Recombinant Prochlorococcus marinus subsp. pastoris ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) in Prochlorococcus marinus and why is it significant for research?

ATP synthase subunit c (atpE) in Prochlorococcus marinus is a critical component of the organism's ATP synthesis machinery. The significance stems from Prochlorococcus being potentially the most abundant photosynthetic organism on the planet, responsible for a substantial fraction of global photosynthesis . The c-subunit forms an oligomeric ring embedded in the thylakoid membrane, where its rotation—driven by proton translocation across the membrane—mechanically couples to ATP synthesis . This coupling mechanism is central to understanding photosynthetic energy conversion in marine ecosystems. Studying atpE from Prochlorococcus provides insights into how this abundant organism has evolutionarily optimized its energy metabolism for survival in oligotrophic ocean environments.

How does the c-ring stoichiometry in Prochlorococcus marinus compare to other photosynthetic organisms?

The c-ring stoichiometry in ATP synthases varies considerably across different organisms, which directly affects the bioenergetic efficiency of ATP production. The ratio of protons translocated to ATP synthesized depends on the number of c-subunits (n) in the oligomeric c-ring (c₍ₙ₎), which is organism-dependent . While the exact c-ring stoichiometry for Prochlorococcus marinus has not been definitively established in the provided literature, this variability is inherently related to the organism's metabolism. The factors that contribute to this stoichiometric variation remain incompletely understood . Comparative analyses between Prochlorococcus and other cyanobacteria, as well as chloroplasts from higher plants (such as spinach with 14 c-subunits), can provide evolutionary insights into how different photosynthetic organisms optimize their ATP production based on their ecological niches.

What expression systems have been successfully used for recombinant production of membrane proteins like ATP synthase subunit c?

For hydrophobic membrane proteins like ATP synthase subunit c, several expression systems have proven effective. The most successful approach documented involves using a fusion protein strategy in E. coli. Specifically, the hydrophobic c₁ subunit can be expressed as a soluble MBP-c₁ fusion protein (maltose binding protein fusion), then cleaved from MBP and purified on a reversed phase column . This strategy enables the soluble expression of eukaryotic membrane proteins in BL21 derivative E. coli cells. For cyanobacterial proteins specifically, various promoter systems have been tested with different efficacies. The Ptrc promoter has shown high expression levels for heterologous proteins in cyanobacteria, although it sometimes lacks tight regulation . Metal-inducible promoters like PpetE, Pcoa, and Psmt offer tighter repression but may yield lower expression levels even when induced .

What are the optimal conditions for recombinant expression of ATP synthase subunit c from Prochlorococcus marinus in E. coli?

For optimal recombinant expression of ATP synthase subunit c from Prochlorococcus marinus in E. coli, a carefully designed experimental protocol is essential. Based on successful approaches with similar proteins, the recommended procedure includes:

• Codon optimization: The atpE gene should be codon-optimized for E. coli expression to enhance translation efficiency, as has been demonstrated with similar membrane proteins .

• Fusion protein design: Express the hydrophobic c-subunit as a fusion with maltose binding protein (MBP) to increase solubility. This approach has successfully produced significant quantities of purified c₁ subunit with correct α-helical secondary structure .

• Expression vector selection: Use a vector with an inducible promoter system that provides tight regulation and high expression. The IPTG-inducible system (Ptrc or optimized variants) has shown up to 360-fold induction in some cyanobacterial systems and may work effectively for expressing cyanobacterial proteins in E. coli .

• Expression conditions: Transform the construct into BL21 derivative E. coli cells, which have been successfully used for similar membrane proteins . Culture at lower temperatures (18-25°C) after induction to allow proper protein folding and reduce inclusion body formation.

• Induction protocol: For IPTG-inducible systems, titrate IPTG concentration between 1 μM and 10,000 μM to determine optimal expression levels, as this range has been effective for cyanobacterial promoters .

What purification strategy provides the highest yield and purity of recombinant atpE protein?

The optimal purification strategy for recombinant ATP synthase subunit c (atpE) from Prochlorococcus marinus combines several techniques to achieve high yield and purity:

• Initial purification of fusion protein: Affinity chromatography using an amylose resin for MBP-fusion proteins allows selective binding of the fusion construct while leaving most bacterial proteins in the flow-through .

• Proteolytic cleavage: After initial purification, cleave the fusion tag using a specific protease (such as TEV or Factor Xa) with optimized buffer conditions to maintain protein solubility during cleavage .

• Reversed-phase chromatography: The cleaved atpE protein can be purified to high homogeneity using reversed-phase column chromatography, which has proven effective for hydrophobic membrane proteins like the c-subunit .

• Quality control steps: Confirm the correct α-helical secondary structure of the purified protein using circular dichroism spectroscopy, as has been done with spinach chloroplast ATP synthase c-subunit .

This multi-step approach has yielded significant quantities of highly purified c₁ subunit with verified secondary structure in previous studies with similar proteins .

How can researchers optimize RBS design for efficient translation of atpE in cyanobacterial expression systems?

Optimizing the ribosome binding site (RBS) for efficient translation of atpE in cyanobacterial expression systems requires careful consideration of several factors:

• RBS sequence compatibility: Cyanobacteria (including PCC 7942, PCC 7120, and PCC 6301) have a 16S rRNA sequence of ACCTCCTTT that pairs with mRNA, suggesting that an optimal RBS would contain AGGAGG in the central region, similar to the consensus prokaryotic sequence .

• Computational prediction tools: Utilize thermodynamic models such as the RBS Calculator, RBS Designer, or UTR Designer to predict translation efficiency. These tools calculate free energies for interactions between mRNA and rRNA and utilize RNA folding software to predict mRNA secondary structure .

• Empirical validation: While computational tools show good correlation with expression levels in E. coli (R² values of 0.84-0.87), they are not perfect for cyanobacteria. Therefore, construct and test a small library of RBS variants to empirically determine the optimal sequence for atpE expression .

• Consideration of mRNA secondary structure: Ensure that the mRNA region around the start codon lacks strong secondary structures that could impede ribosome binding. Software tools can predict these structures and their impact on translation initiation .

• Translation coupling: If appropriate, consider placing atpE downstream of a well-translated gene with optimal spacing to leverage translation coupling effects .

This combined computational and empirical approach can significantly improve translation efficiency for atpE expression in cyanobacterial systems.

What approaches can be used to study the oligomerization properties of recombinant atpE in vitro?

Studying the oligomerization properties of recombinant ATP synthase subunit c (atpE) requires specialized techniques for membrane protein analysis:

• Detergent-based reconstitution: Purified atpE can be reconstituted into detergent micelles (using mild detergents like DDM or CHAPS) to maintain protein solubility while allowing oligomerization. The choice of detergent is critical, as it must maintain the native-like environment for proper c-ring assembly.

• Cross-linking studies: Chemical cross-linking followed by SDS-PAGE or mass spectrometry can identify interacting residues and provide insights into the oligomeric structure. This approach can verify the formation of c-subunit rings and potentially determine the number of subunits per ring.

• Analytical ultracentrifugation: This technique can determine the molecular mass of the oligomeric complex in detergent solution, helping to establish the stoichiometry of c-subunits in the assembled ring.

• Cryo-electron microscopy: Single-particle cryo-EM has emerged as a powerful tool for structural analysis of membrane protein complexes. For the c-ring, this technique can reveal the number of subunits and their arrangement within the oligomeric complex.

• Native mass spectrometry: Native MS can determine the intact mass of the c-ring complex, providing direct evidence of the oligomeric state when the complex is transferred from a detergent or amphipol environment into the gas phase.

These techniques, often used in combination, can provide comprehensive information about the stoichiometry and structure of the oligomeric c-ring formed by recombinant atpE protein.

How can CRISPR interference be utilized to study atpE function in Prochlorococcus marinus?

CRISPR interference (CRISPRi) offers a powerful approach for studying atpE function through targeted gene repression in Prochlorococcus marinus:

• Inducible CRISPRi system design: Develop an inducible CRISPRi system using anhydrotetracycline (aTc) as the inducer, which has been successfully implemented in other cyanobacteria with up to 32-fold induction in PCC 7002 and >1,200-fold induction in PCC 7120 .

• sgRNA design for atpE targeting: Design single guide RNAs (sgRNAs) targeting the promoter region or early coding sequence of atpE. Multiple sgRNAs should be tested to identify those providing optimal repression efficiency.

• dCas9 expression optimization: Express catalytically inactive Cas9 (dCas9) under a promoter that provides appropriate expression levels. Low-level expression is crucial, as high dCas9 levels can cause maximal repression even without inducer presence .

• Titration of repression: Use varying concentrations of aTc (between 0-2,000 ng/mL) to achieve different levels of atpE repression, enabling the study of phenotypic effects across a spectrum of ATP synthase activity levels .

• Phenotypic analysis: Monitor changes in growth rate, photosynthetic efficiency, ATP production, and membrane potential in response to atpE repression. This approach can reveal the physiological consequences of reduced ATP synthase activity.

• Compensatory mechanisms: Analyze transcriptomic and proteomic changes following atpE repression to identify potential compensatory mechanisms that Prochlorococcus employs to maintain energy homeostasis.

What are the current challenges in determining the c-ring stoichiometry in Prochlorococcus marinus, and what novel approaches might overcome these limitations?

Determining the c-ring stoichiometry in Prochlorococcus marinus presents several technical challenges with potential solutions:

• Challenge: Membrane protein crystallization

  • Traditional crystallography is difficult with hydrophobic membrane proteins

  • Solution: Utilize lipidic cubic phase crystallization or cryo-electron microscopy, which has recently yielded high-resolution structures of c-rings from other organisms

• Challenge: Low natural abundance

  • ATP synthase may be expressed at low levels in Prochlorococcus

  • Solution: Develop an overexpression system in the native organism using strong, regulated promoters or express recombinant c-rings in heterologous systems

• Challenge: Assembly verification

  • Ensuring that recombinantly expressed c-subunits assemble into the native oligomeric state

  • Solution: Combine cross-linking mass spectrometry with native mass spectrometry to verify correct assembly and stoichiometry

• Challenge: Environmental adaptation

  • c-ring stoichiometry might vary with environmental conditions

  • Solution: Culture Prochlorococcus under different light, temperature, and nutrient conditions to assess potential stoichiometric plasticity

• Challenge: Genetic intractability

  • Traditional genetic manipulation of Prochlorococcus has been difficult

  • Solution: Apply newer genetic tools like CRISPR/Cas9 for genome editing or CRISPRi for gene regulation to facilitate studies of native atpE

The exact cause of c-ring stoichiometric variation across organisms remains incompletely understood . Novel integrative approaches combining structural biology, molecular dynamics simulations, and evolutionary analysis may help elucidate the factors driving c-ring stoichiometry determination in Prochlorococcus and its ecological significance.

How does the atpE sequence and structure from Prochlorococcus marinus compare with other cyanobacteria and photosynthetic organisms?

The ATP synthase subunit c (atpE) from Prochlorococcus marinus shows interesting evolutionary features when compared to other photosynthetic organisms:

Sequence conservation patterns:

• The transmembrane helices show higher conservation than loop regions

• The essential proton-binding glutamate residue is universally conserved across species

• Prochlorococcus, as the smallest known photosynthetic organism with a highly streamlined genome, may show sequence adaptations reflecting its specialized marine lifestyle

Structural considerations:

• The α-helical secondary structure is maintained across species, reflecting the functional importance of this structural feature

• The c-ring size (number of c-subunits) varies between organisms, affecting the bioenergetic efficiency of ATP production

• This stoichiometric variation is inherently related to the metabolism of each organism

Evolutionary implications:

• Prochlorococcus has undergone genome reduction during evolution, potentially affecting components of its ATP synthase

• Analysis of its atpE sequence may provide insights into adaptations for survival in low-nutrient, high-light marine environments

• The c-ring stoichiometry variability likely represents different evolutionary solutions to balancing ATP synthesis efficiency with cellular energy demands

Comparative genomic and structural studies of atpE across diverse photosynthetic organisms can illuminate how evolutionary pressures have shaped this critical component of the cellular energy production machinery.

What are the differences in expression strategies for atpE between heterotrophic E. coli and photosynthetic Prochlorococcus systems?

Expression strategies for atpE differ significantly between heterotrophic E. coli and photosynthetic Prochlorococcus systems:

While E. coli systems offer higher biomass and potentially higher protein yields, expression in Prochlorococcus or related cyanobacteria may provide advantages for proper folding, assembly, and function of atpE in its native context.

How can heterologous expression systems be optimized for different research objectives involving ATP synthase subunit c?

Optimization strategies for heterologous expression of ATP synthase subunit c vary based on specific research objectives:

• For structural studies:

  • Focus on high protein yield and purity

  • Use fusion tags that can be completely removed without leaving additional residues

  • Express in E. coli with MBP fusion for solubility, followed by reversed-phase chromatography purification

  • Consider adding stabilizing mutations if the protein shows instability

  • Optimize detergent selection for maintaining native-like structure

• For functional reconstitution:

  • Express in systems that maintain proper folding and assembly

  • Optimize the lipid environment for reconstitution experiments

  • Consider co-expression with other ATP synthase subunits to facilitate proper assembly

  • Use milder purification conditions to preserve protein-protein interactions

  • Implement quality control steps to verify correct α-helical secondary structure

• For evolutionary studies:

  • Express c-subunits from multiple species under identical conditions

  • Standardize expression and purification protocols across all variants

  • Include positive controls (e.g., well-characterized c-subunits) for comparison

  • Design chimeric constructs to identify determinants of c-ring stoichiometry

  • Develop assays to quantify oligomerization properties

• For in vivo studies in cyanobacteria:

  • Develop inducible expression systems with tight regulation

  • Optimize promoter strength based on experimental needs

  • For high expression, modified heterologous promoters like Ptrc may be preferable

  • For tight regulation, aTc-inducible systems can provide up to 1,200-fold induction

  • Consider genetic stability issues and monitor for mutations during long-term expression

Each research objective requires specific optimization strategies, and researchers should carefully select the expression system based on their experimental requirements.

What are common pitfalls in recombinant expression of ATP synthase subunit c, and how can they be addressed?

Recombinant expression of ATP synthase subunit c presents several challenges that researchers commonly encounter:

• Protein aggregation and inclusion body formation

  • Problem: The hydrophobic nature of subunit c often leads to aggregation

  • Solution: Express as a fusion with solubility-enhancing partners like MBP ; lower induction temperature to 18-25°C; use specialized E. coli strains designed for membrane proteins

• Low expression levels

  • Problem: Membrane proteins often express poorly in heterologous systems

  • Solution: Optimize codon usage for the expression host ; test multiple promoter systems with different strengths ; optimize the RBS sequence for efficient translation initiation

• Genetic instability

  • Problem: Expression constructs may accumulate mutations that reduce protein production

  • Solution: Sequence verify plasmids before and after expression; use low-copy plasmids; consider inducible systems with tight regulation to minimize selection pressure

• Improper folding

  • Problem: Recombinant protein may not adopt the correct secondary structure

  • Solution: Verify α-helical content using circular dichroism spectroscopy ; optimize membrane-mimetic environments during purification; consider expression in systems with appropriate chaperones

• Inefficient fusion tag cleavage

  • Problem: Protease accessibility to cleavage sites may be limited

  • Solution: Design constructs with extended linkers between the fusion and target protein; optimize cleavage conditions including detergent selection; test multiple proteases

• Poor yield after final purification

  • Problem: Significant loss during purification steps

  • Solution: Optimize each step of the purification protocol; consider alternative chromatography methods; minimize sample handling and transfers

• Promoter leakiness in inducible systems

  • Problem: Background expression before induction

  • Solution: Test improved systems like cLac143/cLac94 (48-fold and 38-fold induction ranges) or optimized aTc systems (up to 230-fold induction)

Addressing these challenges requires systematic optimization and careful monitoring throughout the expression and purification process.

How can researchers troubleshoot issues with c-ring assembly in reconstitution experiments?

Troubleshooting c-ring assembly issues in reconstitution experiments requires a systematic approach to identify and address specific problems:

• Issue: No detectable oligomerization

  • Diagnostic approach: Use size-exclusion chromatography or native PAGE to assess oligomeric state

  • Potential solutions:

    • Optimize detergent type and concentration

    • Test different lipid compositions in reconstitution mixtures

    • Verify protein integrity using mass spectrometry

    • Adjust pH and ionic conditions to promote assembly

    • Consider adding ATP synthase a-subunit, which may stabilize c-ring assembly

• Issue: Incorrect stoichiometry or heterogeneous assemblies

  • Diagnostic approach: Analyze using native mass spectrometry or analytical ultracentrifugation

  • Potential solutions:

    • Modify reconstitution protocol to allow equilibration over longer periods

    • Test reconstitution at different protein-to-lipid ratios

    • Use gentle detergent removal techniques (dialysis or Bio-Beads)

    • Try different pH conditions, as protonation state of key residues may affect assembly

• Issue: Aggregation during reconstitution

  • Diagnostic approach: Monitor using dynamic light scattering or turbidity measurements

  • Potential solutions:

    • Decrease protein concentration during reconstitution

    • Implement step-wise detergent removal

    • Add stabilizing agents like glycerol or specific lipids

    • Use more gentle detergents for initial solubilization

• Issue: Non-functional assembled rings

  • Diagnostic approach: Assess proton translocation activity in liposomes

  • Potential solutions:

    • Verify correct orientation in liposomes using protease protection assays

    • Optimize lipid composition to match native environment

    • Check for post-translational modifications that might be required for function

    • Consider co-reconstitution with other ATP synthase subunits

• Issue: Poor reproducibility

  • Diagnostic approach: Carefully control all variables and document conditions

  • Potential solutions:

    • Standardize protein preparation methods

    • Use consistent detergent and lipid lots

    • Control temperature throughout the reconstitution process

    • Develop quantitative assays to measure assembly efficiency

These troubleshooting approaches address the complex challenges of membrane protein reconstitution and can significantly improve success rates in c-ring assembly experiments.

What strategies can overcome the genetic instability observed when expressing heterologous proteins in cyanobacteria?

Genetic instability is a significant challenge when expressing heterologous proteins in cyanobacteria, but several strategies can mitigate this issue:

• Codon optimization

  • Problem: Non-optimized codons can cause translational stress

  • Solution: Optimize codon usage for the host cyanobacterium while avoiding rare codons; this approach has prevented instability in ethylene-producing strains in PCC 6803 compared to PCC 7942

• Inducible expression systems

  • Problem: Constitutive expression creates continuous selective pressure

  • Solution: Use tightly regulated inducible systems like the optimized aTc induction system (32-fold induction in PCC 7002, >1,200-fold in PCC 7120, 230-fold in PCC 6803) to minimize selective pressure during growth

• Promoter selection

  • Problem: Strong promoters may cause metabolic burden

  • Solution: Test a range of promoter strengths; sometimes moderate promoters provide better stability than maximum-strength promoters

• Neutral genomic integration sites

  • Problem: Integration into sensitive genomic regions can disrupt essential functions

  • Solution: Target neutral integration sites that minimize disruption to the host metabolism

• Toxicity reduction

  • Problem: Protein products or intermediates may be toxic

  • Solution: Include efflux pumps or sequestration mechanisms; co-express chaperones to improve folding; target protein to appropriate subcellular locations

• HIP1 sequence avoidance

  • Problem: Highly iterated palindrome (HIP1) sequences are hotspots for recombination

  • Solution: Analyze and modify gene sequences to remove or alter HIP1-like sequences that have been associated with insertion mutations in ethylene-producing strains

• Regular verification

  • Problem: Mutations can accumulate silently

  • Solution: Implement regular PCR screening and sequencing of transgenes; monitor protein expression/activity; use antibiotic selection carefully

• Strain engineering

  • Problem: Wild-type DNA repair mechanisms may promote recombination

  • Solution: Consider engineering strains with modified recombination pathways to enhance genetic stability

By implementing these strategies, researchers can significantly improve the genetic stability of heterologous protein expression in cyanobacterial systems, enabling more robust and reproducible experiments with recombinant ATP synthase components.

What emerging technologies could enhance our understanding of ATP synthase c-subunit structure and function in Prochlorococcus marinus?

Several cutting-edge technologies are poised to revolutionize our understanding of ATP synthase c-subunit structure and function in Prochlorococcus marinus:

• Cryo-electron tomography (cryo-ET)

  • This technique allows visualization of ATP synthase complexes in their native membrane environment

  • Recent advances in sub-tomogram averaging could reveal the c-ring stoichiometry and arrangement in situ

  • The application to Prochlorococcus cells could provide insights into the native conformation without artificial reconstitution

• Single-molecule biophysics

  • Single-molecule FRET can measure conformational changes during c-ring rotation

  • Magnetic tweezers or optical traps could directly measure the mechanical properties of the c-ring

  • These approaches could reveal how the c-ring's unique properties in Prochlorococcus contribute to its ecological adaptation

• Integrative structural biology

  • Combining multiple structural techniques (X-ray crystallography, cryo-EM, NMR, and mass spectrometry)

  • Computational integration of data from different methods can generate more complete structural models

  • This could reveal subtle structural features unique to Prochlorococcus ATP synthase

• Advanced genetic tools

  • Optimized CRISPR/Cas9 systems for precise genome editing in Prochlorococcus

  • Inducible CRISPRi systems with up to 1,200-fold induction for detailed functional studies

  • These tools would enable precise in vivo manipulation of atpE to study function

• Artificial intelligence for protein design

  • Machine learning approaches can predict the impact of mutations on c-ring assembly and function

  • AI-guided protein engineering could help design variants with altered properties for mechanistic studies

  • These computational approaches could guide experimental design more efficiently

• Improved membrane mimetics

  • Novel nanodiscs or lipid cubic phase systems that better mimic the native thylakoid membrane

  • Customized lipid compositions matching the unique membrane environment of Prochlorococcus

  • These systems could provide more physiologically relevant reconstitution conditions

• High-resolution native mass spectrometry

  • Direct measurement of intact c-rings with bound lipids and interacting proteins

  • Determination of precise stoichiometry and detection of post-translational modifications

  • This could resolve questions about c-ring size variability under different conditions

These emerging technologies, particularly when used in combination, hold tremendous promise for elucidating the unique adaptations of ATP synthase in this ecologically critical marine cyanobacterium.

How might comparative studies of ATP synthase c-subunits across different Prochlorococcus ecotypes inform our understanding of evolutionary adaptation?

Comparative studies of ATP synthase c-subunits across different Prochlorococcus ecotypes offer valuable insights into evolutionary adaptation:

These comparative approaches would significantly enhance our understanding of how this crucial enzyme has been fine-tuned during the evolution and diversification of Prochlorococcus across different ocean environments.

What are the most promising applications of engineered ATP synthase c-subunits for fundamental research in bioenergetics?

Engineered ATP synthase c-subunits offer several promising applications for fundamental bioenergetics research:

• Designer c-rings with altered stoichiometry

  • Engineering c-subunits that assemble into rings with specific numbers of subunits

  • Studying how c-ring size affects proton-to-ATP ratio and bioenergetic efficiency

  • This approach could resolve longstanding questions about the relationship between structure and function in ATP synthases

• Fluorescent protein fusions for real-time visualization

  • Creating functional fluorescent fusions to monitor c-ring dynamics

  • Visualizing ATP synthase distribution, movement, and turnover in living cells

  • These tools could provide unprecedented insights into ATP synthase behavior in vivo

• Site-specific probes for mechanistic studies

  • Introducing unnatural amino acids at specific positions for spectroscopic studies

  • Using click chemistry to attach probes for monitoring conformational changes

  • These approaches could reveal details of the proton translocation and rotary mechanisms

• Chimeric c-subunits

  • Creating hybrid c-subunits combining elements from different species

  • Identifying determinants of species-specific properties such as c-ring size

  • This strategy could help resolve the factors controlling c-ring stoichiometry

• Synthetic ATP synthases with novel properties

  • Engineering c-subunits that respond to alternative energy sources

  • Creating systems with altered ion specificities (e.g., Na⁺ instead of H⁺)

  • These synthetic systems could expand our understanding of energy conversion mechanisms

• Minimal ATP synthase models

  • Designing simplified c-rings to identify essential structural requirements

  • Creating minimal functional systems for detailed mechanistic studies

  • This reductionist approach could clarify fundamental principles of rotary motors

• Inducible systems for in vivo regulation

  • Developing precisely controlled expression systems using optimized inducible promoters

  • Creating conditional mutants for studying ATP synthase function

  • These systems could enable temporal control of ATP synthase activity for studying cellular responses

• Cross-species complementation

  • Expressing engineered Prochlorococcus atpE in other organisms

  • Assessing functional compatibility across evolutionary distances

  • This approach could reveal constraints and flexibilities in the evolution of this essential enzyme

These engineering approaches, facilitated by advances in recombinant expression systems and regulatory tools , promise to significantly advance our fundamental understanding of ATP synthase function and evolution.