Recombinant Pichia pastoris Altered inheritance of mitochondria protein 31, mitochondrial (AIM31)

What is AIM31 and what is its alternative nomenclature in yeast systems?

AIM31 (Altered Inheritance of Mitochondria protein 31) is a mitochondrial protein originally identified in a screen designed to find proteins whose absence caused altered inheritance of mitochondrial DNA. The protein has been renamed RCF1 (Respiratory supercomplex factor 1) based on its functional role . In Pichia pastoris (Komagataella phaffii), AIM31/RCF1 is a member of the conserved hypoxia-induced gene 1 (Hig1) protein family and represents a novel component of the yeast cytochrome bc1-COX supercomplex .

Why is Pichia pastoris used as an expression system for recombinant proteins like AIM31?

Pichia pastoris (also known as Komagataella spp.) has attracted extensive attention as an efficient platform for recombinant protein production due to several advantages over other expression systems . Compared to Saccharomyces cerevisiae, P. pastoris shows distinct physiological adaptations, particularly in response to environmental stresses such as hypoxia, which can actually enhance protein secretion . This makes it particularly valuable for the production of complex proteins like AIM31 that require proper folding and post-translational modifications. Additionally, P. pastoris can grow to high cell densities and has a strong preference for respiratory growth, which is advantageous for large-scale protein production .

What are the optimal conditions for expressing recombinant AIM31 in Pichia pastoris?

For optimal expression of recombinant AIM31 in Pichia pastoris, researchers should consider the following methodological approach:

• Expression system selection: While AIM31 can be expressed in E. coli (as seen in the commercial product) , native expression in P. pastoris may provide better functional characteristics due to proper post-translational modifications.

• Oxygen conditions: Research has shown a beneficial effect of hypoxia on recombinant protein secretion in P. pastoris chemostat cultivations . For AIM31, which is involved in respiratory function, controlling oxygen availability during expression is crucial. Hypoxic conditions can trigger adaptive responses that may enhance protein yield and quality .

• Promoter selection: Strong constitutive promoters (PGAP, PTEF1) should be used when integrating the AIM31 gene into the P. pastoris chromosome for consistent expression .

• Growth parameters: Chemostat cultivation under controlled oxygen conditions (normoxic, oxygen-limiting, or hypoxic) allows for consistent production and comparative studies .

When transitioning from normoxic to hypoxic conditions, P. pastoris shows distinct transcriptional, proteomic, and metabolic flux adaptations in its core metabolism, which can be advantageous for recombinant protein production .

How should AIM31 protein be purified from Pichia pastoris cultures?

For efficient purification of AIM31 from P. pastoris cultures, the following methodological approach is recommended:

• Affinity tag selection: His-tagging the N-terminus of AIM31 facilitates purification via metal affinity chromatography .

• Cell lysis conditions: For mitochondrial proteins like AIM31, careful lysis procedures are essential to maintain protein integrity while releasing the protein from mitochondrial membranes.

• Solubilization method: Different detergents can be used depending on the experimental goals:

  • For maintaining supercomplex associations: mild digitonin solubilization

  • For isolating individual AIM31: Triton X-100 solubilization

• Post-purification processing: After purification, AIM31 protein should be lyophilized and stored as a powder. Upon reconstitution, it should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol added for long-term storage at -20°C/-80°C .

• Quality control: Purity should be assessed using SDS-PAGE (target >90% purity) .

What genetic manipulation techniques are most effective for AIM31 studies in Pichia pastoris?

Several genetic manipulation strategies can be employed for AIM31 studies in P. pastoris, with varying efficiencies:

• CRISPR/Cas9 system: Introduced to P. pastoris in 2016, this system significantly enhances targeting efficiency . For AIM31 studies, stable Cas9 expression through integrative expression combined with specific sgRNAs can achieve near 100% homologous recombination efficiency without deleting Ku70 .

• Homologous recombination (HR) enhancement:

  • P. pastoris naturally favors non-homologous end-joining (NHEJ) over HR

  • Deletion of dnl4 (coding for DNA ligase IV) or Ku70 can increase HR efficiency

  • Overexpression of RAD52 can improve HR efficiency while mitigating the chromosome instability issues associated with Ku70 mutation

  • Using longer homology arms improves targeting specificity

• Marker recycling strategies:

  • FRT/FLP recombinase or Cre/loxp systems for zeocin-resistance marker recycling

  • Acetamidase (amdS) as an effective selection marker with counter-selection using fluoroacetamide

  • CRISPR/Cas9 geneticin plasmids targeting the zeocin resistance gene for marker recycling

• Multi-loci gene integration: CRISPR/Cas9 with efficient gRNA targets enables the integration of multiple gene cassettes simultaneously for comprehensive AIM31 interaction studies .

What is the role of AIM31 in mitochondrial function and respiratory complexes?

AIM31 (RCF1) plays several critical roles in mitochondrial function:

• Cytochrome bc₁-COX supercomplex association: AIM31 is a component of the cytochrome bc₁-COX supercomplex, binding to both the cytochrome bc₁ and COX enzyme domains, though it associates more tightly with the COX complex .

• Interaction with Cox3: AIM31 displays a close physical relationship with Cox3 protein, and this association occurs independently of their final assembly into the COX enzyme. This suggests AIM31 may play a role in the early assembly or stabilization of Cox3 .

• Supercomplex assembly: AIM31, together with AIM38 (RCF2), is required for the correct assembly of the cytochrome bc₁-COX supercomplex. These proteins may act as bridges to support the assembly of the supercomplex state .

• COX enzyme activity: While loss of either AIM31 or AIM38 alone has minimal impact, loss of both proteins significantly affects COX enzyme activity and assembly of the peripheral COX subunits Cox12 and Cox13 .

• Oxygen consumption regulation: As a member of the Hig1 protein family, AIM31 likely plays a role in the regulation and functioning of mitochondrial COX complex, the most abundant oxygen-consuming enzyme of the cell .

How do AIM31 and AIM38 (RCF2) functionally interact in Pichia pastoris?

AIM31 (RCF1) and AIM38 (RCF2) show overlapping but distinct functions in P. pastoris mitochondria:

How does hypoxia affect AIM31 expression and function in Pichia pastoris?

The relationship between hypoxia and AIM31 function in P. pastoris reveals important insights:

• Transcriptional regulation: As a member of the hypoxia-induced gene 1 (Hig1) protein family, AIM31 expression is likely regulated by oxygen availability. P. pastoris shows distinct transcriptional adaptation of its core metabolism to hypoxia .

• Respiratory adaptation: Under hypoxic conditions, P. pastoris undergoes significant changes in its respiratory machinery, including modifications to the cytochrome bc₁-COX supercomplex where AIM31 functions .

• Metabolic shifts: Hypoxia induces important changes in lipid metabolism, stress responses, protein folding, and trafficking in P. pastoris, all of which may affect AIM31 function or depend on its proper operation .

• Species-specific responses: The physiological adaptation of P. pastoris to hypoxia shows distinct traits compared to the model yeast S. cerevisiae, suggesting unique roles for proteins like AIM31 in different yeast species .

• Protein secretion enhancement: Hypoxia has been reported to have a beneficial effect on recombinant protein secretion in P. pastoris, which may involve mitochondrial adaptations mediated by proteins like AIM31 .

How can AIM31 mutational studies inform our understanding of mitochondrial supercomplex assembly?

Systematic mutational analysis of AIM31 can provide valuable insights into mitochondrial supercomplex assembly through the following approaches:

• Domain mapping: Creating truncated versions of AIM31 can help identify which regions are essential for:

  • Binding to the cytochrome bc₁ complex

  • Binding to the COX complex

  • Interaction with Cox3 protein

  • Supercomplex stability

• Site-directed mutagenesis: Targeting conserved residues in AIM31 can reveal:

  • Critical amino acids for protein-protein interactions

  • Residues involved in supercomplex assembly

  • Potential regulatory sites affected by cellular conditions

• Chimeric protein studies: Creating fusion proteins between AIM31 and AIM38 can help determine:

  • Whether functional domains are interchangeable

  • If specific regions confer specificity for certain interactions

  • How structural differences relate to their overlapping functions

• Cross-species complementation: Testing whether AIM31 homologs from other species can rescue AIM31 deletion phenotypes in P. pastoris can reveal:

  • Evolutionarily conserved functions

  • Species-specific adaptations

  • Critical structural features maintained across evolution

• Interaction with assembly factors: Investigating how AIM31 mutations affect interactions with known assembly factors can illuminate:

  • The sequence of assembly events

  • Cooperative vs. independent assembly steps

  • Quality control mechanisms for supercomplex formation

What techniques are most effective for studying AIM31 protein interactions within the respiratory chain complexes?

To effectively study AIM31 interactions within respiratory chain complexes, researchers should consider these advanced methodological approaches:

• Blue Native-PAGE (BN-PAGE): This technique preserves protein-protein interactions and can reveal:

  • The integrity of the cytochrome bc₁-COX supercomplex

  • Changes in supercomplex composition in response to mutations or environmental conditions

  • The distribution of AIM31 across different respiratory complexes

• Affinity purification with mild detergents:

  • Digitonin solubilization maintains supercomplex integrity while allowing purification

  • His-tagged components (cytochrome c₁, Aac2, AIM31 itself) can be used as baits to characterize interaction networks

• In organello protein synthesis:

  • Radiolabeling newly synthesized mitochondrial proteins in isolated mitochondria

  • Affinity purification of AIM31 and associated newly synthesized proteins

  • This approach revealed AIM31's association with newly synthesized Cox3 independent of their final assembly into the COX enzyme

• Proximity labeling approaches:

  • BioID or APEX2 fusion to AIM31 to identify proximal proteins

  • Time-resolved proximity labeling to capture dynamic interaction changes

  • Cross-linking mass spectrometry to map interaction interfaces

• Cryo-electron microscopy:

  • Structural visualization of the supercomplex with and without AIM31

  • Mapping AIM31's position within the supercomplex

  • Identifying conformational changes induced by AIM31 binding

How does the role of AIM31 differ between Pichia pastoris and Saccharomyces cerevisiae?

Understanding the differences in AIM31 function between yeast species provides valuable comparative insights:

• Transcriptional regulation differences:

  • P. pastoris shows distinct transcriptional, proteomic, and metabolic flux adaptations of core metabolism to hypoxia compared to S. cerevisiae

  • This suggests species-specific regulation of genes like AIM31 in response to oxygen availability

• Respiratory metabolism adaptations:

  • P. pastoris has a stronger preference for respiratory growth compared to S. cerevisiae

  • This fundamental metabolic difference likely influences the role and importance of respiratory chain components like AIM31

• Genetic manipulation considerations:

  • P. pastoris relies more heavily on non-homologous end-joining (NHEJ) for DNA repair, while S. cerevisiae favors homologous recombination

  • This affects experimental approaches for studying AIM31 in each organism

• Protein production implications:

  • P. pastoris shows enhanced protein secretion under hypoxic conditions

  • The connection between mitochondrial function (where AIM31 operates) and protein secretion may differ between the species

• Evolutionary conservation analysis:

  • Despite functional similarities, sequence divergence between the species' AIM31 homologs may reveal adaptations to different ecological niches

  • Studying these differences can provide insights into the evolution of mitochondrial supercomplex assembly

What are the main challenges in maintaining AIM31 stability during purification and storage?

Researchers often encounter several challenges when working with AIM31 protein:

• Protein solubility concerns:

  • As a mitochondrial membrane-associated protein, AIM31 can aggregate during purification

  • Recommendation: Use appropriate detergents at optimal concentrations; digitonin for maintaining native interactions or Triton X-100 for higher solubility

• Storage stability issues:

  • AIM31 protein may lose activity during freeze-thaw cycles

  • Recommendation: Store as lyophilized powder; after reconstitution, add 5-50% glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C; avoid repeated freeze-thaw cycles

• Buffer composition effects:

  • Buffer components can significantly impact AIM31 stability

  • Recommendation: Use Tris/PBS-based buffer with 6% Trehalose, pH 8.0 for optimal stability

• Reconstitution procedures:

  • Improper reconstitution can lead to protein aggregation or activity loss

  • Recommendation: Briefly centrifuge vials before opening; reconstitute to 0.1-1.0 mg/mL in deionized sterile water

• Quality control assessments:

  • Verifying functional integrity after purification can be challenging

  • Recommendation: Combine SDS-PAGE purity assessment (>90%) with functional assays measuring interaction with known partners like Cox3

How can researchers overcome genetic manipulation challenges when studying AIM31 in Pichia pastoris?

When genetically manipulating AIM31 in P. pastoris, researchers can address common challenges through these approaches:

• Low homologous recombination efficiency:

  • Challenge: P. pastoris preferentially uses NHEJ over HR for DNA repair

  • Solution: Either delete dnl4 (DNA ligase IV) or Ku70, or overexpress RAD52 to improve HR efficiency

• Random integration issues:

  • Challenge: Foreign DNAs may undergo ectopic integration even with homology arms

  • Solution: Use longer homology arms (>1kb) or introduce the HR machinery from S. cerevisiae with multiplex genome integration

• Multiple genetic modifications limitations:

  • Challenge: Shortage of selective markers limits multiple modifications

  • Solution: Employ marker recycling using FRT/FLP or Cre/loxp systems, or use acetamidase (amdS) with fluoroacetamide counter-selection

• CRISPR/Cas9 optimization:

  • Challenge: Some genes like OCH1 show low mutation efficiency (50%)

  • Solution: Add hydroxyurea during transformation to stop cell division at S/G2 phase where HR is more active than NHEJ

• Chromosome stability concerns:

  • Challenge: Ku70 mutation can negatively impact chromosome terminal stability

  • Solution: Overexpress RAD52 to improve HR efficiency while maintaining chromosome stability

What controls are essential when assessing AIM31 function in respiratory complex assembly studies?

To ensure robust and reproducible results when studying AIM31's role in respiratory complex assembly, the following controls are essential:

• Genetic complementation controls:

  • Include AIM31 deletion strain (ΔAIM31)

  • Include AIM31 deletion strain with reintroduced wild-type AIM31

  • Include double deletion strain (ΔAIM31ΔAIM38) to account for functional redundancy

• Respiratory activity measurements:

  • Compare oxygen consumption rates between wild-type and mutant strains

  • Measure individual complex activities (Complex III and IV) alongside supercomplex activity

  • Assess growth rates under different carbon sources requiring respiratory function

• Protein interaction validations:

  • Perform reverse pull-down experiments (using Cox3 to pull down AIM31)

  • Include negative controls (unrelated mitochondrial proteins)

  • Use size exclusion chromatography to verify complex formations independently

• Expression level considerations:

  • Ensure comparable expression levels between wild-type and tagged versions

  • Use inducible promoters to assess dosage-dependent effects

  • Monitor expression levels across different growth conditions

• Functional rescue experiments:

  • Test whether AIM38 overexpression can rescue AIM31 deletion phenotypes

  • Evaluate whether human homologs can complement yeast AIM31 function

  • Create partial function mutants to map specific functional domains

What are the potential applications of AIM31 research in understanding mitochondrial diseases?

Research on AIM31 has significant implications for understanding mitochondrial diseases through several avenues:

• Respiratory chain supercomplex assembly disorders:

  • AIM31's role in supercomplex assembly suggests its homologs could be involved in human disorders affecting respiration

  • Understanding the molecular mechanisms of AIM31 function may reveal new therapeutic targets for mitochondrial diseases

• Hypoxia response mechanisms:

  • As a member of the hypoxia-induced gene 1 (Hig1) family, AIM31 research provides insights into cellular adaptation to low oxygen

  • This knowledge is relevant to conditions involving tissue hypoxia, including stroke, cardiac ischemia, and cancer

• Mitochondrial DNA inheritance disorders:

  • AIM31's original identification in a screen for altered mitochondrial DNA inheritance suggests it may influence mtDNA stability

  • This connection could illuminate mechanisms in diseases characterized by mtDNA deletions or depletions

• Comparative studies across species:

  • The distinct physiological adaptations of P. pastoris compared to S. cerevisiae offer valuable comparative insights

  • These differences might reveal evolutionary adaptations in mitochondrial function relevant to human disease

• Protein folding and quality control:

  • AIM31's involvement in respiratory complex assembly connects to broader questions of protein quality control

  • This aspect has implications for neurodegenerative diseases involving protein misfolding

How might advanced structural biology techniques advance our understanding of AIM31?

Emerging structural biology approaches could significantly enhance our understanding of AIM31 through:

• Cryo-electron microscopy advances:

  • High-resolution structures of AIM31 within the respiratory supercomplex

  • Visualization of conformational changes during assembly or in response to different metabolic states

  • Mapping of interaction interfaces at atomic resolution

• Integrative structural biology approaches:

  • Combining X-ray crystallography, NMR, SAXS, and computational modeling

  • Creating dynamic models of AIM31's role in supercomplex assembly

  • Predicting effects of mutations on structure and function

• Time-resolved structural studies:

  • Capturing intermediate states during assembly processes

  • Visualizing dynamic interactions between AIM31 and its partners

  • Understanding the temporal sequence of supercomplex formation

• In-cell structural biology:

  • Studying AIM31 structure in its native mitochondrial environment

  • Correlating structural features with functional states in intact cells

  • Observing physiological responses to environmental changes

• Computational structure prediction:

  • Applying AlphaFold2 and similar AI tools to model AIM31 interactions

  • Simulating the dynamics of AIM31-containing complexes

  • Virtual screening for compounds that might modulate AIM31 function

What emerging technologies might revolutionize the study of proteins like AIM31 in Pichia pastoris?

Several cutting-edge technologies are poised to transform research on mitochondrial proteins like AIM31 in P. pastoris:

• Genome editing advancements:

  • Next-generation CRISPR systems with higher specificity and efficiency

  • Base editing and prime editing for precise genetic modifications without double-strand breaks

  • Inducible or tissue-specific gene editing systems adapted for yeast

• Single-cell technologies:

  • Single-cell transcriptomics to study cell-to-cell variation in AIM31 expression

  • Spatial transcriptomics to map mitochondrial gene expression patterns

  • Single-cell proteomics to quantify AIM31 abundance and modifications

• Advanced imaging techniques:

  • Super-resolution microscopy of labeled AIM31 to visualize its distribution within mitochondria

  • Live-cell imaging to track AIM31 dynamics during mitochondrial biogenesis

  • Correlative light and electron microscopy to link AIM31 localization with ultrastructural features

• Proteomics innovations:

  • Thermal proteome profiling to identify AIM31 interactors and their stability

  • Targeted proteomics for absolute quantification of AIM31 and its partners

  • Proximity labeling proteomics to map the spatial environment of AIM31

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Computational modeling of respiratory chain assembly incorporating AIM31

  • Network analysis to position AIM31 within the broader mitochondrial regulatory system