Recombinant Saccharomyces cerevisiae Altered inheritance of mitochondria protein 36, mitochondrial (AIM36)

What is AIM36 and what is its function in Saccharomyces cerevisiae?

AIM36 (Altered inheritance of mitochondria protein 36, mitochondrial) is a protein encoded by the AIM36 gene (YMR157C) in Saccharomyces cerevisiae. It is also known as FMP39 (Found in mitochondria protein 39) . This protein is localized to the mitochondria and plays a role in the proper inheritance of mitochondria during cell division in yeast.

The protein is classified as part of the mitochondrial proteome and has been identified through systematic screens for genes affecting mitochondrial function and inheritance. The specific molecular mechanisms by which AIM36 influences mitochondrial inheritance remain an active area of research, but studies suggest it contributes to mitochondrial membrane organization and dynamics during cell division.

How is recombinant AIM36 protein typically produced for research purposes?

Recombinant AIM36 protein is typically produced using E. coli expression systems. The general methodology involves:

• Cloning the AIM36 gene sequence (codons 41-255, excluding the mitochondrial targeting sequence) into a bacterial expression vector with an N-terminal His-tag .

• Transforming the construct into an E. coli expression strain.

• Inducing protein expression under optimized conditions.

• Purifying the protein using nickel-affinity chromatography based on the His-tag.

• Additional purification steps such as size-exclusion chromatography may be implemented to achieve higher purity.

• The final product is often lyophilized and stored with trehalose as a stabilizing agent .

For optimal storage and handling, the purified protein should be aliquoted and stored at -20°C to -80°C to prevent repeated freeze-thaw cycles that may compromise protein integrity .

What experimental systems are used to study AIM36 function in vivo?

Several experimental systems are employed to study AIM36 function in vivo:

• Gene deletion studies: AIM36 knockout strains are created using homologous recombination to replace the gene with a selectable marker. These strains are then analyzed for alterations in mitochondrial morphology, inheritance patterns, and cellular respiration.

• Fluorescent tagging: AIM36 can be fused with GFP or other fluorescent proteins to track its localization and dynamics in living cells.

• Red mutant hunts: This classical yeast genetics approach can identify mutations affecting AIM36 function or interacting pathways by screening for red colonies that indicate defects in respiratory capacity .

• Yeast mating assays: Confrontation tests using different mating types can help characterize the impact of AIM36 mutations on mitochondrial inheritance during sexual reproduction .

What methodological considerations are important when designing experiments to study AIM36's role in mitochondrial inheritance?

When designing experiments to study AIM36's role in mitochondrial inheritance, researchers should consider:

• Control for strain background effects: Different laboratory yeast strains may have subtle differences in mitochondrial behavior. Always include appropriate wild-type controls of the same genetic background .

• Temporal resolution: Mitochondrial inheritance is a dynamic process. Time-lapse microscopy with appropriate temporal resolution is essential to capture key events.

• Cell cycle synchronization: Since mitochondrial inheritance is coordinated with the cell cycle, methods to synchronize yeast cultures (such as alpha-factor arrest-release) should be considered to obtain coherent observations.

• Quantitative metrics: Develop clear quantitative metrics for assessing mitochondrial inheritance defects, such as:

  • Percentage of daughter cells receiving mitochondria

  • Volume/mass of mitochondria transferred to daughter cells

  • Time required for complete inheritance

• Combinatorial genetic approaches: Consider creating double mutants with genes known to affect mitochondrial dynamics (e.g., fission/fusion machinery) to position AIM36 in the broader network of mitochondrial inheritance.

How can mutations in AIM36 be systematically induced and characterized?

Systematic mutation analysis of AIM36 can be performed using the following methodological approach:

• UV mutagenesis protocol:

  • Prepare yeast cultures at approximately 10^6 cells/ml

  • Perform serial dilutions to achieve approximately 10^3 cells per plate

  • Expose plates to calibrated UV-C radiation for varying durations

  • Incubate plates at 30°C for 2-3 days until colonies are 2-3mm in diameter

  • Screen for phenotypes of interest (e.g., red colonies indicating respiratory defects)

• Targeted mutagenesis approaches:

  • Site-directed mutagenesis of conserved residues

  • Alanine-scanning mutagenesis of functional domains

  • CRISPR-Cas9 genome editing for precise mutations

• Characterization workflow:

  • Growth rate analysis on fermentable vs. non-fermentable carbon sources

  • Mitochondrial morphology assessment using fluorescent markers

  • Complementation tests with known mitochondrial inheritance mutants

  • Mating tests to assess mitochondrial inheritance during sexual reproduction

What approaches are recommended for analyzing AIM36 protein-protein interactions in the mitochondrial context?

Several complementary approaches are recommended for analyzing AIM36 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Express epitope-tagged AIM36 (e.g., His-tag as in commercially available constructs)

  • Solubilize mitochondrial membranes with appropriate detergents

  • Perform pull-down assays followed by mass spectrometry to identify interacting proteins

  • Validate interactions with reciprocal Co-IPs and Western blotting

• Proximity-based labeling:

  • Create fusion proteins of AIM36 with BioID or APEX2

  • Allow in vivo biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

  • This approach captures both stable and transient interactions in the native cellular environment

• Yeast two-hybrid screening:

  • Use the mature form of AIM36 (amino acids 41-255) as bait

  • Screen against cDNA libraries or focused mitochondrial protein libraries

  • Validate hits using orthogonal methods

• Fluorescence resonance energy transfer (FRET):

  • Create fluorescent protein fusions with AIM36 and candidate interactors

  • Measure FRET efficiency in living cells

  • This approach provides spatial information about interactions within mitochondria

When interpreting interaction data, researchers should consider that membrane proteins like AIM36 may require specific conditions to maintain proper folding and interaction capacity during experimental manipulation.

How does AIM36 research in yeast relate to mitochondrial inheritance patterns in higher organisms?

Research on AIM36 in yeast provides valuable insights that can be extrapolated to understand mitochondrial inheritance in higher organisms:

• Conserved mechanisms: While AIM36 itself may not have direct orthologs in mammals, many mechanisms of mitochondrial dynamics and inheritance are conserved across species. Studies in yeast can reveal fundamental principles applicable to human cells.

• Disease relevance: Understanding mitochondrial inheritance in yeast models can inform research on human mitochondrial diseases. Approximately 1 in 5,000 people suffer from mitochondrial diseases, and 1 in 8 women in the general population carries mitochondrial DNA mutations that can be transmitted to offspring .

• Bottleneck phenomenon: Both yeast and human cells exhibit a genetic bottleneck effect during mitochondrial inheritance, where only a subset of the mitochondrial DNA population is transmitted to the next generation. This phenomenon affects the segregation of mitochondrial mutations .

• Maternal inheritance patterns: While yeast has biparental inheritance of mitochondria (though with some bias), humans exhibit strict maternal inheritance. Nevertheless, the cellular machinery controlling mitochondrial segregation during cell division shares common features .

• Selection processes: Both systems show evidence of selection processes acting on mitochondrial genomes during inheritance, which influence the transmission of deleterious mutations .

Researchers studying AIM36 should consider these parallels when designing experiments and interpreting results in the broader context of mitochondrial biology across species.

What are the optimal experimental techniques for characterizing the biochemical properties of recombinant AIM36 protein?

Characterizing the biochemical properties of recombinant AIM36 requires multiple complementary approaches:

• Protein quality assessment:

  • SDS-PAGE to confirm purity (>90% is typical for commercial preparations)

  • Mass spectrometry to verify the intact mass and post-translational modifications

  • Circular dichroism to assess secondary structure integrity

  • Dynamic light scattering to evaluate homogeneity and aggregation state

• Membrane association studies:

  • Liposome binding assays with various lipid compositions mimicking mitochondrial membranes

  • Membrane insertion analysis using protease protection assays

  • Detergent solubility profiling to determine optimal conditions for maintaining native conformation

• Reconstitution experiments:

  • Reconstituting recombinant AIM36 into proteoliposomes

  • Measuring effects on membrane properties (fluidity, curvature)

  • Assessing interactions with other mitochondrial proteins in the reconstituted system

• Functional assays:

  • ATPase activity measurements (if applicable)

  • Membrane potential sensitivity assays

  • Protein folding stability under various pH and salt conditions

For reproducible results, it's critical to follow proper handling protocols including avoiding repeated freeze-thaw cycles and maintaining the protein in appropriate buffer conditions with stabilizing agents such as trehalose .

What are the most effective protocols for isolating and purifying native AIM36 from yeast mitochondria?

Isolating native AIM36 from yeast mitochondria requires a careful sequential approach:

• Yeast culture optimization:

  • Grow Saccharomyces cerevisiae in rich medium with a non-fermentable carbon source (e.g., glycerol) to induce mitochondrial proliferation

  • Harvest cells during logarithmic growth phase when mitochondrial content is highest

• Mitochondrial isolation:

  • Enzymatically digest the cell wall with zymolyase to create spheroplasts

  • Gently lyse spheroplasts using Dounce homogenization

  • Separate mitochondria through differential centrifugation

  • Further purify mitochondria using Percoll gradient centrifugation

• AIM36 extraction and purification:

  • Solubilize mitochondrial membranes with mild detergents (e.g., digitonin or DDM)

  • Perform immunoprecipitation using AIM36-specific antibodies

  • Alternatively, use affinity chromatography if working with tagged versions of AIM36

• Validation of purified protein:

  • Western blot analysis with AIM36-specific antibodies

  • Mass spectrometry analysis to confirm identity

  • Activity assays to verify native conformation

This approach preserves the native state of AIM36 including post-translational modifications and associated binding partners that may be absent in recombinant systems.

How can researchers effectively analyze AIM36 gene expression patterns under different experimental conditions?

To effectively analyze AIM36 gene expression patterns:

• Quantitative RT-PCR (qRT-PCR):

  • Design primers specific to AIM36 (YMR157C) with careful attention to avoid amplification of homologous sequences

  • Select appropriate reference genes for normalization (ACT1, TDH3, and ALG9 are commonly used in yeast)

  • Test expression under different carbon sources, growth phases, and stress conditions

• Northern blot analysis:

  • Provides information about transcript size and alternative splicing

  • Useful for validating qRT-PCR results with an orthogonal method

• RNA-Seq:

  • Offers genome-wide context for AIM36 expression changes

  • Reveals co-regulated genes that may function in the same pathway

  • Identifies potential regulatory elements through correlation analysis

• Reporter gene assays:

  • Clone the AIM36 promoter upstream of a reporter gene (e.g., GFP or lacZ)

  • Measure reporter activity under various conditions to map regulatory elements

  • Perform deletion analysis of the promoter to identify critical regulatory sequences

When analyzing expression data, consider the broader context of mitochondrial biogenesis and inheritance, as AIM36 regulation likely coordinates with other genes involved in these processes.

What methodological approaches can distinguish between direct and indirect effects of AIM36 deletion on mitochondrial function?

Distinguishing between direct and indirect effects of AIM36 deletion requires a multi-faceted approach:

• Acute depletion systems:

  • Employ auxin-inducible degron (AID) tags to rapidly deplete AIM36 protein

  • Use tetracycline-repressible promoters for controlled expression shutdown

  • Compare acute vs. chronic effects to separate primary from adaptive responses

• Complementation strategies:

  • Reintroduce wild-type AIM36 to confirm phenotype rescue

  • Test structure-function relationships with mutant variants

  • Use orthologous genes from related species to assess functional conservation

• Temporal analysis:

  • Establish a detailed timeline of events following AIM36 depletion

  • Early events (minutes to hours) are more likely to represent direct effects

  • Late events (many hours to days) may represent indirect or compensatory responses

• Proximity-based methods:

  • Use APEX2 or BioID fusions to identify proteins in direct physical proximity to AIM36

  • Compare the immediate interaction neighborhood with broader affected pathways

• In vitro reconstitution:

  • Purify component systems and test if AIM36 directly affects biochemical processes

  • Reconstitute minimal systems to test sufficiency for specific functions

This systematic approach helps researchers establish causality rather than mere correlation in the functional analysis of AIM36.

What are the recommended methods for studying AIM36 in the context of mitochondrial genetic bottlenecks?

Studying AIM36 in the context of mitochondrial genetic bottlenecks requires specialized approaches:

• Heteroplasmy establishment:

  • Create yeast strains with mixed populations of wild-type and marked mitochondrial genomes

  • Use mitochondrial-targeted restriction endonucleases or CRISPR systems to induce specific mitochondrial DNA modifications

  • Validate heteroplasmy levels using qPCR or next-generation sequencing

• Single-cell lineage tracking:

  • Isolate individual cells and follow the mitochondrial genotype through multiple generations

  • Use microfluidic devices to trap mother cells and collect daughter cells for analysis

  • Apply fluorescent markers to distinguish different mitochondrial genotypes in living cells

• Quantitative inheritance analysis:

  • Measure the variance in heteroplasmy levels between mother and daughter cells

  • Calculate bottleneck size using statistical models based on the observed segregation patterns

  • Compare bottleneck effects in wild-type versus AIM36 mutant strains

• Integration with human mitochondrial inheritance models:

  • Draw parallels with bottleneck phenomena observed in human mitochondrial inheritance

  • Apply mathematical models developed for human mitochondrial genetics to yeast data

  • Use findings to inform broader understanding of mitochondrial inheritance across species

This integrated approach can reveal whether AIM36 plays a role in controlling the mitochondrial genetic bottleneck, which has important implications for understanding both basic mitochondrial biology and inheritance of mitochondrial diseases.

How can researchers design experiments to elucidate the structure-function relationship of specific AIM36 domains?

To elucidate structure-function relationships in AIM36:

This systematic approach allows researchers to create a detailed map of which protein regions contribute to specific aspects of AIM36 function in mitochondrial inheritance.

What are the most sensitive methods for detecting subtle phenotypes in AIM36 mutant strains?

Detecting subtle phenotypes in AIM36 mutant strains requires highly sensitive methods:

• High-resolution microscopy techniques:

  • Super-resolution microscopy (PALM/STORM, STED) for detailed mitochondrial morphology

  • Live-cell time-lapse imaging with optimized temporal resolution

  • Quantitative image analysis using machine learning algorithms for unbiased detection of subtle morphological changes

• Single-cell analysis:

  • Microfluidic-based single-cell isolation and phenotyping

  • Flow cytometry with mitochondrial dyes to detect heterogeneity in populations

  • Single-cell transcriptomics to identify compensatory responses

• Metabolic profiling:

  • High-sensitivity respirometry to detect minor changes in respiratory capacity

  • Metabolomics to identify shifts in mitochondrial metabolism

  • Isotope labeling to track metabolic flux through specific pathways

• Genetic interaction mapping:

  • Synthetic genetic array (SGA) analysis to identify genetic interactions

  • Chemical-genetic screens to find conditions that enhance subtle phenotypes

  • Dosage suppression screens to identify functional relationships

• Competitive growth assays:

  • Long-term competition experiments between wild-type and mutant strains

  • Barcode sequencing for highly quantitative fitness measurements

  • Growth under varying environmental conditions to reveal condition-specific defects

These approaches can reveal phenotypes that might be missed by conventional assays, providing deeper insight into AIM36 function.

How can researchers effectively apply systems biology approaches to position AIM36 within the broader mitochondrial functional network?

Positioning AIM36 within the broader mitochondrial functional network through systems biology requires:

• Multi-omics integration:

  • Combine proteomics, transcriptomics, and metabolomics data from AIM36 mutants

  • Use computational methods to integrate diverse datasets

  • Apply network analysis to identify functional modules affected by AIM36 disruption

• Genetic interaction mapping:

  • Perform systematic genetic interaction screens (e.g., synthetic genetic array)

  • Create a genetic interaction profile for AIM36

  • Compare with profiles of other mitochondrial genes to identify functional neighborhoods

• Protein interaction network analysis:

  • Map all physical interactions of AIM36 using AP-MS or BioID approaches

  • Extend to second-degree interactions to build a comprehensive network

  • Apply clustering algorithms to identify functional modules

• Dynamic network modeling:

  • Develop mathematical models of mitochondrial inheritance incorporating AIM36

  • Test model predictions experimentally

  • Refine models iteratively based on new experimental data

• Comparative genomics:

  • Analyze conservation patterns of AIM36 and interacting partners across species

  • Identify co-evolution patterns suggesting functional relationships

  • Use evolutionary information to predict function of uncharacterized domains

How might findings from AIM36 research in yeast inform therapeutic approaches for human mitochondrial diseases?

Research on AIM36 in yeast can inform therapeutic approaches for human mitochondrial diseases in several ways:

• Mechanistic insights into mitochondrial inheritance:

  • Understanding fundamental processes of mitochondrial segregation during cell division

  • Identifying conserved machinery that influences mitochondrial DNA transmission

  • Developing methods to modulate inheritance of mitochondrial mutations

• Prediction of disease progression:

  • Improved models of mitochondrial genetic bottlenecks based on yeast studies

  • Better risk assessment for carriers of mitochondrial mutations

  • More accurate counseling for women carrying disease-causing mitochondrial DNA mutations

• Therapeutic target identification:

  • Discovery of conserved pathways that could be targeted in human cells

  • Screening platforms using humanized yeast to identify compounds affecting mitochondrial inheritance

  • Development of strategies to shift heteroplasmy levels away from pathogenic mutations

• Gene therapy approaches:

  • Insights from yeast mitochondrial biology informing mitochondrial gene editing strategies

  • Understanding mitochondrial genetic bottlenecks to optimize timing of therapeutic interventions

  • Identification of critical periods when interventions might be most effective

While AIM36 itself may not have a direct human ortholog, the cellular machinery governing mitochondrial inheritance has significant conservation between yeast and humans, making these translational connections valuable for therapeutic development.

What are the most promising future research directions for understanding AIM36's role in mitochondrial biology?

The most promising future research directions include:

• Structural biology:

  • Solving the high-resolution structure of AIM36

  • Mapping interaction surfaces with binding partners

  • Understanding how AIM36 interacts with mitochondrial membranes

• Integration with mitochondrial dynamics:

  • Investigating AIM36's relationship with mitochondrial fission and fusion machinery

  • Exploring connections to the mitochondrial contact site and cristae organizing system (MICOS)

  • Understanding how AIM36 coordinates with cytoskeletal elements during inheritance

• Single-molecule approaches:

  • Tracking AIM36 molecules in living cells using super-resolution microscopy

  • Measuring binding kinetics and stoichiometry in native contexts

  • Visualizing AIM36's role in mitochondrial membrane organization

• Evolutionary perspectives:

  • Comparative analysis of AIM36 across fungal species with different mitochondrial inheritance patterns

  • Identification of functional analogs in higher eukaryotes

  • Understanding how mitochondrial inheritance machinery has evolved

• Integration with human disease models:

  • Creating humanized yeast systems to study mechanisms relevant to human mitochondrial diseases

  • Using insights from AIM36 to better understand mitochondrial bottleneck phenomena in humans

  • Developing improved predictive models for mitochondrial disease inheritance

These research directions will contribute to a more comprehensive understanding of AIM36's role in mitochondrial biology and potentially reveal new approaches for addressing mitochondrial diseases in humans.