Recombinant Debaryomyces hansenii Probable cytosolic iron-sulfur protein assembly protein 1 (CIA1)

What is Debaryomyces hansenii and why is it significant for biotechnology research?

Debaryomyces hansenii is a halophilic yeast that has gained significant attention due to its unique physiological properties. It is the most common yeast species found in cheese (present in more than 50% of examined cheeses) and possesses remarkable halotolerance . This yeast is being established as a superior cell factory for the green transition, particularly for optimizing the use of industrial side-streams and complex feedstock for biotechnological applications . Its ability to thrive in high-salt environments makes it especially valuable for bioprocessing applications using salty industrial by-products. Additionally, D. hansenii produces killer toxins against pathogenic Candida species, adding to its biotechnological importance . Recent advances in genetic engineering tools, particularly CRISPR-Cas9 systems specifically adapted for D. hansenii, have significantly enhanced our ability to manipulate this organism for research and production purposes .

What is the role of CIA1 in iron-sulfur protein assembly?

CIA1 (Cytosolic Iron-sulfur protein Assembly protein 1) is an essential WD40 repeat protein that functions as a critical component in the assembly of iron-sulfur (Fe/S) clusters in cytosolic and nuclear proteins . Unlike other components in this pathway, CIA1 is not involved in mitochondrial Fe/S protein assembly, demonstrating its specificity to extra-mitochondrial Fe/S protein maturation . Functionally, CIA1 operates in a late step of the Fe/S cluster incorporation process, acting after the P-loop NTPases Cfd1 and Nbp35, as well as the hydrogenase-like protein Nar1 . This was demonstrated by the observation that Nbp35 and Nar1, themselves Fe/S proteins, could assemble their Fe/S clusters in the absence of CIA1, positioning CIA1 downstream in the assembly pathway . Coimmunoprecipitation assays have confirmed a specific interaction between CIA1 and Nar1, providing molecular evidence for its mechanistic role .

How is CIA1 distributed within eukaryotic cells?

CIA1 exhibits a distinct subcellular distribution pattern that provides insights into its biological function. While primarily localized to the nucleus, significant amounts of CIA1 are also present in the cytosol . This distribution has been confirmed through both subcellular fractionation studies and in situ immunofluorescence analyses . When investigated by subcellular fractionation, CIA1 was found to be associated with purified nuclei and also present in soluble fractions (S10 and S100), but not with membranous organelles (P10) . This fractionation pattern resembled that of DNA-associated Pol30, further supporting its nuclear presence . Immunofluorescence microscopy using HA-tagged CIA1 confirmed its predominant nuclear localization with lower concentration in the cytoplasm . This dual localization aligns with CIA1's functional role in both nuclear and cytosolic Fe/S protein assembly.

What are the most effective genetic engineering approaches for D. hansenii?

Genetic engineering of D. hansenii has historically been challenging, but recent advances have dramatically improved transformation efficiencies and gene targeting capabilities. The most effective current approach involves the CRISPR-Cas9 system specifically adapted for D. hansenii . This system allows for precise Cas9-mediated point mutations and marker-free gene deletions via homologous recombination (HR) . A key breakthrough in improving engineering efficiency was the disruption of the non-homologous end joining (NHEJ) pathway by creating a mutant for the KU70 gene, which significantly enhanced gene targeting via HR .

For researchers working with D. hansenii, several specific techniques have proven successful:

• In vivo DNA assembly: Multiple DNA fragments (up to three) containing 30-bp homologous overlapping overhangs can be co-transformed and fused in the correct order in a single step .

• Promoter screening: The TEF1 promoter from Arxula adeninivorans has shown the highest production capability for recombinant proteins in D. hansenii .

• Multiplex gene targeting: The CRISPR-Cas9 tool developed by Strucko et al. (2021) is compatible with targeting multiple genes simultaneously .

These methods represent significant improvements over earlier approaches that suffered from extremely low transformation and gene disruption efficiencies.

How can I assess CIA1 function in D. hansenii experimentally?

To assess CIA1 function in D. hansenii, researchers can adapt several experimental approaches used in model yeasts like S. cerevisiae. The essential functions of CIA1 necessitate creating conditional mutants rather than direct knockouts. Based on established methodologies, I recommend the following experimental workflow:

• Creation of a regulatable mutant strain: Replace the native promoter of CIA1 with a glucose-repressible promoter like GAL1-10. This allows controlled depletion of CIA1 by switching from galactose to glucose-containing media .

• Functional assays for Fe/S cluster assembly:

  • Measure activities of cytosolic Fe/S enzymes like isopropylmalate isomerase (Leu1) and sulfite reductase in cell extracts after CIA1 depletion

  • Compare with control enzymes (e.g., Zn-dependent alcohol dehydrogenase) that should remain unaffected by CIA1 depletion

• Fe/S protein maturation analysis:

  • Perform 55Fe incorporation assays using tagged versions of target Fe/S proteins

  • For CIA1 specifically, Rli1-HA is a good reporter as its function is dependent on CIA1

• Protein-protein interaction studies:

  • Conduct coimmunoprecipitation assays with HA-tagged CIA1

  • Overexpress potential interaction partners (Nar1, Nbp35, Cfd1) to enhance detection

• Phenotypic assays:

  • Monitor ribosomal export using GFP-tagged ribosomal proteins (e.g., Rpl25-GFP)

  • Observe nuclear accumulation of ribosomal subunits after CIA1 depletion

These approaches collectively provide a comprehensive assessment of CIA1 function and its role in the cytosolic Fe/S protein assembly machinery.

What expression systems yield optimal production of recombinant CIA1 in D. hansenii?

Optimizing the expression of recombinant CIA1 in D. hansenii requires careful selection of promoters, terminators, and signal peptides. Based on available research, the following expression system components have demonstrated superior performance:

• Promoters: The TEF1 promoter from Arxula adeninivorans has shown the highest expression levels for recombinant proteins in D. hansenii, particularly in salt-rich environments . This promoter outperforms native D. hansenii promoters for heterologous protein expression.

• Terminators: The CYC1 terminator has been identified as effective for recombinant protein expression in D. hansenii . Proper terminator selection is crucial for mRNA stability and efficient translation.

• Signal peptides: For secreted proteins, optimization of signal peptides is essential. Though specific data for CIA1 secretion is not available in the search results, the general approach of screening multiple signal peptides through the in vivo DNA assembly technique has proven effective .

• Growth conditions: D. hansenii's halotolerance can be leveraged to enhance production. Cultivation in salty-rich by-products both supports D. hansenii's metabolism and hinders competing microorganisms, creating a selective environment for protein production .

• Expression vectors: For laboratory research, both replicative and integrative plasmids with appropriate selection markers (hygromycin B resistance or auxotrophic markers like HIS4) can be used .

The specific approach should be tailored to the research objectives, with consideration for whether CIA1 needs to retain its native subcellular localization for functional studies or if high-yield production is the primary goal.

How does CIA1 interact with other components of the cytosolic iron-sulfur cluster assembly (CIA) machinery?

CIA1's interactions with other CIA machinery components reveal its position and function within the cytosolic Fe/S protein assembly pathway. Experimental evidence demonstrates specific interaction patterns:

The molecular mechanism by which CIA1 facilitates Fe/S cluster transfer from the early-acting CIA factors to target apoproteins remains to be fully elucidated and represents an important area for future research.

What are the consequences of CIA1 dysfunction on cellular metabolism?

CIA1 dysfunction has profound and wide-ranging effects on cellular metabolism due to its essential role in cytosolic and nuclear Fe/S protein assembly. The metabolic consequences of CIA1 depletion can be organized into several interconnected pathways:

• Cytosolic Fe/S enzyme impairment:

  • After complete CIA1 depletion, cytosolic Fe/S enzymes show dramatic activity losses

  • Sulfite reductase activity becomes undetectable

  • Isopropylmalate isomerase (Leu1) activity decreases to approximately 10% of normal levels

  • Non-Fe/S enzymes like Zn-dependent alcohol dehydrogenase remain unaffected, confirming specificity

• Ribosome biogenesis and protein synthesis defects:

  • CIA1 depletion impairs the export of large ribosomal subunits from the nucleus

  • Rpl25-GFP, a marker for large ribosomal subunits, accumulates in the nucleus after ~40 hours of CIA1 depletion

  • This export defect is linked to impaired Fe/S cluster assembly on Rli1, an essential ABC protein required for ribosome biogenesis

• Nuclear processes:

  • The predominant nuclear localization of CIA1 suggests additional roles in nuclear functions

  • Nuclear Fe/S proteins involved in DNA metabolism and genome maintenance likely require CIA1 for maturation

  • Disruption of these processes would impact DNA replication, repair, and transcription

• Growth defects:

  • Complete inhibition of growth occurs upon CIA1 depletion

  • Growth slows considerably after 36 hours of depletion but continues at a reduced rate for at least 64 hours

  • This growth pattern suggests a gradual manifestation of metabolic defects as pre-existing Fe/S proteins become diluted through cell division

These metabolic consequences highlight CIA1's position as a critical node connecting Fe/S protein assembly with fundamental cellular processes including amino acid metabolism, protein synthesis, and genome maintenance.

How does the function of CIA1 in D. hansenii compare to its homologs in other yeast species?

The function of CIA1 shows both conservation and specialization across different yeast species. Comparative analysis reveals important insights into evolutionary adaptation and functional conservation:

While the core function of CIA1 in Fe/S protein assembly appears conserved across yeast species, several species-specific adaptations are notable:

• Structural organization: The natural gene fusion between CFD1 and CIA1 in S. pombe suggests a unique organization of the CIA machinery in this organism, potentially enhancing the efficiency of Fe/S cluster transfer between early and late components .

• Environmental adaptation: In D. hansenii, a halophilic yeast, CIA1 likely functions in an intracellular environment with distinct ionic characteristics. This may necessitate specific adaptations in protein-protein interactions and Fe/S cluster transfer mechanisms .

• Integration with cellular metabolism: The specific Fe/S proteins targeted by CIA1 may vary between species, reflecting metabolic differences. For instance, D. hansenii's unique metabolic adaptations for halotolerance may involve Fe/S proteins with specialized functions .

• Genetic tractability: Until recently, studying CIA1 function in D. hansenii was significantly more challenging than in model yeasts like S. cerevisiae due to limited genetic tools. The development of CRISPR-Cas9 systems for D. hansenii now enables more detailed comparative studies .

These comparisons highlight the value of studying CIA1 across multiple yeast species to gain comprehensive insights into both conserved mechanisms and specialized adaptations in Fe/S protein assembly.

What are common challenges in expressing recombinant CIA1 in D. hansenii and how can they be overcome?

Expressing recombinant CIA1 in D. hansenii presents several challenges that require specific troubleshooting approaches:

• Low transformation efficiency:

  • Challenge: Historically, D. hansenii has shown extremely low transformation efficiency, with early attempts yielding very few transformants .

  • Solution: Implement the latest CRISPR-Cas9 system developed specifically for D. hansenii, which dramatically improves transformation efficiency . Additionally, optimize electroporation parameters and use freshly prepared competent cells for better results.

• Poor homologous recombination:

  • Challenge: Gene targeting via homologous recombination has shown extremely low efficiency in wild-type D. hansenii (e.g., only 4/25 clones showing desired phenotypes in early studies) .

  • Solution: Use strains with disrupted NHEJ pathway (KU70 mutants) to enhance HR efficiency . Increase the length of homology arms (>50 bp) in targeting constructs.

• Protein mislocalization:

  • Challenge: CIA1 has a specific subcellular distribution pattern (nuclear and cytosolic) that may be disrupted in recombinant expression .

  • Solution: Carefully design constructs that preserve native targeting signals. Consider C-terminal tagging approaches that typically have less impact on protein localization than N-terminal tags.

• Expression level optimization:

  • Challenge: Overexpression or insufficient expression of CIA1 may lead to artifactual results or failure to complement CIA1 depletion.

  • Solution: Use the TEF1 promoter from Arxula adeninivorans for high expression or test a panel of promoters with different strengths through the in vivo DNA assembly technique . For conditional expression, the GAL1-10 promoter system has been successfully used for CIA1 in yeast .

• Protein solubility and stability:

  • Challenge: WD40 proteins like CIA1 can have complex folding requirements.

  • Solution: Consider expression as a fusion with solubility-enhancing tags. Optimize growth temperature (lower temperatures often favor proper folding of complex proteins).

• Functional verification:

  • Challenge: Confirming that recombinant CIA1 is functional can be difficult due to its essential nature.

  • Solution: Use complementation assays in a conditional CIA1 mutant strain (e.g., Gal-CIA1) to verify functionality . Additionally, assess interaction with known partners like Nar1 through coimmunoprecipitation .

By addressing these common challenges with the suggested solutions, researchers can significantly improve their success in expressing and studying recombinant CIA1 in D. hansenii.

How can researchers troubleshoot inconsistent results in CIA1 functional assays?

Inconsistent results in CIA1 functional assays can arise from multiple sources. Here's a systematic approach to identify and resolve common issues:

• Incomplete CIA1 depletion or variable expression:

  • Problem: In conditional mutants (e.g., Gal-CIA1), residual CIA1 expression may vary between experiments.

  • Solution: Monitor CIA1 protein levels by Western blot at multiple time points after promoter repression . Standardize the depletion protocol and only perform functional assays when CIA1 levels are consistently below detection limits.

• Variation in Fe/S enzyme activity measurements:

  • Problem: Enzyme assays for Fe/S proteins like Leu1 and sulfite reductase may show high variability.

  • Solution: Include multiple controls including non-Fe/S enzymes (e.g., Zn-dependent alcohol dehydrogenase) . Normalize activity to total protein concentration and verify enzyme protein levels by immunoblotting to distinguish between activity loss and protein degradation.

• 55Fe incorporation assay inconsistencies:

  • Problem: Radiolabeling experiments to track Fe/S cluster assembly may yield variable results.

  • Solution: Standardize cell density, labeling time, and iron concentration. Include positive controls (proteins known to incorporate Fe/S clusters efficiently) and negative controls (mutants of known CIA machinery components).

• Growth condition variations:

  • Problem: D. hansenii's response to salt and other environmental factors may affect experimental reproducibility.

  • Solution: Strictly control media composition, pH, temperature, and salt concentration. For D. hansenii specifically, standardize NaCl concentration as it significantly impacts metabolism .

• Genetic background effects:

  • Problem: Spontaneous suppressor mutations may arise during propagation of CIA1 mutant strains.

  • Solution: Regularly return to frozen stocks of the original strain. Consider creating multiple independent mutant lines and comparing results across them.

• Protein-protein interaction detection issues:

  • Problem: Coimmunoprecipitation of CIA1 interaction partners may yield inconsistent results.

  • Solution: For low-abundance proteins like Nar1, use overexpression as demonstrated in previous studies . Optimize lysis conditions to preserve native protein complexes and include appropriate controls (unrelated HA-tagged proteins).

By systematically addressing these potential issues, researchers can significantly improve the consistency and reliability of CIA1 functional assays in D. hansenii.

What are the most promising applications of engineered D. hansenii expressing recombinant CIA1?

Engineered D. hansenii strains expressing recombinant CIA1 offer several promising research and biotechnological applications:

• Halotolerant expression system for Fe/S proteins:

  • D. hansenii's natural halotolerance combined with optimized CIA1 expression could create a superior platform for producing functional Fe/S proteins in high-salt environments .

  • This system would be particularly valuable for expressing Fe/S proteins that are difficult to produce in conventional hosts due to their sensitivity to oxidative stress.

• Biocontrol applications:

  • D. hansenii naturally produces killer toxins against pathogenic Candida species .

  • Engineering strains with enhanced CIA1 expression could potentially improve the production of these killer toxins by ensuring proper maturation of any Fe/S proteins involved in their biosynthesis.

  • Such strains could be developed as biocontrol agents against fungal pathogens in food preservation or medical applications.

• Valorization of industrial salt-rich by-products:

  • D. hansenii has been proposed as a cell factory for the green transition, particularly for utilizing industrial side-streams .

  • Optimized CIA1 expression could enhance the metabolic capabilities of these strains, improving their efficiency in converting salt-rich waste streams into valuable products.

• Model system for studying Fe/S protein assembly in extreme environments:

  • Engineered D. hansenii provides a unique opportunity to study how the CIA machinery functions under high salt conditions .

  • This could reveal novel insights into the flexibility and environmental adaptation of Fe/S protein assembly pathways.

• Production of stress-resistant enzymes:

  • The combination of D. hansenii's stress tolerance and optimized Fe/S protein assembly could enable production of industrial enzymes with enhanced stability.

  • Fe/S cluster-containing enzymes produced in this system might exhibit improved performance under challenging industrial conditions.

These applications leverage the unique characteristics of D. hansenii while addressing the growing need for sustainable bioprocesses and specialized protein production systems.

What key questions remain unresolved about CIA1 function in D. hansenii?

Despite recent advances, several fundamental questions about CIA1 function in D. hansenii remain unresolved:

• Salt adaptation mechanisms:

  • How does CIA1-mediated Fe/S protein assembly function under the high intracellular ion concentrations typical of D. hansenii?

  • Are there specific adaptations in the CIA machinery that facilitate Fe/S cluster transfer under high-salt conditions?

  • Does CIA1 in D. hansenii interact with halotolerance pathways?

• Target specificity:

  • What is the complete repertoire of Fe/S proteins dependent on CIA1 in D. hansenii?

  • Are there species-specific Fe/S proteins in D. hansenii that support its unique ecological niche?

  • How does target recognition by CIA1 differ from that in conventional model yeasts?

• Structural insights:

  • What is the three-dimensional structure of D. hansenii CIA1?

  • How do potential structural adaptations contribute to function in a halophilic environment?

  • Are there differences in the interaction surfaces between CIA1 and its partners compared to other yeasts?

• Regulatory mechanisms:

  • How is CIA1 expression regulated in response to environmental stresses, particularly salt stress?

  • Is there coordination between mitochondrial and cytosolic Fe/S cluster assembly pathways in D. hansenii?

  • Does CIA1 function change during different growth phases or metabolic states?

• Evolutionary aspects:

  • How has CIA1 evolved in D. hansenii compared to non-halophilic yeasts?

  • Are there specific amino acid substitutions that contribute to salt tolerance?

  • Does the evolutionary history of CIA1 reflect adaptation to D. hansenii's ecological niche?

Addressing these questions would significantly advance our understanding of both the fundamental biology of Fe/S protein assembly and the specific adaptations that allow D. hansenii to thrive in challenging environments.

How might advances in D. hansenii CIA1 research contribute to broader understanding of iron-sulfur cluster assembly mechanisms?

Research on D. hansenii CIA1 has the potential to make significant contributions to our broader understanding of iron-sulfur cluster assembly mechanisms:

• Environmental adaptation of conserved pathways:

  • D. hansenii thrives in high-salt environments that would challenge conventional model organisms .

  • Understanding how the CIA machinery functions under these conditions would reveal fundamental insights into the flexibility and adaptability of Fe/S cluster assembly pathways.

  • This knowledge could inform the design of more robust Fe/S protein expression systems for biotechnological applications.

• Evolutionary diversification of the CIA machinery:

  • Comparative analysis of CIA1 from D. hansenii with homologs from other species can illuminate evolutionary strategies for maintaining essential functions under diverse conditions.

  • The natural CFD1-CIA1 fusion in S. pombe suggests evolutionary plasticity in the organization of CIA components . D. hansenii may reveal additional adaptations.

  • These evolutionary insights could help identify both the conserved core and variable regions of the CIA machinery across eukaryotes.

• Integration with stress response pathways:

  • D. hansenii's adaptation to osmotic stress likely involves coordination between Fe/S protein assembly and stress response pathways.

  • Investigating this coordination could reveal new regulatory mechanisms governing Fe/S cluster assembly under stress.

  • Such knowledge would be relevant to understanding Fe/S protein homeostasis in all eukaryotes under various stress conditions.

• Novel interacting partners:

  • D. hansenii may have evolved specific factors that assist CIA1 function under challenging conditions.

  • Identification of such factors could reveal new components or regulatory mechanisms relevant to the general understanding of Fe/S protein biogenesis.

  • Protein-protein interaction studies in D. hansenii might uncover previously unknown CIA machinery components.

• Fundamental biophysical constraints:

  • Fe/S cluster assembly faces basic challenges including oxygen sensitivity and the need to prevent inappropriate cluster release.

  • Studying how these challenges are addressed in an organism with distinct intracellular physicochemical properties could reveal fundamental principles governing Fe/S cluster transfer and incorporation.

By exploring CIA1 function in this non-conventional yeast, researchers can gain insights that transcend the specific biology of D. hansenii and contribute to our fundamental understanding of an essential cellular process conserved across eukaryotes.

What are the most effective protocols for purifying recombinant CIA1 from D. hansenii?

Purifying recombinant CIA1 from D. hansenii requires specialized approaches that account for both the protein's characteristics and the host's unique properties. Based on established protocols for similar proteins, I recommend the following optimized procedure:

• Expression strategy:

  • Express CIA1 with a C-terminal affinity tag (6xHis or Strep-tag II) to preserve native N-terminal functionality

  • Use the TEF1 promoter from Arxula adeninivorans for high-level expression

  • Culture cells in media containing optimal salt concentration (typically 0.5-1M NaCl) to leverage D. hansenii's halotolerance

• Cell lysis optimization:

  • Harvest cells in mid to late logarithmic phase

  • For mechanical disruption, use glass bead homogenization in a buffer containing:

    • 50 mM Tris-HCl pH 8.0

    • 150 mM NaCl (adjust based on protein stability)

    • 10% glycerol

    • 1 mM DTT or 2 mM β-mercaptoethanol

    • Protease inhibitor cocktail

  • Consider adding 0.1% mild detergent (e.g., Triton X-100) to improve solubilization

• Affinity chromatography:

  • For His-tagged CIA1:

    • Use Ni-NTA resin with imidazole gradient elution (20-250 mM)

    • Include 5-10 mM imidazole in binding buffer to reduce non-specific binding

  • For Strep-tagged CIA1:

    • Use Strep-Tactin resin with desthiobiotin elution

    • This approach may yield higher purity with fewer optimization steps

• Additional purification steps:

  • Size exclusion chromatography to separate monomeric CIA1 from aggregates and confirm proper folding

  • Ion exchange chromatography (if necessary) for removing contaminants with different charge properties

• Special considerations:

  • WD40 proteins like CIA1 can be prone to aggregation; maintain samples at 4°C throughout purification

  • Consider adding low concentrations (1-5 mM) of magnesium or calcium to stabilize the WD40 structure

  • For functional studies, verify that purified CIA1 retains the ability to interact with known partners like Nar1

• Quality control:

  • Assess purity by SDS-PAGE (>90% for most applications)

  • Confirm identity by Western blot and/or mass spectrometry

  • Verify proper folding by circular dichroism spectroscopy

This protocol leverages D. hansenii's natural properties while addressing the specific challenges of working with the CIA1 protein, maximizing the likelihood of obtaining functional protein for biochemical and structural studies.

How can researchers effectively measure and analyze CIA1-dependent iron-sulfur cluster assembly in D. hansenii?

Measuring and analyzing CIA1-dependent iron-sulfur cluster assembly in D. hansenii requires a multi-faceted approach that combines biochemical, spectroscopic, and genetic techniques. The following methodological framework provides a comprehensive strategy:

• Enzyme activity assays:

  • Principle: Fe/S-dependent enzymes require proper cluster assembly for activity

  • Implementation:

    • Measure activities of cytosolic Fe/S enzymes such as isopropylmalate isomerase (Leu1) and sulfite reductase in cell extracts

    • Include non-Fe/S dependent enzymes (e.g., alcohol dehydrogenase) as controls

    • Compare activities in CIA1-depleted cells versus wild-type or complemented cells

  • Analysis: Calculate relative activities normalized to total protein and control enzyme activities

• 55Fe incorporation assays:

  • Principle: Direct measurement of de novo Fe/S cluster synthesis

  • Implementation:

    • Transform D. hansenii with plasmids expressing tagged versions of Fe/S proteins

    • Radiolabel cells with 55Fe under various conditions (wild-type, CIA1-depleted, complemented)

    • Immunoprecipitate tagged proteins and measure associated radioactivity

  • Analysis: Quantify 55Fe incorporation relative to protein levels and normalize to control conditions

• UV-visible absorption spectroscopy:

  • Principle: Fe/S clusters have characteristic absorption spectra

  • Implementation:

    • Purify target Fe/S proteins from CIA1-depleted or wild-type cells

    • Record absorption spectra between 300-700 nm

    • Look for characteristic peaks (typically around 320-420 nm)

  • Analysis: Compare spectra to established standards for properly assembled clusters

• Electron paramagnetic resonance (EPR) spectroscopy:

  • Principle: Provides detailed information about Fe/S cluster oxidation state and environment

  • Implementation:

    • Prepare protein samples under anaerobic conditions

    • Record EPR spectra at various temperatures (typically 4-100K)

    • Compare signals from proteins isolated from CIA1-depleted versus wild-type cells

  • Analysis: Identify characteristic g-values and signal intensities indicative of specific Fe/S cluster types

• Phenotypic assays for Fe/S protein function:

  • Principle: Functional Fe/S proteins are required for specific cellular processes

  • Implementation:

    • Monitor ribosome export using GFP-tagged Rpl25

    • Assess DNA repair efficiency and genome stability

    • Measure growth under conditions requiring specific Fe/S enzymes

  • Analysis: Quantify phenotypic differences between CIA1-depleted and wild-type cells

• Protein-protein interaction studies:

  • Principle: CIA1 functions through interactions with specific partners

  • Implementation:

    • Perform coimmunoprecipitation with HA-tagged CIA1 and potential partners

    • Use bimolecular fluorescence complementation to visualize interactions in vivo

    • Apply proximity-dependent biotin identification (BioID) to identify novel interaction partners

  • Analysis: Quantify interaction strengths and map the CIA1 interaction network