Recombinant Lachancea thermotolerans Altered inheritance of mitochondria protein 36, mitochondrial (AIM36)

What are the known alternative names and gene designations for AIM36?

AIM36 is also referred to as "Found in mitochondria protein 39" (FMP39). The gene encoding this protein in Lachancea thermotolerans has the ordered locus name KLTH0F10164g. When working with genomic databases, searching for both AIM36 and FMP39 designations will ensure comprehensive results .

What is the current understanding of AIM36's function in mitochondrial inheritance?

AIM36 plays a critical role in the inheritance and distribution of mitochondria during cell division. Research suggests that AIM36 contributes to proper mitochondrial segregation during budding, potentially through interactions with the mitochondrial membrane and cytoskeletal elements. Disruption of AIM36 function may lead to altered mitochondrial morphology and inheritance patterns.

When investigating AIM36 function, researchers should consider its role in the context of L. thermotolerans' adaptation to fermentative conditions, where mitochondrial function and inheritance patterns may differ from those observed in standard laboratory yeasts like S. cerevisiae .

What are the recommended methods for expressing and purifying recombinant L. thermotolerans AIM36?

For optimal expression and purification of recombinant L. thermotolerans AIM36:

• Expression system selection: While E. coli systems can be used, yeast expression systems (particularly K. lactis or P. pastoris) often yield better results for mitochondrial proteins with proper folding and post-translational modifications.

• Vector design: Include an appropriate tag (His, GST, or FLAG) to facilitate purification. Consider using the natural sequence from L. thermotolerans strain ATCC 56472 / CBS 6340 / NRRL Y-8284 as reference .

• Purification protocol:

  • Lyse cells in Tris-based buffer with protease inhibitors

  • Perform affinity chromatography based on chosen tag

  • For better purity, follow with size exclusion chromatography

  • Store in Tris-based buffer with 50% glycerol at -20°C, with working aliquots at 4°C for up to one week

• Quality control: Verify protein integrity via SDS-PAGE and Western blotting, with functional assays to confirm activity .

What experimental approaches are most effective for studying AIM36's role in mitochondrial function?

To investigate AIM36's role in mitochondrial function, a multi-faceted approach is recommended:

• Localization studies: Use fluorescent tagging (GFP fusion) to confirm mitochondrial localization in live cells, combined with co-localization studies using established mitochondrial markers.

• Gene knockout/knockdown: CRISPR-Cas9 editing of L. thermotolerans to create AIM36 deletion mutants, followed by phenotypic analysis focusing on:

  • Mitochondrial morphology (using fluorescent microscopy)

  • Mitochondrial inheritance during cell division

  • Respiratory capacity and fermentation efficiency

  • Thermotolerance phenotypes at elevated temperatures (35-37°C)

• Protein-protein interaction studies: Use pull-down assays and co-immunoprecipitation to identify binding partners of AIM36, particularly those related to mitochondrial function and inheritance.

• Transcriptomic analysis: Compare expression patterns of AIM36 under different growth conditions, particularly contrasting fermentative versus respiratory metabolism .

How can researchers effectively compare AIM36 function across different yeast species?

When conducting comparative studies of AIM36 across yeast species:

• Sequence alignment and phylogenetic analysis: Align AIM36 sequences from L. thermotolerans, S. cerevisiae, and other yeasts to identify conserved domains and species-specific variations.

• Complementation studies: Express L. thermotolerans AIM36 in S. cerevisiae AIM36 deletion mutants to assess functional conservation.

• Cross-species localization: Determine if localization patterns are conserved when expressing tagged versions of AIM36 from various species in different host organisms.

• Comparative phenotypic analysis: Examine phenotypes related to mitochondrial inheritance, morphology, and function across species with modifications to AIM36.

• Correlation with ecological niche: Analysis of AIM36 sequence diversity in relation to the ecological origin of strains (anthropized versus wild) may reveal adaptations specific to fermentative environments .

What is the relationship between AIM36 and the thermotolerance phenotype of L. thermotolerans?

The relationship between AIM36 and thermotolerance in L. thermotolerans remains an active area of investigation. Methodological approaches to explore this relationship include:

• Expression analysis: Quantify AIM36 expression levels at different temperatures (30°C, 35°C, 37°C), comparing thermotolerant strains to temperature-sensitive ones. Research has shown that L. thermotolerans strains evolved under bacterial selection pressure can grow at 37°C, whereas ancestral strains are inhibited above 35°C .

• Mutant phenotyping: Compare the temperature sensitivity of AIM36 deletion mutants versus wild-type strains. Document growth curves, viability, and metabolic activity at elevated temperatures.

• Mitochondrial function assessment: Measure respiratory capacity and mitochondrial membrane potential at different temperatures in wild-type versus AIM36 mutants.

• Correlation studies: Analyze whether natural sequence variations in AIM36 across L. thermotolerans isolates correlate with their maximum growth temperatures .

The data suggest that proper mitochondrial function, potentially mediated by AIM36, might be crucial for thermotolerance, especially in strains that have adapted to higher temperatures through evolutionary processes .

How does AIM36 expression vary across different L. thermotolerans subpopulations?

L. thermotolerans exhibits significant genomic diversity across different geographical and ecological niches, with six well-defined subpopulations identified: Americas, Asia, Canada-trees, Europe/Domestic-1, Europe/Domestic-2, and Europe-mix . To investigate AIM36 expression variation:

• Transcriptomic comparison: RNA sequencing data from 23 strains representing these six subpopulations reveals distinct gene expression patterns between wild and anthropized strains. Researchers should examine:

  • Whether AIM36 expression differs between wild (Americas, Asia, Canada-trees) and anthropized (Europe/Domestic) populations

  • If expression correlates with fermentation capacity or lactic acid production

• Regulatory element analysis: Examine promoter regions of AIM36 across subpopulations to identify potential regulatory differences that might explain expression variation.

• Environmental response: Compare AIM36 expression under standard laboratory conditions versus winemaking conditions (synthetic grape must) across different subpopulations .

• Correlation with metabolic profiles: Analyze whether AIM36 expression levels correlate with differences in carbon metabolism, particularly in relation to glycolysis, respiration, and lactic acid production .

How has the AIM36 gene evolved across the Saccharomycetaceae family?

The evolutionary history of AIM36 across the Saccharomycetaceae family provides insights into its functional importance and adaptation. When studying AIM36 evolution:

• Phylogenetic analysis: Construct phylogenetic trees based on AIM36 sequences from multiple yeast species, including L. thermotolerans, S. cerevisiae, K. lactis, and other related yeasts.

• Selection pressure analysis: Calculate dN/dS ratios to determine if AIM36 has undergone purifying selection (conserved function) or positive selection (adaptation) across different lineages.

• Domain conservation: Identify highly conserved regions that likely correspond to essential functional domains versus variable regions that might reflect species-specific adaptations.

• Correlation with mitochondrial genome evolution: Compare AIM36 evolution with changes in mitochondrial genome structure and inheritance patterns across species.

• Post-WGD versus pre-WGD species comparison: Analyze differences in AIM36 between post-Whole Genome Duplication species (like S. cerevisiae) and pre-WGD species (like L. thermotolerans), considering that L. thermotolerans represents an evolutionary lineage that diverged prior to the whole genome duplication event in the Saccharomyces lineage .

What genetic variations in AIM36 exist among different L. thermotolerans strains, and do they correlate with phenotypic differences?

Investigating genetic variations in AIM36 across L. thermotolerans strains requires:

• Whole genome sequencing analysis: Using data from 145 L. thermotolerans strains, researchers can examine:

  • SNPs and indels in the AIM36 coding region

  • Regulatory region variations

  • Correlation of variants with subpopulation structure

• Structure-function correlation: Map identified variations onto the predicted protein structure to assess potential functional implications.

• Phenotype association studies: Correlate genetic variations with:

  • Thermotolerance levels (growth at 35-37°C)

  • Fermentative capacity

  • Mitochondrial morphology and inheritance patterns

  • Lactic acid production

• Environmental adaptation markers: Determine if specific AIM36 variants are enriched in strains from particular environments, especially comparing strains from anthropized (winemaking) environments versus wild habitats .

How can AIM36 be manipulated to enhance thermotolerance in industrial L. thermotolerans strains?

For researchers interested in enhancing thermotolerance through AIM36 manipulation:

• Overexpression strategies: Construct expression vectors with AIM36 under the control of constitutive or inducible promoters:

  • Test various promoter strengths to determine optimal expression levels

  • Create chimeric proteins containing AIM36 variants from highly thermotolerant strains

• Directed evolution approach:

  • Introduce random mutations in AIM36 using error-prone PCR

  • Screen for improved thermotolerance in L. thermotolerans

  • Sequence successful variants to identify beneficial mutations

• Co-evolution methodology: Following the approach described by Hranilovic et al. (2018), subject L. thermotolerans to bacterial selection pressure over multiple generations (approximately 400 generations) to naturally select for enhanced thermotolerance, then analyze AIM36 sequences from the evolved strains .

• CRISPR-based engineering: Target specific residues in AIM36 for modification based on comparative analysis with thermophilic organisms.

• Assessment protocols: Standardized methods to evaluate:

  • Growth rate at elevated temperatures (35-37°C)

  • Fermentation efficiency under temperature stress

  • Metabolic profile analysis, focusing on lactic acid production

  • Mitochondrial morphology and function

What methodological approaches are recommended for investigating the role of AIM36 in lactic acid production by L. thermotolerans?

L. thermotolerans is notable for its high lactic acid production during fermentation, particularly in anthropized strains. To explore AIM36's potential role in this process:

• Expression correlation analysis:

  • Compare AIM36 expression levels between high and low lactic acid-producing strains

  • Analyze temporal expression patterns during fermentation, particularly at peak lactic acid production (around 30 hours into fermentation)

• Metabolic flux analysis:

  • Use 13C-labeled glucose to trace carbon flow through glycolysis and into lactic acid

  • Compare flux distributions in wild-type versus AIM36 modified strains

• Redox balance investigation:

  • Measure NAD+/NADH ratios in relation to AIM36 expression

  • Assess whether AIM36 influences redox cofactor regeneration pathways

• Protein interaction studies:

  • Identify potential interactions between AIM36 and enzymes involved in pyruvate metabolism

  • Investigate whether AIM36 interacts with lactate dehydrogenase or related enzymes

• Co-expression network analysis:

  • Construct gene co-expression networks to identify genes whose expression patterns correlate with AIM36

  • Focus on correlations with genes involved in glycolysis, pyruvate metabolism, and lactic acid production

Researchers should note that anthropized L. thermotolerans strains show increased glycolytic flux and differential expression of genes related to sugar metabolism and fermentation, which may intersect with AIM36 function .

What are the recommended protocols for investigating AIM36's role in mitochondrial-nuclear crosstalk?

To explore AIM36's potential role in mitochondrial-nuclear communication:

• Subcellular fractionation and localization:

  • Isolate pure mitochondrial, nuclear, and cytosolic fractions

  • Determine AIM36 distribution across these compartments under different conditions

  • Use super-resolution microscopy to visualize potential contact sites

• Proximity-dependent labeling:

  • Use BioID or APEX2 fused to AIM36 to identify proximal proteins

  • Focus on proteins that shuttle between mitochondria and nucleus

• Transcription factor interaction studies:

  • Investigate whether AIM36 interacts with transcription factors or signaling molecules

  • Perform ChIP-seq after AIM36 perturbation to identify affected nuclear gene targets

• Retrograde signaling assessment:

  • Monitor expression of nuclear genes involved in mitochondrial biogenesis after AIM36 depletion

  • Analyze how AIM36 manipulation affects the cell's response to mitochondrial stress

• Genomic integration with mitochondrial studies:

  • Compare nuclear and mitochondrial DNA variations in relation to AIM36 sequence diversity

  • Investigate whether AIM36 variants affect mitochondrial DNA inheritance or integrity

The evolutionary divergence of L. thermotolerans prior to the whole genome duplication event may have resulted in distinct mechanisms for mitochondrial-nuclear communication compared to post-WGD species like S. cerevisiae .

What are the current limitations in AIM36 research in L. thermotolerans and how might they be addressed?

Current research on AIM36 in L. thermotolerans faces several challenges:

• Limited genetic tools: While genomic data is available, genetic manipulation systems for L. thermotolerans are less developed than for model yeasts.

  • Solution: Adapt CRISPR-Cas9 systems specifically optimized for L. thermotolerans, focusing on codon optimization and appropriate promoters

• Protein structural information gap: No crystal structure exists for AIM36.

  • Solution: Employ cryo-EM or computational structural biology approaches to predict structural features

• Functional redundancy: Potential overlapping functions with other mitochondrial proteins complicates phenotypic analysis.

  • Solution: Create multiple gene deletions and employ synthetic genetic array approaches

• Metabolic complexity: The interactions between mitochondrial function, lactic acid production, and thermotolerance are multifaceted.

  • Solution: Implement systems biology approaches integrating transcriptomics, proteomics, and metabolomics data

• Strain variation: The high genetic diversity across L. thermotolerans strains creates difficulties in generalizing findings.

  • Solution: Establish a reference panel of strains representing the six major subpopulations for standardized comparative studies

What emerging technologies might advance our understanding of AIM36 function in the coming years?

Emerging technologies likely to advance AIM36 research include:

• Single-cell technologies:

  • Single-cell RNA-seq to capture cell-to-cell variation in AIM36 expression

  • Single-cell proteomics to understand protein level heterogeneity

• Advanced imaging techniques:

  • Super-resolution microscopy combined with specific AIM36 labeling

  • Live-cell imaging to track AIM36 dynamics during mitochondrial inheritance

• CRISPR-based screening:

  • Genome-wide CRISPR screens to identify genetic interactions with AIM36

  • CRISPRi/CRISPRa for tunable control of AIM36 expression

• Long-read sequencing:

  • Improved assembly of L. thermotolerans genomes across diverse strains

  • Better characterization of structural variations affecting AIM36

• Protein structure prediction:

  • AI-based structure prediction tools like AlphaFold to model AIM36 structure

  • Molecular dynamics simulations to understand structural adaptations to temperature

• Synthetic biology approaches:

  • Creation of minimal mitochondrial systems to isolate AIM36 function

  • Designer mitochondrial networks to test inheritance mechanisms

These technologies will enable researchers to build a more comprehensive understanding of how AIM36 contributes to the unique biological properties of L. thermotolerans, particularly its adaptation to various environmental niches and its biotechnological potential.