Recombinant Schizosaccharomyces pombe Cytochrome c (cyc1)

What is the gene structure of S. pombe cytochrome c (cyc1)?

The cytochrome c gene of S. pombe has been cloned using the Saccharomyces cerevisiae iso-1-cytochrome c gene as a molecular hybridization probe. DNA sequencing has confirmed the previously determined protein sequence with only two exceptions . The gene contains all necessary regulatory signals for expression, as demonstrated by successful heterologous expression in S. cerevisiae mutants lacking functional cytochrome c . The gene structure includes promoter elements, coding regions, and regulatory sequences that enable its proper expression and function in the cellular context.

How is cytochrome c (cyc1) expression regulated in S. pombe?

Cytochrome c expression in S. pombe is subject to complex regulatory mechanisms, particularly in response to oxygen levels. Research has shown that cyc1 is among the genes repressed during hypoxia . This repression is enhanced in Ofd2Δ strains compared to wild-type cells, suggesting that the histone H2A dioxygenase Ofd2 plays a role in modulating the hypoxic repression of oxidative phosphorylation genes including cyc1 . Additionally, iron availability impacts gene expression in S. pombe through the iron sensor Fep1, which mediates transcriptional repression of iron transport genes under high iron conditions . This regulatory network ensures appropriate expression of respiratory components like cytochrome c under varying environmental conditions.

What expression systems are optimal for recombinant S. pombe cytochrome c production?

Based on research with other recombinant proteins, several expression systems can be adapted for S. pombe cytochrome c production. For heterologous expression, both S. cerevisiae and Pichia pastoris represent viable options, with each offering distinct advantages. S. cerevisiae is well-established with extensive genetic tools and proven success with cytochrome c expression . For S. pombe cytochrome c expression in its native host, standard methods and techniques for fission yeast genetic manipulations can be employed using vectors such as pREPNT81 for N-terminal tagging .

The optimal approach depends on research objectives:

• For structural studies: E. coli with specialized vectors for high yield

• For functional studies in yeast: Native S. pombe or S. cerevisiae systems

• For post-translational modification studies: Yeast expression systems

What vector systems and promoters are recommended for cyc1 expression?

For recombinant expression in S. pombe, plasmids containing genomic fragments generated by PCR can be inserted into vectors such as pREPNT81 to generate N-terminal tagged proteins (e.g., Flag-tagged Ofd2), as demonstrated in related research . For bacterial expression, pET series vectors (e.g., pET30a for hexa-histidine tagging) have been successfully applied to related proteins .

When selecting promoters, consider:

• For high-level constitutive expression: Strong promoters like ADH1 or TDH3 in S. cerevisiae

• For inducible expression: The nmt1 promoter in S. pombe, which is repressed by thiamine

• For achieving physiological expression levels: Native cyc1 promoter with its regulatory elements

What purification strategies yield the highest purity recombinant cytochrome c?

Based on established protocols for cytochrome c and related proteins, an effective purification strategy would include:

• Affinity chromatography: Using N-terminal tags such as hexa-histidine or FLAG tags for initial capture

• Ion-exchange chromatography: Exploiting the basic nature of cytochrome c

• Size exclusion chromatography: For final polishing and buffer exchange

For optimal results, cell lysis conditions should be carefully controlled, using buffers containing protease inhibitors (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS) . For heme-containing proteins like cytochrome c, additional considerations include maintaining reducing conditions to preserve the native state of the protein.

What methods are most effective for analyzing cytochrome c expression levels?

Several complementary approaches provide robust analysis of cytochrome c expression:

• Reverse transcriptase and quantitative PCR (RT-qPCR): This technique effectively quantifies mRNA levels of cyc1 under different conditions. Primers specific to cyc1 can be designed and quantification performed using the SYBR-green PCR mix on instruments such as the ABI 7300 . Data analysis using the Pfaffl method allows calculation of changes in mRNA levels relative to control genes .

• Microarray analysis: For genome-wide expression studies, microarray platforms such as the S. pombe 72K array service can identify expression patterns of cyc1 alongside other genes, providing context for its regulation within broader cellular responses .

• Protein detection: Western blotting with antibodies against cytochrome c or its tags (if recombinantly expressed with epitope tags) enables protein-level quantification.

How can researchers assess the functional activity of recombinant cytochrome c?

Functional assessment of recombinant cytochrome c can be performed through:

• Complementation assays: Testing the ability of recombinant S. pombe cytochrome c to restore respiratory growth in S. cerevisiae mutants lacking functional cytochrome c, as demonstrated in previous research .

• Spectroscopic analysis: UV-visible spectroscopy to analyze the characteristic absorption spectra of properly folded cytochrome c with correctly incorporated heme.

• Electron transfer assays: Measuring the electron transfer capabilities using cytochrome c oxidase as an electron acceptor.

• Circular dichroism: To assess proper protein folding and secondary structure elements.

What experimental approaches can determine the subcellular localization of cytochrome c?

To determine subcellular localization:

• Fluorescent protein fusion: Creating GFP-fusions with cytochrome c for live-cell imaging, similar to approaches used for studying Fep1-GFP localization in S. pombe .

• Cell fractionation: Separating mitochondria, cytosol, and nuclear fractions followed by western blotting to detect cytochrome c distribution.

• Immunofluorescence microscopy: Using specific antibodies against cytochrome c or epitope tags in fixed cells.

• Chromatin immunoprecipitation (ChIP): While typically used for DNA-binding proteins, this technique has been adapted for studying protein-protein interactions and could be modified to study cytochrome c associations with mitochondrial components .

How does oxygen availability affect cyc1 expression in S. pombe?

Cytochrome c (cyc1) in S. pombe shows significant repression during hypoxia, as confirmed by both microarray analysis and RT-qPCR validation . Specifically:

• When S. pombe cells are subjected to hypoxic conditions (<1% O₂), cyc1 expression is downregulated along with other oxidative phosphorylation genes .

• This hypoxic repression is enhanced in strains lacking the histone H2A dioxygenase Ofd2 (Ofd2Δ strains), suggesting that Ofd2 plays a role in modulating the hypoxic response of these genes .

• The experimental approach to study this phenomenon involves:

  • Culturing cells in anaerobic conditions using systems such as the AnaeroGen system

  • Harvesting cells after defined exposure periods (e.g., 90 minutes)

  • Extracting RNA and performing microarray or RT-qPCR analysis

  • Comparing expression between normoxic and hypoxic conditions across different genetic backgrounds

What is the relationship between iron metabolism and cytochrome c in S. pombe?

While the search results don't directly address the relationship between iron metabolism and cytochrome c in S. pombe, we can infer important connections based on the biology of cytochrome c:

• Cytochrome c contains heme, an iron-containing cofactor essential for its electron transport function.

• In S. pombe, iron-dependent gene regulation is mediated by the transcription factor Fep1, which binds to chromatin in an iron-dependent manner . Under high iron conditions, Fep1 represses genes involved in iron uptake.

• Given cytochrome c's dependence on iron for function, its expression likely intersects with iron-regulatory networks, though the specific regulatory mechanisms would require experimental verification.

• Chromatin immunoprecipitation (ChIP) assays could be adapted to investigate potential Fep1 binding to the cyc1 promoter region, similar to studies of iron-responsive promoters .

How do mutations in the cyc1 gene affect cell viability and respiration?

The functional importance of cytochrome c is demonstrated by experiments showing that S. pombe cytochrome c can complement S. cerevisiae mutants lacking functional cytochrome c, restoring their ability to grow on non-fermentable carbon sources . This suggests:

• Mutations impairing cytochrome c function would likely compromise respiratory growth while potentially allowing fermentative growth.

• Despite amino acid differences between S. pombe and S. cerevisiae cytochrome c proteins, the core functional elements are conserved enough to maintain activity across species .

• Experimental approaches to study the effects of cyc1 mutations would include:

  • Site-directed mutagenesis of key residues

  • Expression of mutant proteins in cytochrome c-deficient strains

  • Assessment of respiratory growth on non-fermentable carbon sources

  • Measurement of oxygen consumption rates

  • Analysis of mitochondrial membrane potential

How can site-directed mutagenesis of S. pombe cyc1 provide insights into function-structure relationships?

Site-directed mutagenesis represents a powerful approach to probe structure-function relationships in cytochrome c:

• Key targets for mutagenesis include:

  • Heme-binding residues (typically Cys, His, Met)

  • Surface residues involved in interaction with cytochrome c oxidase or reductase

  • Residues that differ between S. pombe and S. cerevisiae cytochrome c

• Experimental design would involve:

  • QuickChange site-directed mutagenesis or similar techniques to generate specific mutations, similar to the approach used for creating the Ofd2H132A mutation

  • Expression of mutant proteins in appropriate host systems

  • Functional assays to assess electron transfer activity

  • Structural analysis through spectroscopic methods

• The complementation system with S. cerevisiae cytochrome c-deficient strains provides an excellent platform for assessing the functional impact of mutations .

What are the challenges in crystallizing recombinant S. pombe cytochrome c?

While the search results don't specifically address crystallization of S. pombe cytochrome c, crystallization of heme proteins presents several general challenges:

• Heme group instability: The heme cofactor can be lost or modified during purification, affecting protein homogeneity.

• Oxidation state heterogeneity: Cytochrome c can exist in reduced (Fe²⁺) or oxidized (Fe³⁺) states, potentially creating mixed populations that complicate crystallization.

• Surface properties: The highly charged surface of cytochrome c can promote nonspecific interactions.

• Methodological approaches to address these challenges include:

  • Careful control of redox conditions during purification

  • Use of reducing agents appropriate for heme proteins

  • Screening a wide range of crystallization conditions with varying pH, ionic strength, and precipitants

  • Surface engineering through site-directed mutagenesis to promote crystal contacts

How does recombinant S. pombe cytochrome c compare with native protein in terms of structural and functional properties?

A thorough comparison between recombinant and native S. pombe cytochrome c would consider:

• Post-translational modifications: Native cytochrome c undergoes several modifications, including heme attachment and potential processing of the N-terminus. Recombinant systems must faithfully reproduce these modifications.

• Folding and stability: Expression systems influence protein folding pathways, potentially affecting thermal stability and resistance to denaturation.

• Spectroscopic properties: UV-visible, CD, and NMR spectroscopy can reveal differences in heme environment and protein conformation.

• Functional activity: Electron transfer kinetics with physiological partners would provide the most relevant functional comparison.

• Experimental approaches would include:

  • Side-by-side purification of native and recombinant proteins

  • Comparative biochemical and biophysical characterization

  • Mass spectrometry to identify post-translational modifications

  • Enzymatic activity assays under standardized conditions

What are the key differences between S. pombe cytochrome c and cytochrome c proteins from other organisms?

Cytochrome c is highly conserved across species but displays important variations:

• Sequence divergence: The amino acid sequence of S. pombe cytochrome c differs from that of S. cerevisiae, though these differences do not drastically affect function . These differences represent evolutionary adaptations that may influence interaction specificity, stability, or regulatory properties.

• Codon usage: Analysis of the S. pombe cytochrome c gene revealed that the nonrandom distribution of silent third base differences observed between the two cytochrome c genes of S. cerevisiae does not extend to the S. pombe cytochrome c gene . This suggests distinct evolutionary constraints on codon selection.

• Regulatory elements: The S. pombe cytochrome c gene contains all regulatory signals required for expression in S. cerevisiae , indicating conservation of core regulatory mechanisms despite evolutionary divergence.

• A comparative analysis approach would include:

  • Multiple sequence alignment of cytochrome c proteins from diverse species

  • Phylogenetic analysis to map evolutionary relationships

  • Structural modeling to identify conserved structural features versus species-specific adaptations

  • Cross-species complementation experiments to test functional conservation

Can S. pombe expression systems offer advantages for difficult-to-express recombinant proteins compared to S. cerevisiae?

Both S. pombe and S. cerevisiae offer distinct advantages as expression hosts:

• Growth characteristics: P. pastoris (now reclassified as Komagataella phaffii) prefers respiratory growth without accumulation of ethanol and acetate, enabling high cell density cultures up to 200 g/L . While this specific comparison is with P. pastoris, S. pombe similarly has a more respiratory metabolism than S. cerevisiae, potentially offering advantages for protein expression.

• Protein processing: S. pombe's protein processing machinery may be more suitable for certain classes of proteins, particularly those from higher eukaryotes, as S. pombe's cell cycle and splicing mechanisms more closely resemble those of higher eukaryotes.

• Glycosylation patterns: S. pombe produces different glycosylation patterns compared to S. cerevisiae, which may be advantageous for certain proteins.

• Selection criteria for choosing between these systems should consider:

  • Protein complexity and post-translational modification requirements

  • Growth medium preferences and culture conditions

  • Available genetic tools and expression vectors

  • Desired yield and scalability

What model systems can best evaluate the apoptotic function of recombinant S. pombe cytochrome c?

While the search results don't directly address cytochrome c's role in apoptosis in S. pombe, several experimental systems could be adapted:

• Heterologous systems: Mammalian cell-free systems depleted of endogenous cytochrome c could be reconstituted with recombinant S. pombe cytochrome c to assess its ability to activate caspases.

• Yeast apoptosis models: While classical apoptosis as understood in mammalian systems differs in yeast, S. cerevisiae and S. pombe display forms of programmed cell death. Expression of recombinant variants in these systems could provide insights into conserved functions.

• Hybrid systems: Recombinant S. pombe cytochrome c could be microinjected or delivered to mammalian cells depleted of endogenous cytochrome c to test functional conservation.

• The experimental approach would include:

  • Purification of recombinant S. pombe cytochrome c to high homogeneity

  • Development of assays measuring downstream apoptotic events

  • Comparison with cytochrome c from organisms with well-characterized apoptotic pathways

  • Structural analysis of interaction interfaces with apoptotic proteins