins.

• Pexophagy pathway involvement: ATG27 may participate in recognition and selective degradation of peroxisomes. In P. pastoris, pexophagy requires core autophagy machinery plus specialized factors like Atg30 . ATG27 would likely interact with these components to facilitate peroxisome engulfment and delivery to the vacuole.

• Membrane source regulation: ATG27 cycles between organelles and potentially regulates membrane sources for both pathways, coordinating with ATG9 to facilitate membrane delivery to growing isolation membranes.

For experimental investigation, researchers should employ selective autophagy assays:

• Monitoring Ape1 processing for the Cvt pathway

• Tracking peroxisome marker degradation (like Pex14-GFP) for pexophagy

• Using electron microscopy to visualize intermediate structures in ATG27 mutant strains

How do the N-glycosylation patterns of recombinant ATG27 differ between A. gossypii and other expression systems?

The N-glycosylation patterns of recombinant ATG27 likely exhibit significant differences between A. gossypii and other expression systems, impacting protein function and utility. A. gossypii has been noted to hyperglycosylate heterologous proteins less extensively than S. cerevisiae, particularly regarding N-glycans . This characteristic offers potential advantages for recombinant protein production where excessive glycosylation might adversely affect function.

For recombinant ATG27 specifically, these differences may manifest as:

• Glycan composition: A. gossypii likely produces less elaborate mannose-rich outer chains compared to S. cerevisiae, resulting in more compact glycan structures on ATG27.

• Site occupancy: The efficiency of N-glycosylation at specific sequons within the ATG27 sequence may vary between expression systems, affecting the heterogeneity of the final product.

• Functional implications: Less extensive glycosylation in A. gossypii may better preserve native membrane protein interactions of ATG27, potentially enhancing its functionality in experimental applications.

To characterize these differences, researchers should consider:

What experimental approaches can resolve contradictory data regarding ATG27's role in autophagy versus vacuolar protein sorting?

Resolving contradictory data regarding ATG27's role in autophagy versus vacuolar protein sorting (VPS) requires sophisticated experimental approaches that can distinguish between direct and indirect effects. Based on similar challenges encountered with other autophagy-related proteins, researchers should consider:

• Conditional expression systems: Implementing the newly identified carbon source-regulatable promoters in A. gossypii to create strains with tunable ATG27 expression levels. This approach allows for temporal separation of autophagy and VPS functions by inducing or repressing ATG27 at specific experimental phases.

• Domain-specific mutations: Creating point mutations or domain deletions in ATG27 that selectively disrupt either autophagy or VPS functions while preserving the other. This requires detailed structural analysis to identify functional domains.

• Multiple functional assays: Employing parallel assays that separately evaluate:

  • Autophagy: GFP-Atg8 processing, nitrogen starvation viability

  • VPS: CPY sorting, vacuolar protein localization

  • Membrane trafficking: Atg9 cycling, retromer function

• Interaction network analysis: Conducting comprehensive protein-protein interaction studies comparing ATG27 interactors under different conditions:

• Real-time imaging: Utilizing advanced microscopy techniques to track ATG27 localization dynamics in living cells under various conditions, potentially revealing condition-specific behavior patterns.

• Interspecies complementation: Testing whether ATG27 from organisms with well-characterized functions can rescue specific defects in A. gossypii atg27Δ strains, providing insights into conserved and divergent functions.

• Epistasis analysis: Systematically creating double mutants of atg27Δ with mutations in either autophagy-specific or VPS-specific genes to determine pathway relationships through phenotypic analysis.

By implementing these approaches, researchers can systematically dissect the multifunctional nature of ATG27 and resolve apparent contradictions in experimental data.