Recombinant Scheffersomyces stipitis Cytochrome oxidase assembly protein 3, mitochondrial (COA3)

What is Scheffersomyces stipitis Cytochrome oxidase assembly protein 3, mitochondrial (COA3)?

Scheffersomyces stipitis Cytochrome oxidase assembly protein 3, mitochondrial (COA3) is a mitochondrial protein encoded by the COA3 gene (also known as PICST_32500) in the yeast species Scheffersomyces stipitis. The protein has a key role in the assembly of cytochrome c oxidase (COX) protein complex within the mitochondrial respiratory chain. The full amino acid sequence of this protein is: MAVIGAPKGHDRYRDPKTHQMTPALYRVRAPFFWKNTIGLAICTAIPLGVYMYTLHMLSKDEFGDIPIPPISDTELTKLKKEYEASKNQN . This 90-amino acid protein functions primarily in the mitochondria, where it participates in essential processes related to cellular respiration and energy production.

How does COA3 function in Scheffersomyces stipitis compared to homologs in other organisms?

COA3 in Scheffersomyces stipitis functions similarly to its homologs in other organisms as a regulator of cytochrome oxidase assembly. In yeast species like S. stipitis, COA3 works in conjunction with other assembly factors like Cox14 to regulate the expression of Cox1, a core component of the cytochrome oxidase complex. This regulation occurs through the formation of assembly intermediates with newly synthesized Cox1 and association with translational activators like Mss51 . The fundamental role of COA3 in mitochondrial function appears to be conserved across species, though with organism-specific adaptations.

In humans, COA3 (also known as CCDC56) performs similar functions in cytochrome c oxidase assembly but has additional implications in disease contexts. For instance, human COA3 has been linked to cancer progression, particularly in non-small cell lung cancer (NSCLC), where its overexpression promotes mitochondrial fragmentation and metastasis through mechanisms involving DRP1 phosphorylation . This suggests that while the core mitochondrial assembly function is conserved, the broader biological implications of COA3 may vary significantly across species.

What expression systems are most effective for producing recombinant S. stipitis COA3?

For recombinant production of S. stipitis COA3, Escherichia coli expression systems are commonly used due to their high yield and relative simplicity. The methodology typically involves:

• Cloning the COA3 gene sequence into a suitable expression vector containing an appropriate promoter (T7 is common for mitochondrial proteins)

• Transformation into a compatible E. coli strain (BL21(DE3) or similar derivatives)

• Induction of protein expression using IPTG

• Cell lysis and protein extraction, often under native conditions to maintain protein folding

• Purification using affinity chromatography, typically with a His-tag fusion construct

For more native-like post-translational modifications, yeast expression systems can be advantageous. Pichia pastoris or Saccharomyces cerevisiae expression platforms may provide better folding and processing of mitochondrial proteins like COA3. The recombinant protein is typically stored in a Tris-based buffer with 50% glycerol for stability at -20°C or -80°C for extended storage .

What are the optimal conditions for studying COA3 interactions with Cox1 and other assembly factors?

To effectively study COA3 interactions with Cox1 and other assembly factors, researchers should consider the following methodological approaches:

• Immunoprecipitation assays: Using antibodies against COA3 or tagged versions of the protein to pull down interacting proteins, followed by Western blotting or mass spectrometry to identify binding partners. This approach has been successfully used to demonstrate how Coa3 and Cox14 form assembly intermediates with newly synthesized Cox1 .

• Blue Native PAGE: To visualize intact protein complexes containing COA3, Cox1, and other assembly factors under non-denaturing conditions. This technique is particularly useful for tracking the assembly state of respiratory chain complexes.

• In organello translation assays: To monitor the synthesis of mitochondrially encoded proteins like Cox1 in the presence or absence of COA3. This approach can reveal the role of COA3 in translational regulation.

The buffer composition is critical, typically requiring:

• pH 7.2-7.4 phosphate or Tris-based buffers

• 150-300 mM NaCl

• 1-5% glycerol for stability

• Mild detergents (0.5-1% digitonin or 0.1% n-dodecyl-β-D-maltoside) for membrane protein solubilization

• Protease inhibitor cocktails to prevent degradation

Temperature conditions should be carefully controlled, with interaction studies typically performed at 4°C to preserve complex integrity, while functional assays may require physiological temperatures (25°C for yeast proteins or 30°C for S. stipitis-specific experiments).

How can researchers effectively analyze COA3's impact on mitochondrial function in S. stipitis?

Analyzing COA3's impact on mitochondrial function in S. stipitis requires a multi-parameter approach:

• Respiratory capacity measurement: Oxygen consumption rates can be measured using respirometry or oxygen electrode systems to determine the effect of COA3 deletion or overexpression on mitochondrial respiration.

• Mitochondrial membrane potential assessment: Fluorescent dyes like JC-1, TMRM, or DiOC6 can be used to measure membrane potential changes resulting from altered COA3 activity.

• ATP production assays: Luciferase-based assays can quantify ATP levels to assess the impact of COA3 on energy production.

• Reactive oxygen species (ROS) detection: Fluorescent probes like DCFDA can measure ROS production as an indicator of mitochondrial dysfunction.

• Mitochondrial morphology analysis: Confocal microscopy with mitochondrial stains can visualize changes in mitochondrial network morphology. This approach has revealed that COA3 can influence mitochondrial fragmentation through mechanisms involving DRP1 phosphorylation, as seen in human cells .

What purification strategies yield the highest quality recombinant S. stipitis COA3 for functional studies?

Obtaining high-quality recombinant S. stipitis COA3 for functional studies requires careful attention to purification strategies:

• Affinity chromatography: His-tag purification is commonly the first step, using nickel or cobalt resins with imidazole gradients for elution. For S. stipitis COA3, low imidazole concentrations (20-50 mM) in wash buffers and higher concentrations (250-300 mM) for elution typically yield good results.

• Size exclusion chromatography: This second purification step helps remove aggregates and provides the protein in a more native-like state. For small proteins like COA3 (approximately 10 kDa), Superdex 75 or similar columns are appropriate.

• Ion exchange chromatography: Can be used as an additional purification step, particularly if the recombinant protein has distinctive charge properties.

Critical considerations include:

• Maintaining a mild detergent throughout purification (0.03-0.05% DDM or similar) to prevent aggregation

• Including reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to maintain thiol groups

• Adding glycerol (10-20%) to stabilize the protein

• Performing all steps at 4°C to minimize degradation

The purified protein should be stored in a Tris-based buffer with 50% glycerol at -20°C for short-term use or at -80°C for extended storage . Repeated freeze-thaw cycles should be avoided to maintain protein integrity.

How does COA3 contribute to the metabolic flexibility of Scheffersomyces stipitis in different carbon sources?

Scheffersomyces stipitis is known for its unique ability to metabolize various sugars, including those found in molasses and lignocellulosic biomass. While COA3's direct role in this metabolic flexibility hasn't been fully characterized, its function in mitochondrial respiration has significant implications for the organism's carbon metabolism:

• S. stipitis depends heavily on respiratory metabolism, even under high sugar conditions, unlike Saccharomyces cerevisiae which readily undergoes fermentation. The cytochrome oxidase complex, which COA3 helps assemble, is crucial for this respiratory capacity.

• When S. stipitis is metabolically engineered to produce compounds like resveratrol from different sugars, the composition of sugars (sucrose, glucose, fructose) significantly affects intracellular metabolite accumulation and ATP/AMP ratios . These energetic parameters are directly influenced by mitochondrial function, suggesting that COA3's role in maintaining functional cytochrome oxidase may impact the cell's ability to efficiently utilize different carbon sources.

• The tight regulation of mitochondrial gene expression, including the COX1 feedback loop that COA3 participates in , likely allows S. stipitis to adapt its respiratory capacity to changing carbon conditions.

Understanding COA3's contribution to metabolic flexibility could provide insights for optimizing S. stipitis for biotechnological applications, such as the production of resveratrol from molasses, where the strain has demonstrated production of 1076 ± 167 mg/L using sugarcane molasses containing 120 g/L of total sugars .

What mechanisms explain the correlation between COA3 expression levels and cellular energy metabolism?

The correlation between COA3 expression levels and cellular energy metabolism can be explained through several molecular mechanisms:

• Electron transport chain assembly regulation: COA3, together with Cox14, regulates the assembly of cytochrome c oxidase by forming complexes with newly synthesized Cox1 and controlling the availability of the translational activator Mss51 . This regulatory mechanism creates a negative feedback loop that balances the production of respiratory chain components with their assembly, directly impacting cellular respiration and ATP production.

• Mitochondrial morphology modulation: COA3 has been shown to influence mitochondrial morphology by affecting the phosphorylation state of dynamin-related protein 1 (DRP1). In human cells, COA3 promotes DRP1 phosphorylation at Ser616, which enhances mitochondrial fragmentation . This morphological change has been linked to metabolic reprogramming, particularly a shift toward glycolysis.

• Metabolic pathway crosstalk: Changes in COA3 expression can alter the balance between oxidative phosphorylation and glycolysis. Research in human cancer cells has shown that COA3 overexpression promotes aerobic glycolysis, partly through DRP1-mediated mitochondrial fragmentation .

The following table summarizes the experimental evidence for these mechanisms:

How can genomic modifications of COA3 be leveraged to enhance biofuel or bioproduct production in S. stipitis?

Strategic genomic modifications of COA3 could potentially enhance biofuel or bioproduct production in S. stipitis through several approaches:

• Fine-tuning respiratory capacity: Modulating COA3 expression levels could optimize the balance between respiration and fermentation, potentially redirecting carbon flux toward desired products. This approach would require careful calibration, as both overexpression and complete deletion might be detrimental.

• Engineering stress tolerance: Optimizing mitochondrial function through COA3 modifications could enhance cellular tolerance to stressors encountered during industrial fermentation (e.g., high product concentrations, inhibitory compounds in biomass hydrolysates).

• Coupling with metabolic engineering strategies: Combining COA3 modifications with other metabolic engineering approaches could create synergistic effects. For example, in a S. stipitis strain metabolically engineered for resveratrol production, optimizing COA3 expression might improve the utilization of molasses as a substrate .

• Targeting carbon catabolite repression: S. stipitis shows carbon catabolite repression when grown on mixed sugars, with sucrose consumption suppressed by the coexistence of glucose and fructose . Understanding and potentially modifying how mitochondrial function interacts with these regulatory mechanisms could enhance simultaneous utilization of multiple sugars.

Implementation of these strategies would require:

• CRISPR-Cas9 or similar techniques for precise genomic editing

• Promoter engineering to achieve controlled expression levels

• Fermentation optimization under industrially relevant conditions

• Metabolomic analysis to confirm the redirection of metabolic fluxes

What functional differences exist between COA3 in S. stipitis and its homologs in other yeast species?

Comparative analysis of COA3 across yeast species reveals both conserved functions and species-specific adaptations:

• Core conservation of cytochrome oxidase assembly function: The fundamental role of COA3 in forming assembly intermediates with newly synthesized Cox1 and regulating mitochondrial translation appears to be conserved across yeast species . This conservation reflects the essential nature of respiratory chain assembly.

• Metabolic context differences: S. stipitis is a Crabtree-negative yeast with strong preference for respiratory metabolism, while Saccharomyces cerevisiae is Crabtree-positive with ready fermentation under aerobic conditions. These metabolic differences likely place different evolutionary pressures on COA3 function.

• Regulatory network variations: The precise interactions between COA3, Cox14, and translational activators like Mss51 may differ between species, reflecting adaptations to different ecological niches and metabolic strategies.

• Protein structure variations: While the core functional domains of COA3 are conserved, sequence variations exist between species. These differences may affect protein-protein interactions, stability, or regulatory mechanisms.

A distinctive feature of S. stipitis metabolism is its ability to utilize a wide range of sugars and its unique carbon catabolite repression patterns, where sucrose consumption is suppressed by the coexistence of glucose, fructose, and even ethanol . These metabolic characteristics may have driven species-specific adaptations in mitochondrial regulatory proteins like COA3.

How does the structure-function relationship of COA3 contribute to its role in mitochondrial dynamics?

The structure-function relationship of COA3 plays a crucial role in its ability to influence mitochondrial dynamics:

• Transmembrane domain: COA3 is a mitochondrial transmembrane protein, with its membrane-spanning regions likely critical for its localization and interaction with other membrane-bound components of the respiratory chain assembly machinery.

• Interaction domains: Specific regions of COA3 mediate its binding to Cox1 and other assembly factors. These interaction domains are essential for the formation of assembly intermediates that sequester translational activators like Mss51, creating the negative feedback loop that regulates Cox1 expression .

• Regulatory elements: In human cells, COA3 influences mitochondrial fragmentation by affecting DRP1 phosphorylation . Though the direct molecular mechanism remains unclear, specific structural elements of COA3 must facilitate this regulatory function, either through direct interaction with kinases/phosphatases or through indirect effects on signaling pathways.

• Conservation and variability: Sequence analysis reveals that certain regions of COA3 are highly conserved across species, likely representing functional domains essential for its core roles in cytochrome oxidase assembly. Other regions show greater variability, potentially reflecting species-specific functions or regulatory mechanisms.

The amino acid sequence of S. stipitis COA3 (MAVIGAPKGHDRYRDPKTHQMTPALYRVRAPFFWKNTIGLAICTAIPLGVYMYTLHMLSKDEFGDIPIPPISDTELTKLKKEYEASKNQN) contains hydrophobic segments consistent with membrane integration, along with charged and polar residues that likely mediate protein-protein interactions. Structural studies using techniques like NMR or cryo-EM would be valuable for further elucidating these structure-function relationships.

What are the technical challenges in studying COA3's role in mitochondrial translation regulation?

Investigating COA3's role in mitochondrial translation regulation presents several technical challenges:

• Mitochondrial isolation quality: Obtaining pure, functional mitochondria is essential but challenging. Contamination with other cellular components can confound results, particularly in assays measuring protein synthesis or assembly.

• Protein complex stability: The assembly intermediates containing COA3, Cox14, and newly synthesized Cox1 are often labile and can dissociate during experimental manipulation. Optimization of detergent types, concentrations, and buffer conditions is critical for maintaining complex integrity during isolation and analysis.

• Temporal dynamics: The assembly process is dynamic, with transient interactions occurring at different stages. Capturing these temporal dynamics requires sophisticated pulse-chase methodologies or time-resolved structural techniques.

• Distinguishing direct and indirect effects: When COA3 is deleted or overexpressed, separating direct effects on translation from indirect consequences of altered mitochondrial function can be challenging. This requires careful experimental design with appropriate controls.

• Tissue and condition specificity: The regulatory mechanisms may vary depending on cellular context, metabolic state, or environmental conditions. This variability necessitates studies under diverse conditions to fully understand COA3's regulatory role.

Advanced approaches to address these challenges include:

• Proximity labeling techniques (BioID, APEX) to identify transient interaction partners

• Ribosome profiling adapted for mitochondrial translation

• Cryo-electron microscopy for structural analysis of assembly intermediates

• Systems biology approaches integrating transcriptomics, proteomics, and metabolomics data

How might insights from human COA3 cancer research inform studies of S. stipitis COA3 function?

Research on human COA3's role in cancer, particularly non-small cell lung cancer (NSCLC), provides valuable insights that could inform studies of S. stipitis COA3:

• Metabolic reprogramming mechanisms: Human COA3 overexpression promotes aerobic glycolysis in NSCLC cells, contributing to the Warburg effect characteristic of many cancers . This suggests that COA3 may have broader metabolic regulatory functions beyond cytochrome oxidase assembly. Studies in S. stipitis could investigate whether COA3 similarly influences the balance between respiratory and fermentative metabolism, particularly under different carbon source conditions.

• Mitochondrial dynamics regulation: In human cells, COA3 promotes mitochondrial fragmentation by enhancing DRP1 phosphorylation at Ser616 and its recruitment to mitochondria . This connection between a cytochrome oxidase assembly factor and mitochondrial morphology regulation was unexpected. Similar mechanisms might exist in S. stipitis, potentially linking respiratory chain assembly with mitochondrial dynamics in response to metabolic changes.

• Post-translational modification networks: The finding that human COA3 affects DRP1 phosphorylation suggests involvement in kinase/phosphatase signaling networks . Similar regulatory networks might exist in S. stipitis, potentially involving different targets relevant to its unique metabolism.

• microRNA regulation: Human COA3 expression is regulated by miR-338-3p, which is downregulated in NSCLC . While the specifics of post-transcriptional regulation likely differ in S. stipitis, this highlights the importance of investigating regulatory mechanisms controlling COA3 expression.

A comparative approach studying both human and S. stipitis COA3 could provide valuable insights into both fundamental mitochondrial biology and the evolutionary adaptations of these proteins for different cellular contexts.

What novel experimental approaches could reveal unexpected functions of COA3 in cellular metabolism?

Several innovative experimental approaches could uncover unexpected functions of COA3 in cellular metabolism:

• Spatiotemporal proteomics: Techniques like proximity labeling combined with mass spectrometry (BioID, APEX) could identify transient or context-specific COA3 interaction partners under different metabolic conditions. This approach might reveal connections to unexpected metabolic pathways or regulatory mechanisms.

• Metabolic flux analysis with stable isotopes: 13C-labeling combined with metabolomics could track how COA3 modifications affect carbon flow through central metabolism. This would be particularly valuable for understanding how COA3 influences S. stipitis' utilization of different carbon sources, such as the sugars found in molasses .

• Single-cell approaches: Single-cell transcriptomics or proteomics could reveal cell-to-cell variability in COA3 expression and its correlation with metabolic states, potentially uncovering subpopulation-specific functions.

• In situ structural biology: Techniques like cryo-electron tomography could visualize COA3-containing complexes within their native mitochondrial environment, potentially revealing structural roles beyond protein-protein interactions.

• Synthetic biology approaches: Creating chimeric proteins or orthogonal systems where COA3 function can be precisely controlled (e.g., through optogenetics or chemical genetics) could help dissect its mechanisms of action.

• Multi-omics integration: Combining transcriptomics, proteomics, metabolomics, and lipidomics data from COA3 perturbation experiments could provide a systems-level view of its functional impact, potentially highlighting unexpected metabolic connections.

These approaches might reveal roles for COA3 beyond cytochrome oxidase assembly, such as involvement in mitochondrial stress responses, metabolic adaptation to changing nutrient conditions, or coordination of nuclear and mitochondrial gene expression. As seen with human COA3's unexpected role in cancer progression , such studies may uncover novel functions with both biological significance and potential biotechnological applications.

How does current understanding of COA3 inform both basic mitochondrial biology and biotechnological applications?

The current understanding of COA3 bridges fundamental mitochondrial biology and practical biotechnological applications in several significant ways:

• Respiratory chain assembly mechanism insights: Studies of COA3's role in cytochrome oxidase assembly have revealed sophisticated regulatory mechanisms, including a negative feedback loop where the translational activator Mss51 is sequestered by assembly intermediates containing newly synthesized Cox1, COA3, and Cox14 . This elegant coordination of translation and assembly represents a fundamental principle in mitochondrial biogenesis that may apply broadly across eukaryotes.

• Metabolic engineering implications: Understanding how COA3 influences respiratory capacity and energy metabolism in S. stipitis provides valuable insights for metabolic engineering efforts. This knowledge could inform strategies to optimize this yeast for biofuel or high-value chemical production from renewable feedstocks like molasses .

• Evolutionary adaptation mechanisms: Comparative studies of COA3 across species highlight how mitochondrial regulatory mechanisms have evolved to support different metabolic strategies. S. stipitis' preference for respiratory metabolism and ability to utilize diverse sugars may have driven specific adaptations in its mitochondrial assembly machinery, including COA3.

• Biomedical relevance: The finding that human COA3 overexpression contributes to cancer progression underscores how mitochondrial assembly factors can influence health and disease beyond their core functions. This has potential implications for both diagnostic and therapeutic approaches.

By connecting fundamental mechanisms of mitochondrial biogenesis with species-specific metabolic strategies and potential applications, COA3 research exemplifies how basic science can inform biotechnological innovation and biomedical understanding.

What integrative research approaches would most effectively advance understanding of COA3 function across species?

Advancing understanding of COA3 function across species would benefit from integrative research approaches that combine multiple disciplines and methodologies:

• Comparative genomics and structural biology: Systematic comparison of COA3 sequences, structures, and interaction networks across diverse species could reveal both conserved functional domains and lineage-specific adaptations. This approach would benefit from integrating computational predictions with experimental structural determination.

• Evolutionary systems biology: Reconstructing the evolution of mitochondrial assembly pathways, including COA3's role, could provide insights into how these systems adapted to different metabolic strategies across the eukaryotic tree of life.

• Multi-organism functional genomics: Parallel studies of COA3 function in model organisms representing different metabolic strategies (e.g., S. cerevisiae, S. stipitis, human cells) could identify both conserved and species-specific aspects of its biology. CRISPR-based approaches could enable systematic functional characterization across species.

• Interdisciplinary collaboration: Bringing together expertise from biochemistry, structural biology, systems biology, metabolic engineering, and evolutionary biology would provide complementary perspectives on COA3 function.

• Technology integration: Combining cutting-edge technologies like cryo-EM, metabolic flux analysis, and multi-omics approaches would provide a more comprehensive understanding of how COA3 influences cellular physiology.

This integrative approach would not only advance understanding of COA3 specifically but could also yield broader insights into mitochondrial evolution, the coordination of nuclear and mitochondrial genomes, and the adaptation of energy metabolism to different ecological niches and biotechnological contexts.