Recombinant Lachancea thermotolerans Autophagy-related protein 32 (ATG32)

What is ATG32 and what is its primary function in Lachancea thermotolerans?

ATG32 is a transmembrane protein that functions as the primary receptor for mitophagy in yeasts including Lachancea thermotolerans . The protein contains a cytosolic domain that interacts with core autophagy machinery, specifically Atg8 and Atg11, which are essential components for selective autophagy . This interaction recruits the autophagy machinery to the mitochondrial surface, initiating the formation of isolation membranes around targeted mitochondria . ATG32 essentially acts as an autophagic degron - a specific polypeptide sequence that marks proteins or organelles for autophagy-dependent degradation . The protein's structure includes transmembrane domains that anchor it to the mitochondrial outer membrane, with its cytosolic domain extending into the cytoplasm where it can interact with autophagy-related proteins .

What experimental methods are commonly used to study ATG32 expression and function?

Several methodological approaches are employed to study ATG32 expression and function:

• Promoter activity assays: Using reporter constructs like pPROM-ATG32-β-galactosidase, where the reporter gene lacZ is expressed from the ATG32 promoter, allowing measurement of promoter activity under different conditions .

• Protein level quantification: Western blotting with antibodies against epitope-tagged ATG32 (commonly V5 or 6xHIS tags) to quantify protein levels during different growth phases or treatments .

• Protein-protein interaction studies: Co-immunoprecipitation and pull-down assays to identify binding partners of ATG32, particularly its interactions with the core autophagy machinery components Atg8 and Atg11 .

• Organelle targeting assays: Experiments where the cytosolic domain of ATG32 is artificially targeted to different organelles to test its ability to recruit autophagy machinery and induce selective autophagy .

• Mass spectrometry: LC-MS/MS analysis to identify post-translational modifications such as ubiquitination sites in ATG32 .

• Mitophagy assays: Using fluorescent markers to track mitochondrial degradation in wild-type versus ATG32-deficient strains .

How does ubiquitination regulate ATG32 function during mitophagy in yeasts?

Ubiquitination plays a crucial role in regulating ATG32 expression levels and activity during mitophagy. Research has identified that during the stationary phase of growth and during starvation, ATG32 protein levels decrease despite increased promoter activity, suggesting post-translational regulation . Treatment with MG-132, a proteasome inhibitor, counteracts this protein loss, indicating that the ubiquitin-proteasome system actively degrades ATG32 during conditions that normally induce mitophagy . Specific ubiquitination sites have been identified in ATG32, with Lysine 282 being confirmed through LC-MS/MS analysis as a site that receives the characteristic glycine-glycine (GG) tag remaining after tryptic proteolysis of ubiquitinated proteins . This evidence suggests a regulatory mechanism where ubiquitination of ATG32 modulates its availability and potentially its activity during mitophagy . The timing of this ubiquitination appears critical - it may initially activate ATG32 by inducing conformational changes that expose binding sites for autophagy proteins, followed by subsequent ubiquitination events that target it for proteasomal degradation, potentially as a feedback mechanism to control the extent of mitophagy .

Can ATG32 from L. thermotolerans induce mitophagy in heterologous systems, and what are the experimental considerations?

While direct experimental evidence for L. thermotolerans ATG32 function in heterologous systems is not explicitly detailed in the provided search results, insights can be drawn from studies with S. cerevisiae ATG32. The cytosolic domain of ATG32 appears sufficient to target autophagy machinery to mitochondria and even to other organelles when artificially anchored to them . When the cytosolic domain of ATG32 was anchored to peroxisomes, it successfully promoted autophagy-dependent peroxisome degradation (pexophagy), suggesting that ATG32 contains a modular degron-like element compatible with multiple organelle autophagy systems .

For experimental considerations when using L. thermotolerans ATG32 in heterologous systems:

• Expression optimization: Codon optimization may be necessary when expressing L. thermotolerans genes in distant hosts.

• Binding partner conservation: Verify that the heterologous system contains compatible versions of Atg8 and Atg11 or their homologs.

• Post-translational modifications: Consider whether the heterologous system can perform the necessary post-translational modifications for ATG32 function.

• Membrane targeting: Ensure proper localization to the mitochondrial outer membrane by retaining the transmembrane domain or using an appropriate targeting sequence for the host system.

• Assay development: Design specific assays to quantify the induced mitophagy, potentially using fluorescently labeled mitochondria and measuring their degradation rate.

What is the role of ATG32 in L. thermotolerans adaptation to environmental stress?

Lachancea thermotolerans is known to survive in stressful environments, including high ethanol conditions in wine fermentation . ATG32-mediated mitophagy likely plays a crucial role in this stress adaptation, similar to its function in other yeasts. During environmental stress, damaged mitochondria accumulate and can produce harmful reactive oxygen species (ROS) . ATG32-mediated mitophagy selectively removes these damaged organelles, maintaining mitochondrial quality control .

L. thermotolerans has been found in diverse environments including grape must, fruits, lake water, cocoa fermentation, and olive paste . This ecological versatility suggests strong adaptive mechanisms, potentially involving mitophagy regulation. The yeast's ability to assimilate trehalose is particularly notable, as trehalose metabolism has been linked to stress response in yeasts . While this connection needs further investigation in Lachancea spp., in Saccharomyces spp., trehalose metabolism is an indicator of a yeast's ability to survive stressful conditions .

Some L. thermotolerans strains have been found surviving in wine at the end of fermentation when co-fermented with Saccharomyces, indicating ability to withstand the stressful environment . This survival may depend on proper mitochondrial homeostasis maintained through ATG32-mediated mitophagy. Additionally, some Lachancea yeasts produce toxins that eliminate fungi and inhibit bacteria, providing another potential mechanism for outcompeting other microorganisms in stressful environments .

How should researchers design experiments to investigate ATG32-protein interactions in L. thermotolerans?

When investigating ATG32-protein interactions in L. thermotolerans, researchers should consider the following methodological approach:

• Protein expression and purification:

  • Clone the L. thermotolerans ATG32 gene with appropriate tags (e.g., V5, 6xHIS) for purification and detection

  • Express the protein in a suitable system (yeast or bacterial expression)

  • Purify using affinity chromatography (e.g., Ni-NTA columns for His-tagged proteins)

• Interaction validation assays:

  • Yeast two-hybrid screening to identify potential interaction partners

  • Co-immunoprecipitation to confirm interactions in vivo

  • Pull-down assays with purified proteins to validate direct interactions

  • BiFC (Bimolecular Fluorescence Complementation) to visualize interactions in living cells

• Structural studies:

  • X-ray crystallography of ATG32 peptides with binding partners, particularly the ATG8-family interacting motif (AIM)

  • NMR spectroscopy for flexible protein regions

• Functional validation:

  • Mutagenesis of predicted binding interfaces followed by interaction assays

  • In vitro reconstitution of minimal interaction complexes

  • Mitophagy assays using fluorescently-labeled mitochondria to assess functional outcomes of interactions

• Data analysis considerations:

  • Control for non-specific binding by including appropriate negative controls

  • Quantify binding affinities using techniques like isothermal titration calorimetry or surface plasmon resonance

  • Consider the dynamic nature of interactions during different growth conditions

What considerations are important when designing assays to measure mitophagy mediated by ATG32 in L. thermotolerans?

When developing assays to measure ATG32-mediated mitophagy in L. thermotolerans, researchers should consider:

• Growth conditions and mitophagy induction:

  • Stationary phase growth in respiratory media (e.g., glycerol) induces mitophagy naturally

  • Nitrogen starvation can also be used to induce mitophagy

  • Rapamycin treatment mimics starvation conditions and can trigger mitophagy

• Mitochondrial markers:

  • Fluorescent proteins targeted to mitochondria (e.g., mito-GFP) to track mitochondrial mass

  • Mitochondrial proteins can be tagged and their degradation monitored by western blotting

  • Specific dyes like MitoTracker can be used to assess mitochondrial mass by microscopy or flow cytometry

• Autophagy inhibition controls:

  • Include atg1Δ, atg5Δ, or other autophagy-deficient strains as negative controls

  • Chemical inhibitors of autophagy (e.g., 3-methyladenine) can serve as additional controls

• Vacuolar degradation assessment:

  • Visualize delivery of mitochondria to vacuoles using fluorescent markers that maintain signal in the vacuolar lumen

  • Co-localization studies with vacuolar membrane markers (e.g., Vph1-mCherry)

• Quantification methods:

  • Microscopy-based quantification of mitochondrial mass or degradation

  • Biochemical assays measuring activity of mitochondrial enzymes

  • Flow cytometry to measure mitochondrial mass in cell populations

• Time course considerations:

  • Mitophagy is a dynamic process, so measurements at multiple time points are essential

  • Consider the growth phase of the culture, as ATG32 levels change during different phases

How should contradictory results in ATG32 expression studies be analyzed and reconciled?

When faced with contradictory results in ATG32 expression studies, researchers should systematically analyze potential sources of variance and apply the following methodological approaches:

• Examine experimental conditions:

  • Growth media composition and carbon source significantly impact ATG32 expression and mitophagy induction

  • Growth phase is critical as ATG32 protein levels decrease during stationary phase despite increased promoter activity

  • Strain background differences can cause significant variation in expression patterns

  • Temperature conditions may affect expression differently in L. thermotolerans (which is thermotolerant as its name suggests) compared to other yeasts

• Distinguish between transcriptional and post-transcriptional regulation:

  • Compare promoter activity assays (e.g., using β-galactosidase reporter constructs) with protein level measurements

  • A discrepancy between transcript levels and protein abundance suggests post-transcriptional regulation

  • Consider proteasomal degradation, as ATG32 is subject to ubiquitination and proteasomal turnover

• Analytical reconciliation approaches:

  • Create a comprehensive table comparing experimental conditions across contradictory studies

  • Perform time-course experiments to capture the dynamic nature of ATG32 expression

  • Use multiple detection methods (e.g., fluorescence microscopy, western blotting, mass spectrometry) to validate findings

  • Implement statistical methods appropriate for time-series data

• Data visualization for clarification:

  • Present contradictory data in a unified format for direct comparison

  • Use heat maps to visualize ATG32 expression across different conditions and time points

• Consider functional outcomes:

  • Correlate ATG32 expression levels with mitophagy efficiency

  • Low protein levels with high mitophagy rates may indicate that activated ATG32 is rapidly degraded after initiating mitophagy

What bioinformatic approaches are most effective for identifying potential ATG32 homologs across yeast species?

For identifying potential ATG32 homologs across yeast species, particularly in less-studied organisms like L. thermotolerans, the following bioinformatic approaches are most effective:

• Sequence-based homology searches:

  • BLASTP searches using known ATG32 sequences against fungal proteomes

  • PSI-BLAST for detecting remote homologs using position-specific scoring matrices

  • HMMer profiles built from multiple sequence alignments of known ATG32 proteins

• Domain and motif identification:

  • Search for conserved functional motifs like the Atg8-family interacting motif (AIM)

  • Identify transmembrane domains characteristic of mitochondrial outer membrane proteins

  • Look for conserved ubiquitination sites, particularly around Lysine 282 in S. cerevisiae ATG32

• Structural prediction approaches:

  • Use AlphaFold or similar tools to predict protein structures

  • Compare predicted structures with known ATG32 structures or models

  • Identify structurally conserved regions that may maintain function despite sequence divergence

• Synteny analysis:

  • Examine conservation of gene order and chromosomal context around the ATG32 locus

  • Syntenic relationships often provide evidence for orthology even when sequence similarity is moderate

• Phylogenetic analysis:

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Include multiple autophagy receptor proteins to identify true ATG32 orthologs

  • Use reconciliation methods to distinguish orthologs from paralogs

• Functional prediction validation:

  • Analyze expression patterns in available transcriptomic data

  • Look for co-expression with other mitophagy and autophagy genes

  • Examine upregulation under conditions known to induce mitophagy

How does ATG32 function in L. thermotolerans compare to its role in other non-conventional yeasts?

ATG32 functions as a mitophagy receptor across yeast species, but comparative analyses reveal important distinctions in L. thermotolerans and other non-conventional yeasts:

• Evolutionary context:

  • The Lachancea genus was only properly classified in 2003 using multi-gene sequencing strategies

  • For almost 100 years, Lachancea strains were mislabeled, with L. fermentati placed in the Z. fermentati genus since 1928

  • This taxonomic history suggests that our understanding of protein function across these species is still developing

• Environmental adaptation:

  • L. thermotolerans is found in diverse environments including grape must, fruits, lake water, cocoa fermentation, and olive paste

  • This ecological versatility suggests potential specialization of mitophagy mechanisms to different environmental stressors

  • L. thermotolerans can survive in wine at the end of fermentation when co-fermented with Saccharomyces, indicating robust stress tolerance potentially involving mitophagy

• Metabolic context:

  • L. thermotolerans is known to produce lactic acid, which increases titratable acids - a trait valued in wine production from hot regions

  • The interplay between this metabolic characteristic and mitochondrial function may influence ATG32 regulation

  • L. thermotolerans ferments both glucose and sucrose but typically leaves residual sugars, indicating different metabolic regulation compared to Saccharomyces

  • The ability to assimilate trehalose in L. thermotolerans may be linked to stress tolerance through mechanisms potentially involving mitochondrial quality control

• Functional conservation:

  • The core mechanism of ATG32 as an autophagic degron likely remains conserved across yeasts

  • The bipartite interaction with Atg8 and Atg11 prior to isolation membrane formation appears to be a fundamental mechanism conserved from yeast to humans

  • The ability of the ATG32 cytosolic domain to recruit autophagy machinery to different organelles suggests functional flexibility that may vary between species

• Regulatory differences:

  • Post-translational regulation, particularly ubiquitination patterns, may differ between species

  • The dynamics of ATG32 expression during different growth phases or stress conditions might be species-specific

  • The molecular significance of ATG32 is broadly conserved, but regulatory fine-tuning likely adapts to the ecological niche of each yeast species

What experimental approaches can determine the specificity of L. thermotolerans ATG32 compared to S. cerevisiae ATG32?

To determine the specificity of L. thermotolerans ATG32 compared to S. cerevisiae ATG32, researchers should employ the following experimental approaches:

• Complementation studies:

  • Express L. thermotolerans ATG32 in S. cerevisiae atg32Δ mutants to assess functional complementation

  • Create chimeric proteins with domains swapped between the two species' ATG32 proteins to identify species-specific functional regions

  • Quantify mitophagy restoration in these complementation systems under various stress conditions

• Binding partner analysis:

  • Compare the affinity of each ATG32 variant for Atg8 and Atg11 from both species

  • Conduct competitive binding assays to assess preference

  • Perform structural studies (X-ray crystallography or cryo-EM) of the binding interfaces to identify species-specific interaction determinants

• Regulatory comparison:

  • Analyze ubiquitination patterns of both ATG32 proteins under identical conditions

  • Compare promoter regulation using reporter constructs in both native and heterologous contexts

  • Examine half-life and degradation kinetics of both proteins

• Environmental response profiling:

  • Subject both ATG32 proteins to a range of environmental stressors (temperature, pH, ethanol, osmotic stress)

  • Measure mitophagy efficiency under each condition

  • Identify conditions where performance diverges significantly between the two proteins

• Interactome mapping:

  • Perform immunoprecipitation coupled with mass spectrometry to identify all binding partners

  • Compare interactomes between species to find unique interactions

  • Validate functional significance of species-specific interactions