Recombinant Schizosaccharomyces pombe Cytochrome oxidase assembly protein 1 (coa1)

What is Cytochrome oxidase assembly protein 1 (coa1) in Schizosaccharomyces pombe?

Cytochrome c oxidase assembly factor 1 (coa1) in Schizosaccharomyces pombe is a protein encoded by the coa1 gene (also known as SPBC16E9.03c). It functions as an assembly factor for cytochrome c oxidase (complex IV), which is the terminal enzyme in the mitochondrial respiratory chain. According to the iPTMnet database, coa1 is identified with UniProt accession number O14320 and protein ID COA1_SCHPO. The protein plays a crucial role in the biogenesis and assembly of complex IV components, thereby maintaining proper mitochondrial respiratory function in fission yeast . Unlike some other mitochondrial proteins that have been extensively characterized, coa1 represents a specific adaptation in the respiratory chain assembly pathway of S. pombe that may differ from its counterparts in other organisms.

How does coa1 differ from Shy1 in S. pombe mitochondrial function?

While both coa1 and Shy1 are involved in cytochrome c oxidase assembly in S. pombe, they perform distinct functions:

Shy1 contains a conserved SURF1 domain that links to the biogenesis of complex IV and shares high structural similarity with its homologs in Saccharomyces cerevisiae and humans. Research has shown that Shy1 physically interacts with structural subunits and assembly factors of complex IV. Interestingly, Rip1, a subunit of complex III, can also co-immunoprecipitate with Shy1, suggesting its involvement in the assembly of mitochondrial respiratory chain supercomplexes . Unlike its homologs in other species, deletion of shy1 in S. pombe does not completely disrupt respiratory chain assembly, indicating potential compensatory mechanisms in fission yeast.

What are the common methods for expressing recombinant proteins in S. pombe?

The expression of recombinant proteins in S. pombe typically employs the following methodologies:

• Promoter selection: The nmt1 promoter (no message in thiamine) is commonly used for controlled expression, as it can be repressed by thiamine addition and induced upon thiamine removal. For constitutive expression, modified versions of nmt1 or other promoters like adh1 are utilized .

• Expression vectors: Both integrative (e.g., pCAD1) and episomal (e.g., pREP1) vectors are available. Integrative vectors allow for stable genomic integration, while episomal vectors typically provide higher copy numbers and expression levels .

• Integration strategies:

  • Single-copy integration into specific loci (like leu1)

  • Multi-copy integration for increased expression levels

  • Combination approaches using both genomic integration and episomal expression

• Transformation methods:

  • Lithium acetate transformation

  • Electroporation

  • Cryocompetent cell preparation

Researchers have successfully used these approaches to achieve secretion of recombinant human proteins in S. pombe with titers up to 5 mg/L for complex proteins like single-chain antibody fragments . For optimal results, codon optimization for S. pombe, inclusion of appropriate signal sequences, and careful consideration of culture conditions are essential for successful recombinant protein expression.

What methodologies are most effective for purifying functional recombinant coa1 protein from S. pombe?

Purification of functional recombinant coa1 from S. pombe requires specialized approaches due to its membrane association and involvement in protein complexes:

• Expression strategy optimization:

  • Use of strong, inducible promoters like nmt1 with thiamine control

  • Addition of affinity tags (His6, FLAG, or TAP) at either N- or C-terminus, with optimal tag positioning determined empirically to avoid interference with function

  • Co-expression with interaction partners to enhance stability

• Cell disruption and membrane fraction isolation:

  • Gentle cell lysis using glass beads or enzymatic methods

  • Differential centrifugation to isolate mitochondrial fractions (typically 10,000×g for crude mitochondria)

  • Further purification of mitochondria using sucrose gradient centrifugation

• Solubilization optimization:

  • Testing multiple detergents (digitonin, DDM, Triton X-100) at various concentrations

  • Inclusion of stabilizing agents such as glycerol (10-15%) and protease inhibitors

  • Maintenance of proper ionic strength and pH throughout purification

• Chromatographic purification:

  • Initial capture using affinity chromatography (IMAC for His-tagged proteins)

  • Secondary purification using ion exchange or size exclusion chromatography

  • Optional: Blue Native PAGE for isolation of intact protein complexes

• Functional validation:

  • Assessment of proper folding using circular dichroism

  • Verification of protein-protein interactions with known partners

  • Activity assays measuring ability to complement coa1 deletion strains

When performing these procedures, it's critical to maintain mitochondrial integrity during early purification steps and to preserve native protein interactions by using mild solubilization conditions. Researchers should optimize each step for the specific properties of coa1, as standardized protocols may require significant modification.

How can researchers study the interaction between coa1 and other mitochondrial proteins in S. pombe?

Multiple complementary approaches can be employed to study coa1 interactions with other mitochondrial proteins:

• Co-immunoprecipitation (Co-IP):

  • Express epitope-tagged coa1 (HA, FLAG, or Myc) in S. pombe

  • Solubilize mitochondria with mild detergents (0.5-1% digitonin)

  • Immunoprecipitate with tag-specific antibodies

  • Identify interacting partners by mass spectrometry or Western blotting

  • Validate interactions with reciprocal Co-IP

• Blue Native PAGE analysis:

  • Isolate mitochondria and solubilize with appropriate detergents

  • Separate native complexes on gradient (3-12%) blue native gels

  • Identify complex components using second-dimension SDS-PAGE

  • Compare complex formation in wild-type versus coa1 deletion strains

This approach has been successfully used for studying Shy1, which showed interactions with complex IV components and Rip1 from complex III, suggesting involvement in supercomplex formation .

• Proximity-dependent labeling:

  • Create fusions of coa1 with BioID or TurboID

  • Allow in vivo biotinylation of proximal proteins

  • Isolate biotinylated proteins using streptavidin pulldown

  • Identify interacting partners by mass spectrometry

• Fluorescence microscopy approaches:

  • Create fluorescent protein fusions (GFP, mCherry)

  • Study co-localization patterns with known mitochondrial markers

  • Employ FRET or BiFC to visualize direct protein-protein interactions

• Genetic interaction screening:

  • Generate synthetic genetic arrays using coa1 deletion strains

  • Identify genetic interactions through growth phenotype analysis

  • Validate protein-protein interactions of candidates using biochemical methods

For all approaches, appropriate controls are essential, including testing interactions under different respiratory conditions (fermentative vs. respiratory) and comparing to known interaction partners of cytochrome oxidase assembly factors.

What role does coa1 play in the assembly of cytochrome c oxidase in S. pombe compared to other model organisms?

The role of coa1 in cytochrome c oxidase assembly exhibits both conservation and divergence across species:

In S. pombe, the cytochrome c oxidase assembly pathway appears to have unique features compared to other organisms. While the molecular details remain incompletely characterized, studies of related assembly factors like Shy1 (SURF1 homolog) provide insights. Unlike its homologs in S. cerevisiae and humans, deletion of shy1 in S. pombe does not critically disrupt respiratory chain assembly, suggesting compensatory mechanisms specific to fission yeast .

The study of coa1 in S. pombe has been complicated by several factors:

• The fission yeast mitochondrial genome organization differs from S. cerevisiae

• S. pombe can grow under purely fermentative conditions, making respiratory mutants viable

• Some assembly factors show functional redundancy in fission yeast

Research approaches to elucidate coa1 function should include:

• Comparative genomic analysis across yeast species

• BN-PAGE analysis of respiratory complexes in wild-type and coa1 deletion strains

• Transcriptome and proteome analysis to identify compensatory pathways

• Detailed characterization of protein-protein interactions using techniques described in question 2.2

This comparative approach will help identify both conserved mechanisms and species-specific adaptations in cytochrome c oxidase assembly.

How do mutations in coa1 impact mitochondrial function and cellular respiration in S. pombe?

Mutations in coa1 can affect mitochondrial function and cellular respiration in S. pombe through multiple mechanisms:

• Impact on Complex IV assembly:

  • Decreased assembly of complete cytochrome c oxidase

  • Accumulation of assembly intermediates

  • Altered stoichiometry of complex subunits

• Respiratory chain dysfunction:

  • Reduced oxygen consumption rates

  • Decreased electron transfer capacity

  • Compromised proton pumping efficiency

  • Potential compensatory upregulation of alternative respiratory pathways

• Cellular consequences:

  • Increased production of reactive oxygen species (ROS)

  • Activation of mitochondrial stress response pathways

  • Altered mitochondrial membrane potential

  • Changes in mitochondrial morphology and dynamics

• Metabolic adaptations:

  • Shift toward fermentative metabolism

  • Altered expression of genes involved in carbon metabolism

  • Changes in mitochondrial protein import

  • Potential activation of retrograde signaling pathways

These effects can be measured using the following methodologies:

• Oxygen consumption analysis using oxygen electrodes or plate-based respirometry

• Membrane potential measurements using fluorescent dyes (TMRM, JC-1)

• Assessment of ROS production using specific probes (DCF, MitoSOX)

• Analysis of mitochondrial morphology using fluorescence microscopy

• Evaluation of metabolic profiles using mass spectrometry

By studying how different mutations in coa1 affect these parameters, researchers can gain insights into the specific roles of different protein domains in mitochondrial function and identify potential mechanisms of compensation in S. pombe.

What approaches can be used to study the contribution of coa1 to mitochondrial respiratory supercomplex formation?

The contribution of coa1 to mitochondrial respiratory supercomplex formation can be studied using several advanced approaches:

• Blue Native PAGE analysis:

  • Isolate mitochondria from wild-type and coa1-deleted strains

  • Solubilize using digitonin (typically 4g/g protein) to preserve supercomplexes

  • Separate on gradient (3-12%) blue native gels

  • Identify complexes using in-gel activity assays or antibody detection

  • Quantify differences in supercomplex formation and stability

Similar approaches have revealed that Shy1 may be involved in respiratory chain supercomplex assembly, as Rip1 (a complex III component) can co-immunoprecipitate with Shy1 .

• Cryo-electron microscopy:

  • Purify intact respiratory supercomplexes from wild-type and mutant strains

  • Determine structural differences using single-particle cryo-EM

  • Identify specific interfaces affected by coa1 absence

• Complexome profiling:

  • Separate mitochondrial protein complexes using blue native PAGE

  • Cut gel into equal fractions and perform mass spectrometry on each fraction

  • Compare the migration patterns of respiratory chain components between wild-type and coa1 mutants

  • Identify shifts in complex/supercomplex distribution

• Functional assessment of supercomplex activity:

  • Measure substrate channeling efficiency using flux analysis

  • Quantify electron transfer rates between complexes

  • Assess ROS production at supercomplex interfaces

  • Compare respiratory capacity of intact versus disrupted supercomplexes

• Time-resolved assembly studies:

  • Use pulse-chase labeling of mitochondrially-encoded subunits

  • Track incorporation into individual complexes versus supercomplexes

  • Compare assembly kinetics between wild-type and coa1 mutants

These approaches, particularly when used in combination, can provide comprehensive insights into how coa1 contributes to the formation, stability, and function of respiratory supercomplexes in S. pombe mitochondria.

How can CRISPR-Cas9 genome editing be optimized for studying coa1 function in S. pombe?

CRISPR-Cas9 genome editing in S. pombe for coa1 functional studies requires specific optimizations:

While CRISPR-Cas9 systems have been adapted for S. pombe, efficiency may be lower than in other organisms. Researchers should consider combining CRISPR with traditional homologous recombination approaches and perform careful screening to identify correctly edited clones.

How can researchers resolve contradictory results when studying coa1 function in different strain backgrounds?

Contradictory results when studying coa1 across different S. pombe strain backgrounds require systematic troubleshooting:

• Strain background characterization:

  • Perform whole-genome sequencing of contradictory strains

  • Identify polymorphisms that may affect mitochondrial function

  • Examine nuclear-mitochondrial compatibility in hybrid strains

  • Verify genetic markers to rule out strain mix-ups or contamination

• Standardization approaches:

  • Back-cross strains to a common reference background (multiple generations)

  • Create isogenic strains differing only in the coa1 locus

  • Standard culture conditions (media composition, growth phase, temperature)

  • Deploy identical experimental protocols across laboratories

• Statistical and experimental design considerations:

  • Increase biological and technical replicates

  • Use power analysis to determine appropriate sample sizes

  • Implement blinded analysis when possible

  • Consider batch effects in data interpretation

• Systematic resolution approaches:

  • Cross-complementation experiments between contradictory strains

  • Identification of genetic modifiers through suppressor screens

  • Creation of double mutants to test genetic interactions

  • Detailed phenotypic characterization under varied conditions

• Comprehensive validation:

  • Test phenotypes using multiple methodological approaches

  • Implement genetic rescue experiments with defined alleles

  • Verify protein expression and localization in all strains

  • Document growth history and handling of strains

When contradictions persist, researchers should consider that they may reveal strain-specific adaptation mechanisms or genetic interactions. These differences can be leveraged to identify new components or regulatory mechanisms in the cytochrome oxidase assembly pathway.

What are best practices for experimental design when investigating coa1 interactions with mitochondrial proteins?

Robust experimental design for studying coa1 interactions requires careful planning:

• Control selection and validation:

  • Positive controls: Include known interaction partners (e.g., cytochrome oxidase subunits)

  • Negative controls: Use unrelated mitochondrial proteins or cytosolic proteins

  • Tag-only controls: Express epitope tags alone to identify tag-specific artifacts

  • Reciprocal tagging: Tag potential interaction partners to confirm bidirectional interaction

• Experimental conditions optimization:

  • Growth phase standardization (log vs. stationary)

  • Media selection (fermentative vs. respiratory conditions)

  • Stress conditions (oxidative stress, mitochondrial inhibitors)

  • Temperature variations to identify conditional interactions

• Technical considerations:

  • Detergent selection and concentration for membrane protein solubilization

  • Crosslinking parameters if using chemical crosslinking

  • Salt concentration variations to distinguish strong vs. weak interactions

  • Appropriate buffer composition to maintain physiological conditions

• Statistical approach:

  • Define significance thresholds for interaction scoring

  • Implement appropriate normalization methods

  • Use quantitative measures rather than binary outcomes when possible

  • Consider biological variability in replicate planning

• Validation hierarchy:

  • Primary screen: Co-IP or affinity purification

  • Secondary validation: Reciprocal pulldown or Y2H

  • Functional validation: Genetic interaction or phenotypic analysis

  • Direct interaction confirmation: In vitro binding assays with purified components

By implementing these best practices, researchers can generate more reliable and reproducible data on coa1 interactions, reducing the likelihood of false positives while increasing confidence in true interaction partners.

How can researchers differentiate between direct and indirect effects of coa1 mutations on mitochondrial function?

Differentiating direct from indirect effects of coa1 mutations requires multi-layered experimental approaches:

• Temporal analysis:

  • Time-course experiments following coa1 disruption

  • Identification of primary (early) versus secondary (late) effects

  • Pulse-chase labeling to track assembly kinetics

  • Inducible systems to control the timing of coa1 depletion

• Biochemical proximity assessment:

  • In vitro reconstitution with purified components

  • Direct binding assays with recombinant proteins

  • Crosslinking-mass spectrometry to map interaction interfaces

  • Structural analysis of protein complexes

• Genetic dissection approaches:

  • Domain-specific mutations to separate functions

  • Suppressor screens to identify compensatory pathways

  • Epistasis analysis with interacting genes

  • Allele-specific interactions to confirm direct relationships

• Quantitative modeling:

  • Network analysis of protein-protein interactions

  • Flux balance analysis of metabolic changes

  • Kinetic modeling of assembly pathways

  • Correlation analysis between molecular and functional phenotypes

• Multi-omics integration:

  • Compare transcriptome, proteome, and metabolome changes

  • Distinguish primary targets from downstream responses

  • Time-resolved profiling to capture cascade effects

  • Cell-type or condition-specific effects

For example, when studying the effects of coa1 on cytochrome c oxidase assembly, researchers should first determine whether coa1 physically interacts with complex IV subunits (as shown for Shy1 ), then assess whether assembly intermediates accumulate immediately after coa1 depletion, and finally determine if observed phenotypes can be rescued by overexpression of direct interaction partners.

Comparative Analysis Questions

When adapting protocols from S. cerevisiae to study coa1 in S. pombe, researchers should consider several critical methodological differences:

• Genetic manipulation protocols:

  • S. pombe has higher homologous recombination efficiency but requires longer homology arms

  • Different selectable markers and promoter systems are optimal for each species

  • S. pombe transformation typically requires different conditions (electroporation parameters, lithium acetate concentrations)

  • CRISPR-Cas9 systems require species-specific optimization

• Growth and media considerations:

  • S. pombe has different optimal growth temperatures (30°C vs. 30-32°C for S. cerevisiae)

  • Carbon source response differs (glucose repression mechanisms vary)

  • S. pombe cell wall composition differs, affecting enzymatic digestion protocols

  • Different media formulations for inducing respiratory vs. fermentative growth

• Mitochondrial isolation and analysis:

  • Cell disruption methods require optimization (different cell wall properties)

  • Different detergent concentrations for mitochondrial solubilization

  • Modified gradient conditions for mitochondrial purification

  • Adapted blue native PAGE conditions for respiratory complex separation

• Protein interaction studies:

  • S. pombe requires different epitope tag systems for optimal expression

  • Crosslinking conditions need to be re-optimized

  • Codon usage optimization differs between species

  • Immunoprecipitation buffers require adjustment for S. pombe proteins

• Respiratory analysis techniques:

  • Modified oxygen consumption measurement parameters

  • Different inhibitor concentrations for respiratory chain components

  • Adapted assay conditions for enzymatic activities

  • Species-specific antibodies for Western blot analysis

For example, when studying protein-protein interactions, S. cerevisiae protocols often use GAL promoters for inducible expression, whereas S. pombe typically employs the nmt1 promoter system with thiamine regulation . Similarly, mitochondrial isolation from S. pombe may require modified enzymatic digestion due to differences in cell wall composition compared to S. cerevisiae.

What bioinformatic approaches can identify conserved functional domains in coa1 across different species?

Multiple bioinformatic approaches can be integrated to identify conserved functional domains in coa1:

• Sequence-based comparative methods:

  • Multiple sequence alignment (MSA) of coa1 homologs across species

  • Identification of conserved motifs using MEME Suite and similar tools

  • Calculation of conservation scores across alignment positions

  • Detection of signature sequences specific to functional clades

• Structure prediction approaches:

  • Secondary structure prediction across aligned sequences

  • Tertiary structure modeling using AlphaFold or similar tools

  • Identification of conserved structural elements despite sequence divergence

  • Mapping conservation scores onto predicted structural models

• Evolutionary analysis:

  • Phylogenetic tree construction to establish evolutionary relationships

  • Analysis of selection pressure (dN/dS ratios) across protein regions

  • Identification of co-evolving residues that maintain functional interactions

  • Detection of lineage-specific adaptations that may reflect functional changes

• Functional domain prediction:

  • Search for known domains using SMART, Pfam, or InterPro databases

  • Prediction of transmembrane regions and signal sequences

  • Identification of potential post-translational modification sites

  • Detection of protein-protein interaction motifs

• Integrative approaches:

  • Correlation of conserved regions with experimental mutation data

  • Integration of proteomic data to identify interaction interfaces

  • Comparison with structurally characterized homologs in other systems

  • Network-based approaches to identify functionally linked protein domains

What are the optimal conditions for expressing and purifying recombinant S. pombe coa1 in heterologous systems?

Optimizing the expression and purification of recombinant S. pombe coa1 in heterologous systems requires careful consideration of several parameters:

• Expression system selection:

• Construct design considerations:

  • Codon optimization for the expression host

  • Addition of purification tags (His6, GST, MBP) with appropriate linkers

  • Inclusion of TEV or similar protease sites for tag removal

  • Potential removal of hydrophobic transmembrane segments

  • Design of soluble domain constructs if full-length proves challenging

• Expression condition optimization:

  • Temperature screening (typically lower temperatures improve folding)

  • Induction parameters (inducer concentration, timing, duration)

  • Media composition (rich vs. minimal, supplements)

  • Co-expression with chaperones or assembly partners

  • Additives to stabilize membrane proteins (glycerol, specific detergents)

• Purification strategy:

  • Gentle cell lysis methods to preserve protein structure

  • Detergent screening for membrane protein extraction

  • Multi-step purification combining affinity, ion exchange and size exclusion

  • Buffer optimization to maintain stability throughout purification

  • Addition of stabilizing ligands or cofactors

• Quality control metrics:

  • Size exclusion chromatography to verify monodispersity

  • Circular dichroism to assess secondary structure

  • Thermal shift assays to optimize buffer conditions

  • Functional assays to confirm biological activity

  • Mass spectrometry to verify protein identity and modifications

For membrane proteins like coa1, detergent selection is particularly critical. A systematic screen of detergents (DDM, digitonin, LMNG, etc.) at different concentrations should be performed to identify conditions that extract the protein while preserving its native conformation and activity.

How can researchers effectively monitor the assembly of cytochrome c oxidase in S. pombe?

Multiple complementary techniques can be employed to effectively monitor cytochrome c oxidase assembly in S. pombe:

• Biochemical approaches:

  • Blue Native PAGE to separate intact complexes and assembly intermediates

  • In-gel activity assays using cytochrome c and DAB as substrates

  • Pulse-chase labeling of mitochondrially-encoded subunits

  • Immunoprecipitation of assembly intermediates using subunit-specific antibodies

  • Gradient sucrose or glycerol density centrifugation to separate complexes

• Spectroscopic methods:

  • Reduced minus oxidized spectra to quantify cytochrome content

  • Measurement of absorbance at 605 nm to quantify heme a3 incorporation

  • Resonance Raman spectroscopy to assess metal center configuration

  • EPR spectroscopy to analyze copper center incorporation

• Functional assays:

  • Polarographic measurement of oxygen consumption (Clark electrode)

  • High-resolution respirometry using Oroboros or Seahorse systems

  • Specific activity assays using isolated mitochondria

  • Membrane potential measurements using fluorescent dyes

  • ROS production assessment using specific probes

• Molecular biology approaches:

  • Quantitative PCR to assess mitochondrial gene expression

  • Proteomic analysis of purified mitochondrial fractions

  • Monitoring tagged versions of assembly factors (e.g., Shy1, coa1)

  • RNA-seq to identify transcriptional responses to assembly defects

• Microscopy techniques:

  • Fluorescently tagged assembly factors to track localization

  • Super-resolution microscopy to visualize assembly complexes

  • Electron microscopy to assess mitochondrial ultrastructure

  • Live cell imaging to monitor assembly dynamics

To establish a comprehensive understanding of complex IV assembly, researchers should combine multiple approaches. For example, Blue Native PAGE analysis has successfully revealed that the S. pombe assembly factor Shy1 may be involved in respiratory chain supercomplex formation, as demonstrated by co-immunoprecipitation with the complex III component Rip1 .

What are the key considerations for designing a CRISPR-Cas9 gene editing strategy to study coa1 function?

Designing an effective CRISPR-Cas9 gene editing strategy for coa1 functional studies requires careful planning:

• Target site selection criteria:

  • Identify critical functional domains through sequence conservation analysis

  • Target coding regions near the 5' end to maximize disruption

  • Avoid regions with secondary structures that may impede Cas9 binding

  • Select target sites with minimal potential off-targets in the S. pombe genome

  • Consider PAM availability (NGG for SpCas9) within the target region

• Guide RNA design optimization:

  • Use S. pombe-specific guide RNA design tools that account for genome composition

  • Design multiple guides for each target to increase success probability

  • Verify guide efficiency through preliminary validation experiments

  • Include appropriate RNA polymerase III promoters (e.g., U6) for guide expression

  • Consider chemical modifications to improve stability if using synthetic guides

• Delivery system considerations:

  • Select appropriate vectors for S. pombe (episomal or integrative)

  • Balance expression levels to minimize toxicity while maximizing efficiency

  • Consider transient vs. stable Cas9 expression based on experimental goals

  • Optimize transformation protocols for high efficiency delivery

  • Include appropriate selection markers for screening

• Repair template design:

  • For knock-in mutations, include homology arms of at least 500 bp on each side

  • Incorporate silent mutations in the PAM or seed region to prevent re-cutting

  • Consider adding selection markers for positive identification of edited cells

  • Design uniquely identifying primers spanning the edit site for screening

  • For complex modifications, consider step-wise editing approaches

• Screening and validation strategies:

  • Design PCR screening with primers flanking the edit site

  • Use restriction enzyme digestion if the edit creates/removes a site

  • Implement Sanger sequencing to confirm precise edits

  • Verify expression/knockout at protein level via Western blotting

  • Functional validation through phenotypic assays

For studying coa1 specifically, researchers might consider creating an array of mutations, including:

• Complete knockout to assess essentiality

• Tagged versions for localization and interaction studies

• Domain-specific mutations to dissect function

• Conditional alleles using auxin-inducible degron tags

By carefully designing each element of the CRISPR-Cas9 system, researchers can achieve efficient and precise genetic modifications to study coa1 function in S. pombe.