Recombinant Lachancea thermotolerans Altered inheritance of mitochondria protein 39, mitochondrial (AIM39)

What is Lachancea thermotolerans and what makes it significant for research?

Lachancea thermotolerans is a non-Saccharomyces yeast species that has gained significant research interest due to its unique metabolic capabilities. It is particularly notable for its ability to convert sugars to lactic acid during alcoholic fermentation, which can improve the stability and balance of wines with high sugar content . L. thermotolerans represents an important evolutionary position in yeast phylogeny, having diverged after the appearance of anaerobic capability (estimated 125-150 million years ago) but before the whole-genome duplication event . This species shows extreme tolerance to high osmotic pressures and can grow in sugar concentrations up to 60% . Its adaptation to various environments, particularly enological (wine-related) settings, makes it valuable for studying yeast evolution and domestication processes .

What is the Altered inheritance of mitochondria protein 39 (AIM39) and what is its significance?

AIM39 (Altered inheritance of mitochondria protein 39) is a mitochondrial protein from Lachancea thermotolerans with the Uniprot identifier C5DDY0 . While the specific function of AIM39 in L. thermotolerans requires further research, its name suggests involvement in mitochondrial inheritance processes. The protein is encoded by the AIM39 gene (KLTH0C04708g) . The significance of studying AIM39 lies in understanding mitochondrial dynamics and inheritance in non-conventional yeasts, particularly since L. thermotolerans shows highly conserved mitochondrial genomes with extremely low intraspecific divergence rates (π = 0.0014) . This conservation suggests strong purifying selection or an exceptionally low mutation rate in mitochondrial genes, making AIM39 an interesting target for evolutionary and functional studies.

How does L. thermotolerans fit taxonomically among other yeast species?

L. thermotolerans is the type species of the genus Lachancea, which was previously included within the Kluyveromyces clade . Taxonomically, the Lachancea genus occupies a significant evolutionary position as it represents the first lineage after the loss of respiratory chain complex I, which occurred after the split of the Saccharomyces-Lachancea and Kluyveromyces-Eremothecium lineages approximately 125-150 million years ago . This evolutionary event enabled the emergence of the long-term Crabtree effect and the ability to grow under anaerobic conditions . The species was formerly known as Kluyveromyces thermotolerans before taxonomic reclassification . Studies of the Lachancea clade show greater diversity compared to species in the Saccharomyces clade, providing valuable insights into yeast evolution and adaptation .

What protocols are recommended for expressing recombinant L. thermotolerans AIM39?

For reliable expression of recombinant L. thermotolerans AIM39, researchers should consider the following methodological approach:

• Expression system selection: E. coli-based systems (BL21(DE3) or Rosetta strains) are commonly used for initial expression trials. For proper folding of mitochondrial proteins, consider yeast expression systems like Pichia pastoris.

• Vector design: Include appropriate tags (His, GST, or MBP) to facilitate purification. The tag type should be determined based on the specific experimental requirements .

• Expression conditions: Optimize temperature (typically 16-25°C for mitochondrial proteins), inducer concentration, and expression duration to maximize protein yield while maintaining proper folding.

• Buffer composition: For mitochondrial proteins like AIM39, use buffers containing 50% glycerol and Tris-based systems optimized for protein stability .

• Storage considerations: Store purified protein at -20°C or -80°C for extended storage. Avoid repeated freeze-thaw cycles, and maintain working aliquots at 4°C for up to one week .

The complete amino acid sequence of AIM39 is available, which facilitates primer design for cloning and expression studies .

What methods can be used to study AIM39's role in mitochondrial function?

Several complementary approaches can be employed to investigate AIM39's role in mitochondrial function:

• Gene knockout/knockdown studies: CRISPR-Cas9 or RNA interference can be used to reduce or eliminate AIM39 expression, followed by phenotypic analysis including:

  • Mitochondrial morphology assessment using fluorescent microscopy

  • Mitochondrial inheritance patterns during cell division

  • Respiratory capacity measurements

• Protein localization: Fluorescent protein tagging or immunofluorescence to confirm mitochondrial localization and determine specific sub-mitochondrial compartmentalization.

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify binding partners

  • Yeast two-hybrid screening

  • Proximity labeling approaches

• Functional complementation: Testing whether AIM39 from L. thermotolerans can complement phenotypes of mitochondrial inheritance mutants in model organisms like S. cerevisiae.

• Comparative genomics: Analyze the conservation of AIM39 across Lachancea species and correlate with mitochondrial inheritance patterns .

Since L. thermotolerans mitochondrial genomes are highly conserved with extremely low divergence rates , comparative approaches across strains may provide insights into critical functional domains of AIM39.

How might AIM39 contribute to L. thermotolerans' adaptation to fermentation environments?

L. thermotolerans shows specific adaptations to fermentation environments, particularly winemaking conditions . While the direct role of AIM39 in these adaptations remains to be fully elucidated, several mechanisms can be hypothesized and tested:

• Stress response regulation: Fermentation environments impose multiple stresses (osmotic, oxidative, ethanol). AIM39 may contribute to mitochondrial stability under these conditions, as mitochondrial function in L. thermotolerans has been shown to exhibit strong responses to anaerobic conditions and mixed cultures .

• Metabolic adaptation: L. thermotolerans is known for its ability to produce lactic acid during fermentation . AIM39 could potentially influence this metabolic pathway through effects on mitochondrial function, as the transcriptional analysis has shown that carbohydrate metabolism is significantly influenced under fermentation conditions .

• Cellular wall integrity: Under anaerobic fermentation conditions, L. thermotolerans activates genes for biogenesis and stabilization of the cell wall through β-glucan synthesis . If AIM39 affects mitochondrial signaling, it may indirectly contribute to this response.

• Population-level adaptation: The highly conserved nature of mitochondrial genomes in L. thermotolerans despite diverse ecological origins suggests strong selective pressure. AIM39's potential role in mitochondrial inheritance may contribute to maintaining advantageous mitochondrial genotypes during adaptation to fermentation environments.

Experimental approaches combining transcriptomics, proteomics, and functional assays under various fermentation conditions could help elucidate AIM39's specific contributions to these adaptive processes.

What are the challenges in structural characterization of L. thermotolerans AIM39?

Structural characterization of mitochondrial proteins like AIM39 presents several methodological challenges:

• Protein purification complexities:

  • Maintaining protein stability during extraction and purification

  • Ensuring proper folding of the mitochondrial protein when expressed recombinantly

  • Current protocols recommend storage in 50% glycerol in Tris-based buffer

• Membrane association challenges: If AIM39 interacts with mitochondrial membranes, structural studies may require:

  • Detergent screening for optimal solubilization

  • Lipid nanodisc reconstitution

  • Cryo-EM rather than crystallography approaches

• Post-translational modifications: Identifying and preserving any PTMs that may be critical for AIM39 function.

• Expression region considerations: The annotated expression region for AIM39 is amino acids 32-320 , suggesting potential processing of the full-length protein.

• Comparative structural biology: The low divergence rate in L. thermotolerans mitochondrial genomes suggests strong structural conservation, which may provide insight into critical structural domains.

Researchers should consider an integrated approach utilizing X-ray crystallography, NMR, and cryo-EM, along with computational modeling based on the available amino acid sequence to overcome these challenges.

What does mitochondrial genome conservation in L. thermotolerans reveal about AIM39 evolution?

The extraordinarily conserved mitochondrial genome of L. thermotolerans provides important context for understanding AIM39 evolution:

• Purifying selection: Mitochondrial genomes in L. thermotolerans show extremely low intraspecific divergence rates (π = 0.0014) . This suggests strong purifying selection acting on mitochondrial proteins, likely including AIM39.

• Functional constraints: The high conservation of mitochondrial coding sequences despite variation in intergenic regions indicates strong functional constraints on mitochondrial proteins. This suggests AIM39 may have a critical role that does not tolerate substantial sequence variation.

• Evolutionary timeline: The most recent genomic studies confirm that L. thermotolerans mitochondrial genomes have undergone few rearrangements during evolution . This stability provides a relatively unchanging genomic context for AIM39 function.

• Niche adaptation implications: Despite L. thermotolerans strains being isolated from diverse environments, mitochondrial conservation persists . This suggests AIM39's function may be independent of niche-specific adaptations, instead serving a core mitochondrial function.

• Species-specific features: Comparative analysis with related Lachancea species can highlight L. thermotolerans-specific sequence features of AIM39 that might correlate with the species' unique metabolic capabilities, such as lactic acid production .

Research approaches combining phylogenetics, population genomics, and functional analysis of AIM39 variants could further elucidate the evolutionary forces shaping this protein.

How can genome-wide association studies (GWAS) be applied to understand AIM39 function in different L. thermotolerans strains?

GWAS approaches offer powerful tools for relating genomic variation to phenotypic differences across L. thermotolerans strains:

• Study design considerations:

  • Population sampling should include strains from diverse environments (particularly wine-related vs. natural habitats)

  • Phenotyping should focus on mitochondrial inheritance, stress tolerance, and metabolic traits

  • Whole-genome sequencing should be employed rather than targeted approaches, given the potential for epistatic interactions

• Analytical approaches:

  • Standard GWAS methodologies must be adapted for haploid organisms

  • Population structure must be accounted for, as L. thermotolerans shows geographic and niche-based differentiation

  • The extremely low mitochondrial sequence variation means nuclear variants affecting AIM39 function may be more informative

• Integration with functional data:

  • Transcriptomic data showing differential expression under fermentation conditions

  • Proteomic analysis of AIM39 interactors

  • Metabolomic profiling, particularly focusing on lactic acid production

• Validation strategies:

  • Genetic modification of identified variants

  • Functional complementation tests

  • In vitro biochemical assays with recombinant proteins

Given the recent whole-genome sequencing studies of L. thermotolerans revealing adaptations to winemaking environments , GWAS approaches could reveal whether nuclear or mitochondrial AIM39 variants contribute to these adaptations.

What quality control measures are essential when working with recombinant L. thermotolerans AIM39?

When working with recombinant AIM39, researchers should implement the following quality control measures:

• Protein identity verification:

  • Mass spectrometry analysis to confirm protein mass

  • Peptide mapping to verify sequence coverage

  • Western blotting with specific antibodies

• Purity assessment:

  • SDS-PAGE with densitometry analysis (target >90% purity)

  • Size-exclusion chromatography to detect aggregates

  • Endotoxin testing for applications in cell culture systems

• Functional validation:

  • Activity assays (once the specific function is determined)

  • Thermal shift assays to assess protein stability

  • Circular dichroism to confirm proper folding

• Storage stability monitoring:

  • Aliquot at recommended storage conditions (-20°C or -80°C)

  • Avoid repeated freeze-thaw cycles as recommended in product documentation

  • Maintain working aliquots at 4°C for maximum one week

• Batch consistency:

  • Implement lot-to-lot comparison protocols

  • Maintain reference standards

  • Document production parameters

These quality control measures ensure experimental reproducibility and reliability when studying AIM39 structure and function.

How can isotope labeling techniques be applied to study AIM39 interactions and dynamics?

Isotope labeling offers powerful approaches for investigating AIM39 protein interactions and dynamics:

• NMR-based structural studies:

  • 15N/13C labeling of recombinant AIM39 for backbone and side-chain assignments

  • Selective amino acid labeling to focus on specific regions of interest

  • TROSY-based experiments for examining AIM39 in membrane-mimetic environments

• Interaction mapping:

  • Chemical cross-linking combined with mass spectrometry (CXMS)

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify interaction interfaces

  • FRET-based approaches with strategic fluorophore placement

• In vivo dynamics:

  • Pulse-chase experiments with heavy isotope-labeled amino acids

  • Dynamic SILAC to measure protein turnover rates

  • Targeted mass spectrometry methods (PRM/MRM) for quantifying AIM39 variants

• Metabolic interactions:

  • 13C-glucose labeling to trace metabolic pathways potentially influenced by AIM39

  • Particularly relevant given L. thermotolerans' unique lactic acid production capability

  • Integration with metabolic flux analysis

• Localization studies:

  • Correlative light and electron microscopy with isotope labels

  • Nanoscale secondary ion mass spectrometry (NanoSIMS) imaging

These methodologies can help elucidate how AIM39 functions within the context of L. thermotolerans' unique mitochondrial biology and metabolic adaptations to fermentation environments.