Recombinant Kluyveromyces lactis Altered inheritance of mitochondria protein 36, mitochondrial (AIM36)

What is AIM36 and what is its role in yeast mitochondria?

AIM36 (Altered inheritance of mitochondria protein 36) is a mitochondrial protein that plays critical roles in mitochondrial morphology and inheritance. It functions primarily in three key areas:

• Fission promotion: AIM36 (ortholog of Saccharomyces cerevisiae Mdm36) facilitates mitochondrial division by anchoring Dnm1 (dynamin-related protein) and Num1 (cell cortex protein) at fission sites. Deletion mutants (Δaim36) exhibit hyperconnected mitochondrial networks.

• Cortical tethering: It mediates attachment of mitochondria to the cell periphery, enabling tension generation needed for Dnm1-mediated membrane scission.

• Fusion antagonism: AIM36 counteracts mitochondrial fusion proteins like Fzo1, maintaining proper organelle morphological balance.

Functionally, deletion of AIM36 leads to mitochondrial aggregation and impaired motility, while overexpression can suppress defects in Δnum1 strains, confirming their functional overlap.

Why is Kluyveromyces lactis used as a model organism for mitochondrial research?

Kluyveromyces lactis serves as an excellent alternative yeast model to Saccharomyces cerevisiae for several compelling reasons:

• Respiratory metabolism dominance: Unlike S. cerevisiae, which is predominantly fermentative, K. lactis is adapted to aerobiosis and its respiratory system does not undergo glucose repression . This makes it particularly valuable for mitochondrial studies.

• FDA-approved GRAS status: K. lactis has Generally Regarded As Safe (GRAS) status approved by the Food and Drug Administration (FDA), making it suitable for various biotechnological applications .

• Unique physiological properties: K. lactis exhibits distinctly different physiological strategies compared to S. cerevisiae, particularly in the balance between respiration and fermentation .

• Comparative analysis potential: As noted by researchers, "With the recent development of powerful molecular genetic tools, Kluyveromyces lactis has become an excellent alternative yeast model organism for studying the relationships between genetics and physiology" .

What are the recommended protocols for cloning and expressing recombinant AIM36 in K. lactis?

Based on current research methodologies, the following protocol has proven effective for recombinant AIM36 expression:

• DNA isolation and gene amplification:

  • Isolate genomic DNA from K. lactis strain (e.g., MTCC 458) using a commercial DNA isolation kit

  • Amplify the aim36 gene using PCR with gene-specific primers

• Cloning strategy:

  • Clone the amplified gene into a pKLAC1 vector system with appropriate restriction sites (commonly NdeI/XhoI)

  • Include N-terminal 6xHis-tag for purification purposes

• Transformation and selection:

  • Transform competent K. lactis cells using electroporation (1.5 kV, 25 μF, 200 Ω)

  • Allow cells to recover in YPGlu medium for 30 min at 30°C with agitation

  • Select transformants on YCB agar medium containing 0.005 M acetamide

  • Incubate plates for 4 days at 30°C

• Expression conditions:

  • Cultivate transformants in YP medium with 2% glucose as starter culture

  • Transfer to YPGal medium (4% galactose) for induction

  • Grow at 30°C for optimal protein expression

• Verification:

  • Confirm positive clones by gene-specific PCR amplification, restriction digestion, and sequencing

What purification strategy yields the highest purity of recombinant AIM36?

The following multi-step purification process yields recombinant AIM36 with >90% homogeneity:

• Cell harvest and initial processing:

  • Harvest cells by centrifugation (6,000 × g for 20 min)

  • Filter supernatant sequentially through 0.8- and 0.2-μm cellulose acetate filters

• Ammonium sulfate precipitation:

  • Precipitate proteins with ammonium sulfate (90% saturation) at 4°C for 90 min

  • Centrifuge at 10,000 × g

• Affinity chromatography:

  • Apply to Ni-NTA column for His-tagged protein binding

  • Wash with buffer containing low imidazole concentration

  • Elute with increased imidazole concentration in multiple fractions

• Size exclusion chromatography:

  • Pool purified fractions and further purify by gel filtration chromatography

  • Confirm purity by SDS-PAGE (target: >95% homogeneity)

• Storage conditions:

  • Store purified protein at 4°C in buffer containing Tris (25-50 mM), NaCl (100 mM), and glycerol (10-15%)

  • For long-term storage, add 50% glycerol and store aliquots at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles

Commercial recombinant AIM36 preparations typically achieve purity greater than 90% as determined by SDS-PAGE .

What are the optimal storage conditions to maintain AIM36 stability and activity?

For optimal stability and activity of recombinant AIM36, the following storage conditions are recommended:

• Short-term storage (up to one month):

  • Store at 4°C in buffer containing:

    • 25-50 mM Tris

    • 100 mM NaCl

    • 10-15% glycerol

    • pH 7.0-7.4

• Long-term storage:

  • Store at -20°C/-80°C with 50% glycerol as cryoprotectant

  • Aliquot into small volumes to avoid repeated freeze-thaw cycles

• Reconstitution guidelines:

  • Centrifuge vials briefly before opening to bring contents to the bottom

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration before aliquoting

• Stability considerations:

  • Repeated freeze-thaw cycles significantly reduce protein activity

  • Working aliquots should be maintained at 4°C for no more than one week

What experimental approaches can be used to study AIM36's role in mitochondrial fission?

Several complementary approaches can be employed to study AIM36's function in mitochondrial fission:

• Gene deletion/knockdown studies:

  • CRISPR interference (CRISPRi) for rapid and efficient depletion of AIM36

  • Analysis of synthetic negative effects in combination with deletion of other genes (e.g., TOM70)

• Fluorescence microscopy:

  • Visualization of mitochondrial morphology using fluorescent proteins targeted to mitochondria

  • Time-lapse imaging to observe fission events in real-time

  • Comparison between wild-type and Δaim36 mutants to quantify differences in fission frequency and mitochondrial network connectivity

• Protein localization studies:

  • Immunofluorescence to detect endogenous AIM36

  • Co-localization with known fission site markers (Dnm1, Num1)

  • Analysis at different cell cycle stages to detect temporal regulation

• Proteomics approaches:

  • Affinity purification of ER and mitochondria coupled with mass spectrometry

  • Identification of proteins enriched in specific subcellular compartments upon loss of AIM36

  • Whole cell proteome analysis to detect changes in mitochondrial protein levels

Research has shown that "loss of both contact sites [including AIM36-related contacts] led to a strong decrease of many mitochondrial proteins in the whole cell proteome" .

How can protein-protein interactions of AIM36 be effectively investigated?

To investigate AIM36 protein interactions, researchers can employ these methodologies:

• Co-immunoprecipitation (Co-IP):

  • Use anti-His antibodies to pull down His-tagged AIM36

  • Identify binding partners by Western blot or mass spectrometry

  • Confirm interactions under various physiological conditions

• Yeast two-hybrid (Y2H) assays:

  • Screen for potential interaction partners using AIM36 as bait

  • Validate positive interactions with complementary methods

  • Map interaction domains through truncation mutants

• Proximity-dependent biotin identification (BioID):

  • Express AIM36 fused to a biotin ligase

  • Identify proteins in close proximity through biotinylation

  • Analyze biotinylated proteins by mass spectrometry

• Surface plasmon resonance (SPR):

  • Measure binding kinetics between purified AIM36 and potential partners

  • Determine association/dissociation constants

  • Evaluate effects of mutations on binding efficiency

• Pull-down assays with recombinant proteins:

  • Use purified recombinant AIM36 to capture interaction partners

  • Particularly useful for studying interactions with Dnm1 and Num1

Studies have revealed that AIM36 interacts with key mitochondrial fission machinery components, and these "protein interaction assays [are used for] identifying binding partners (e.g., Dnm1, Num1) via pull-down experiments".

What phenotypic changes are observed in mitochondria upon AIM36 deletion or overexpression?

Deletion or overexpression of AIM36 results in distinct mitochondrial phenotypes:

AIM36 Deletion (Δaim36):

• Morphological changes:

  • Hyperconnected mitochondrial networks

  • Reduction in fission events

  • Mitochondrial aggregation

• Functional defects:

  • Impaired mitochondrial motility

  • Altered distribution of mitochondria within cells

  • Possible impacts on respiratory capacity

• Molecular consequences:

  • Strong decrease in levels of many mitochondrial proteins

  • Enrichment of specific proteins (mainly inner membrane proteins) on the ER

AIM36 Overexpression:

• Suppression of mutant phenotypes:

  • Rescues defects in Δnum1 strains

  • Confirms functional overlap with Num1

• Enhanced fission:

  • Increased mitochondrial fragmentation

  • Antagonism of fusion proteins like Fzo1

• Potential stress responses:

  • Changes in reactive oxygen species (ROS) levels

  • Altered expression of stress-response genes

These observations help establish AIM36 as a critical factor in maintaining proper mitochondrial morphology and distribution within yeast cells.

How does AIM36 function differ between K. lactis and S. cerevisiae?

AIM36 function shows both similarities and differences between K. lactis and S. cerevisiae:

Research indicates that "AIM36 functions differ between K. lactis and S. cerevisiae, necessitating cautious cross-species comparisons". Additionally, "the nature of the hypoxic transcriptional response in K. lactis differed notably from S. cerevisiae" , which may influence AIM36 function in different cellular contexts.

What homology does AIM36 share with proteins in other organisms?

AIM36 shows interesting evolutionary relationships across different organisms:

• Yeast species homologs:

  • High conservation among Saccharomyces and Kluyveromyces species

  • Present in Lachancea thermotolerans (formerly Kluyveromyces thermotolerans)

  • Variably conserved in other fungi with species-specific adaptations

• Functional homologs in distant species:

  • Shares roles with Trypanosoma brucei pATOM36 in mitochondrial outer membrane protein assembly

  • Substrate specificity differs between yeast AIM36 and trypanosome pATOM36

• Domain conservation:

• Structural features:

  • Contains transmembrane domains for anchoring to mitochondrial membranes

  • Includes protein-protein interaction motifs for binding partners like Dnm1 and Num1

The cross-species conservation of AIM36 function suggests its fundamental importance in mitochondrial dynamics, while the differences highlight evolutionary adaptations to specific cellular environments.

How can advanced microscopy techniques be applied to study AIM36's role in mitochondrial membrane dynamics?

Advanced microscopy approaches offer powerful tools for investigating AIM36 function:

• Super-resolution microscopy:

  • STED (Stimulated Emission Depletion) microscopy to resolve AIM36 localization beyond diffraction limit

  • PALM/STORM imaging to map precise distribution at mitochondrial fission sites

  • Quantitative analysis of AIM36 clustering during different phases of fission

• Live-cell imaging:

  • High-speed confocal microscopy to capture dynamic events

  • FRAP (Fluorescence Recovery After Photobleaching) to measure AIM36 mobility

  • Optogenetic approaches to trigger AIM36 recruitment to specific locations

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence imaging of AIM36 with ultrastructural analysis

  • Map AIM36 distribution to specific membrane domains and contact sites

  • Visualize membrane deformation during AIM36-mediated fission events

• Förster resonance energy transfer (FRET):

  • Measure nanoscale proximity between AIM36 and interaction partners

  • Detect conformational changes during active fission

  • Quantify interaction dynamics in real-time

• Cryo-electron tomography:

  • Visualize AIM36-containing complexes in near-native state

  • Reconstruct 3D architecture of fission sites

  • Identify structural changes in mitochondrial membranes during fission

These approaches would enhance understanding of how AIM36 mediates "mitochondrial attachment to the cell periphery, enabling tension generation for Dnm1-mediated membrane scission".

What are the methodological approaches for studying AIM36's role at ER-mitochondria contact sites?

To investigate AIM36's function at ER-mitochondria contact sites, researchers can employ these methodologies:

• Contact site isolation and characterization:

  • Density gradient centrifugation to isolate ER-mitochondria contact fractions

  • Immunoblotting to detect AIM36 in these fractions

  • Proteomics analysis to identify additional components

• In situ proximity labeling:

  • APEX2 or BioID fusions to AIM36 to identify proteins in proximity at contact sites

  • Split-BioID approaches to specifically label proteins at ER-mitochondria interfaces

  • Mass spectrometry identification of labeled proteins

• Synthetic biology approaches:

  • Engineered tethers to artificially increase ER-mitochondria contacts

  • Assessment of AIM36 recruitment to these synthetic contacts

  • Functional complementation studies with artificial tethers in Δaim36 cells

• CRISPRi-based depletion studies:

  • "Using an inducible CRISPR interference (CRISPRi) system" to "rapidly and efficiently deplete" contact site components like Mdm34

  • Analysis of "synthetic negative effect in combination with a deletion of TOM70"

  • Evaluation of impacts on mitochondrial protein distribution

• Quantitative contact site measurements:

  • Split fluorescent protein systems that emit signal only at contact sites

  • FRET-based sensors to measure contact site distances

  • Temporal correlation with mitochondrial fission events

Research has shown that "ER-mitochondria contact sites are critical for the transfer of proteins from the ER to mitochondria" , and AIM36 likely plays an important role in this process.

How might recombinant AIM36 be utilized for structural biology studies?

Recombinant AIM36 provides valuable material for structural investigations:

These approaches are facilitated by recombinant AIM36's properties as it "serves as a substrate for crystallography or NMR due to its solubility and tag". Structural studies would provide critical insights into how AIM36 mediates its multiple roles in mitochondrial dynamics.

How does AIM36 contribute to mitochondrial protein import and assembly?

AIM36 plays several roles in mitochondrial protein import and assembly:

• ER-mitochondria protein transfer:

  • Facilitates transfer of specific proteins from the ER to mitochondria

  • May be particularly important for inner membrane proteins

  • Functions alongside other factors at ER-mitochondria contact sites

• Outer membrane protein assembly:

  • Shares functional similarities with Trypanosoma brucei pATOM36

  • May assist in assembly of specific outer membrane proteins

  • Shows species-specific substrate preferences

• Impact on import machinery:

  • Affects mitochondrial distribution, which influences import efficiency

  • May regulate import machinery components through protein-protein interactions

  • Creates microenvironments conducive to efficient protein import

• ER-SURF pathway involvement:

  • Participates in the ER-SURF (ER-surface) pathway for mitochondrial protein import

  • "ER-SURF pathway uses ER-mitochondria contact sites"

  • "Membrane contact sites are critical for the transfer of proteins from the ER to mitochondria"

Research has demonstrated that "using affinity purification of ER and mitochondria in conjunction with mass spectrometry... a specific set of mitochondrial proteins are enriched on the ER upon loss of [contact sites], which mainly were proteins of the inner membrane" , suggesting AIM36's importance in this process.

What are the emerging therapeutic applications of AIM36 research?

While primarily focused on basic research, AIM36 studies have potential therapeutic implications:

• Neurodegenerative disease insights:

  • Mitochondrial dynamics dysfunction is implicated in Parkinson's, Alzheimer's, and other neurodegenerative diseases

  • AIM36 research may illuminate conserved mechanisms of mitochondrial fission/fusion regulation

  • Could identify new therapeutic targets for diseases with impaired mitochondrial dynamics

• Metabolic disorder connections:

  • Mitochondrial function is central to cellular metabolism

  • Understanding AIM36's role may reveal new approaches to metabolic disorders

  • Particularly relevant given K. lactis' distinct respiratory metabolism

• Cancer research applications:

  • Altered mitochondrial dynamics is a hallmark of many cancers

  • AIM36 homologs in human cells may represent unexplored cancer targets

  • Knowledge from yeast models could inform human mitochondrial research

• Drug development potential:

  • Recombinant AIM36 can serve as a platform for screening small molecule modulators

  • Identified compounds might be developed into therapeutics for mitochondrial disorders

  • "While not yet explored, mitochondrial dynamics proteins like AIM36 could inform therapies for fission/fusion disorders"

As research progresses, "AIM36 studies enhance understanding of mitochondrial efficiency", which may ultimately contribute to developing interventions for human mitochondrial diseases.

What future research directions might yield the most significant insights into AIM36 function?

Several promising research avenues could substantially advance understanding of AIM36:

• Systems biology approaches:

  • Integration of proteomics, genomics, and metabolomics data

  • Network analysis of AIM36 interactions across different conditions

  • Computational modeling of mitochondrial dynamics with and without AIM36

• Comparative analysis across species:

  • Systematic comparison of AIM36 function in respiratory vs. fermentative yeasts

  • Identification of human functional analogs

  • Evolutionary analysis of mitochondrial fission machinery across eukaryotes

• Single-cell studies:

  • Analysis of cell-to-cell variability in AIM36 expression and function

  • Correlation with mitochondrial network states

  • Live-cell tracking of fission/fusion dynamics in relation to AIM36 levels

• Technical innovations:

  • Development of AIM36-specific biosensors to track activity in vivo

  • CRISPR-based screening for genetic interactions

  • Optogenetic tools to manipulate AIM36 function with spatiotemporal precision

• Translational research:

  • Investigation of AIM36 homologs in human cells

  • Screening for small molecules that modulate AIM36 activity

  • Exploration of potential therapeutic applications

These approaches would build upon current understanding that "AIM36 is critical for mitochondrial morphology and inheritance" and could reveal new fundamental insights into mitochondrial biology with potential applications in medicine and biotechnology.