Recombinant Schizosaccharomyces pombe Uncharacterized protein C23H4.13c (SPAC23H4.13c)

What is currently known about the uncharacterized protein SPAC23H4.13c in Schizosaccharomyces pombe?

SPAC23H4.13c is an uncharacterized protein in the fission yeast Schizosaccharomyces pombe. While specific information about this particular protein is limited in current literature, it is part of the S. pombe proteome that continues to be systematically studied. Based on genomic context analysis, it may be located in proximity to other characterized genes on chromosome I, such as SPAC23H4.18c (pip1/rbx1), which is involved in heterochromatin assembly . As an uncharacterized protein, its precise function, subcellular localization, and interaction partners remain to be fully elucidated through dedicated experimental approaches.

What prediction tools are most reliable for initial characterization of SPAC23H4.13c?

For initial in silico characterization of SPAC23H4.13c, researchers should employ a combination of complementary prediction tools:

• Sequence homology analysis: BLAST against characterized proteins across species

• Protein domain prediction: InterPro, Pfam, and SMART to identify conserved domains

• Secondary structure prediction: PSIPRED, JPred

• Subcellular localization prediction: DeepLoc, WoLF PSORT

• Post-translational modification sites: NetPhos (phosphorylation), NetNGlyc/NetOGlyc (glycosylation)

• Protein-protein interaction networks: STRING database integration

The reliability of these predictions should be assessed by consensus across multiple tools and validated experimentally. For novel S. pombe proteins, comparing predictions with data from related characterized proteins in the SIR2/HST gene family or other known S. pombe proteins can provide context for interpretation .

What expression systems are optimal for producing recombinant SPAC23H4.13c protein?

The optimal expression system for recombinant SPAC23H4.13c depends on research objectives and downstream applications. Here's a comparative analysis of expression systems:

For initial characterization, an E. coli system with appropriate tags (His6, GST) is recommended, followed by validation in the native S. pombe system using techniques similar to those employed in studies of other S. pombe proteins like hst4+ .

What purification strategy yields the highest purity of recombinant SPAC23H4.13c while maintaining protein functionality?

A multi-step purification approach is recommended to achieve high purity while preserving functionality:

• Initial capture: Affinity chromatography using N-terminal or C-terminal tags (His6 or GST)

• Intermediate purification: Ion exchange chromatography based on the protein's theoretical isoelectric point

• Polishing: Size exclusion chromatography to separate monomeric protein from aggregates and contaminants

Critical considerations for maintaining functionality:

• Use mild detergents if the protein is predicted to be membrane-associated

• Include protease inhibitors throughout purification

• Maintain buffer conditions similar to S. pombe cellular environment (pH 6.5-7.0)

• Consider including stabilizing agents (glycerol, reducing agents)

• Assess protein integrity by circular dichroism or thermal shift assays

This approach parallels successful strategies used for other S. pombe proteins studied in chromatin modification contexts .

What CRISPR-Cas9 strategies are most effective for generating SPAC23H4.13c knockout strains in S. pombe?

For generating SPAC23H4.13c knockout strains using CRISPR-Cas9 in S. pombe, consider these methodological approaches:

• Guide RNA design:

  • Target unique sequences within the SPAC23H4.13c open reading frame

  • Avoid sequences with off-target potential in the S. pombe genome

  • Design at least 3-4 guide RNAs for redundancy and validation

• Delivery method:

  • Plasmid-based expression of Cas9 and guide RNA under control of S. pombe promoters

  • Ribonucleoprotein (RNP) complex delivery via electroporation

• Homology-directed repair template:

  • Include 500-1000 bp homology arms flanking the target site

  • Incorporate a selectable marker (e.g., his3+ as used in hst4Δ strains )

  • Consider including unique restriction sites for verification

• Validation approach:

  • PCR confirmation of correct integration

  • Sequencing of integration junctions

  • RT-PCR to confirm absence of mRNA expression

  • Western blotting if antibodies are available

This approach aligns with established S. pombe genetic manipulation protocols and can be modeled after successful gene disruption strategies used for other genes like hst4+ .

How should researchers design phenotypic screens to identify cellular functions of SPAC23H4.13c?

A comprehensive phenotypic screening approach should include:

• Growth and morphology analysis:

  • Growth curve analysis in standard and stress conditions

  • Microscopic examination for morphological abnormalities

  • Cell cycle progression analysis (similar to hst4Δ strains which show growth defects and abnormal morphology )

• Genetic interaction screens:

  • Synthetic genetic array (SGA) analysis

  • Double mutant construction with known pathway components

  • Suppressor screens to identify compensatory mechanisms

• Stress response characterization:

  • Temperature sensitivity (25°C, 30°C, 36°C)

  • DNA damaging agents (UV, MMS, hydroxyurea)

  • Microtubule-destabilizing drugs (similar to hst4Δ sensitivity )

  • Oxidative stress (H₂O₂, menadione)

• Chromatin function assessment:

  • Silencing assays using reporter genes at telomeres and centromeres

  • Chromosome loss rate measurement

  • Analysis of heterochromatin-specific markers

  • Potential examination of silencing defects as observed in hst4Δ mutants

• Molecular phenotypes:

  • Transcriptome analysis (RNA-seq)

  • Chromatin immunoprecipitation to identify genomic binding sites

  • Protein-protein interaction studies (immunoprecipitation, yeast two-hybrid)

This systematic approach parallels successful strategies used to characterize other initially uncharacterized S. pombe proteins, particularly those involved in chromatin regulation like heterochromatin assembly factors .

What imaging techniques provide the most accurate assessment of SPAC23H4.13c subcellular localization?

For comprehensive subcellular localization analysis of SPAC23H4.13c:

• Fluorescent protein tagging strategies:

  • C-terminal vs. N-terminal GFP/mCherry fusion constructs

  • Integration at the endogenous locus to maintain native expression levels

  • Verification that the tag doesn't disrupt protein function through complementation assays

• Advanced microscopy techniques:

  • Confocal microscopy for basic localization

  • Super-resolution microscopy (PALM/STORM, SIM) for detailed subnuclear structures

  • Live-cell imaging to capture dynamic localization changes

  • FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility

• Co-localization studies:

  • Nuclear markers (DAPI, histone-mCherry)

  • Nucleolar markers if nucleolar enrichment is suspected (similar to Hst4p )

  • Heterochromatin markers if involved in silencing (HP1/Swi6)

  • Co-staining with known interaction partners

• Immunofluorescence approaches:

  • Generation of specific antibodies against SPAC23H4.13c

  • Epitope tagging (HA, Myc, FLAG) for commercial antibody use

  • Fixation protocol optimization for S. pombe cells

The integration of these approaches would provide robust localization data, potentially revealing nuclear or nucleolar localization patterns similar to those observed for other chromatin-associated proteins in S. pombe .

How can researchers investigate protein-protein interactions of SPAC23H4.13c in vivo?

To comprehensively investigate protein-protein interactions of SPAC23H4.13c in vivo:

• Affinity purification-mass spectrometry (AP-MS):

  • Tandem affinity purification (TAP) tagging of SPAC23H4.13c

  • Single-step purification using GFP-Trap or FLAG immunoprecipitation

  • Crosslinking-assisted purification for transient interactions

  • SILAC or TMT labeling for quantitative interaction analysis

• Proximity-dependent labeling techniques:

  • BioID fusion to identify proteins in close proximity

  • APEX2 fusion for rapid labeling and temporal studies

  • Split-BioID for conditional interaction mapping

• Fluorescence-based interaction assays:

  • Förster Resonance Energy Transfer (FRET)

  • Bimolecular Fluorescence Complementation (BiFC)

  • Fluorescence Cross-Correlation Spectroscopy (FCCS)

• Genetic interaction approaches:

  • Yeast two-hybrid screening

  • Synthetic genetic array analysis

  • Suppressor screens to identify functional relationships

• Validation strategies:

  • Reciprocal co-immunoprecipitation experiments

  • Direct binding assays with purified components

  • Functional assays to test biological relevance of interactions

These approaches should be conducted under various conditions (e.g., different cell cycle stages, stress conditions) to capture dynamic interaction networks. For proteins potentially involved in heterochromatin assembly, interactions with known components of silencing machinery should be specifically investigated .

What approaches are recommended for investigating the potential role of SPAC23H4.13c in heterochromatin formation?

To systematically investigate SPAC23H4.13c's potential role in heterochromatin formation:

• Chromatin silencing assays:

  • Reporter gene silencing at heterochromatic regions (telomeres, centromeres, mating-type locus)

  • qRT-PCR analysis of transcript levels from naturally silenced regions

  • Comparison with known heterochromatin assembly mutants (similar to analyses performed with hst4Δ )

• Chromatin immunoprecipitation (ChIP) studies:

  • ChIP-seq to map SPAC23H4.13c genomic binding sites

  • ChIP for heterochromatin markers (H3K9me, Swi6/HP1) in wild-type vs. mutant strains

  • Sequential ChIP to identify co-occupancy with other heterochromatin factors

• Chromosome function assays:

  • Minichromosome stability assays (using constructs like Ch16 )

  • Centromere function assessment through microtubule-destabilizing drug sensitivity

  • Chromosome segregation analysis during mitosis

• Molecular interaction studies:

  • Interaction with known heterochromatin assembly factors (e.g., pip1/rbx1, pob3, psc3 )

  • Biochemical assays for potential enzymatic activities (e.g., histone modification)

  • Genetic epistasis analysis with established heterochromatin pathway components

• Genomic stability assessment:

  • Analysis of genomic DNA fragmentation (similar to hst4Δ phenotype )

  • Measurement of mutation rates and types

  • DNA damage response pathway activation

This multifaceted approach would establish whether SPAC23H4.13c functions similarly to other proteins involved in heterochromatin assembly in S. pombe .

How can researchers resolve contradictory data about SPAC23H4.13c function from different experimental approaches?

When faced with contradictory data regarding SPAC23H4.13c function:

• Systematic assessment of experimental conditions:

  • Create a detailed comparison table of methodological differences

  • Evaluate strain background variations (h+/h- mating types, auxotrophic markers)

  • Compare growth conditions (media composition, temperature, growth phase)

  • Assess construct design differences (tag position, linker sequences)

• Orthogonal validation approaches:

  • Use multiple independent techniques to test the same hypothesis

  • Employ both gain-of-function and loss-of-function approaches

  • Validate key findings in different strain backgrounds

  • Use complementary in vivo and in vitro approaches

• Resolution strategies for specific contradiction types:

  • Localization discrepancies: Compare fixation methods, tag interference, expression levels

  • Functional conflicts: Consider genetic background suppressors, redundant pathways

  • Interaction disagreements: Compare stringency of binding conditions, transient vs. stable interactions

• Experimental design to directly address contradictions:

  • Create allelic series with point mutations to separate functions

  • Use conditional degron systems for temporal control

  • Employ domain deletion constructs to isolate specific functions

  • Implement rescue experiments with heterologous proteins or domains

• Data integration approach:

  • Develop unifying hypotheses that accommodate seemingly contradictory observations

  • Consider context-dependent functions based on cell cycle, stress conditions

  • Evaluate partial redundancy with other proteins

  • Build comprehensive models with clearly defined confidence levels

This systematic approach to resolving contradictions parallels strategies used in characterizing complex functions of other chromatin-associated proteins in S. pombe .

Evolutionary and Comparative Analysis

For predicting potential enzymatic activities of SPAC23H4.13c:

• Sequence-based enzymatic function prediction:

  • Identification of catalytic motifs through multiple sequence alignments

  • Comparison with known enzyme families and superfamilies

  • Active site prediction based on conserved residue patterns

  • Analysis of structural features associated with specific enzyme classes

• Structural prediction and analysis:

  • Ab initio or homology-based 3D structure prediction

  • Structural alignment with characterized enzymes

  • Active site pocket identification and characterization

  • Ligand docking simulations to predict potential substrates

• Integrative approaches:

  • Gene neighborhood analysis to identify functionally related genes

  • Co-expression network analysis to find genes with similar expression patterns

  • Protein-protein interaction network analysis to identify associations with known enzymes

  • Metabolic pathway gap analysis to identify missing enzymatic functions

• Machine learning techniques:

  • Enzyme function prediction using deep learning models

  • Feature extraction from sequence and predicted structure

  • Transfer learning from well-characterized enzyme families

  • Confidence scoring of predictions based on multiple models

• Experimental validation strategies:

  • Design of activity assays based on predictions

  • Mutagenesis of predicted catalytic residues

  • Substrate screening approaches

  • Metabolomic profiling of knockout strains

This comprehensive bioinformatic approach would generate testable hypotheses about potential enzymatic functions, particularly if SPAC23H4.13c shares any structural similarities with characterized enzyme families involved in chromatin modification or other cellular processes .

What are the most common technical challenges when working with SPAC23H4.13c and how can they be addressed?

Common technical challenges when working with uncharacterized S. pombe proteins like SPAC23H4.13c include:

• Expression and solubility issues:

  • Challenge: Low expression or inclusion body formation

  • Solution: Optimize codon usage for expression system, use solubility tags (SUMO, MBP), test different expression temperatures, employ cell-free expression systems

• Antibody generation problems:

  • Challenge: Poor immunogenicity or cross-reactivity

  • Solution: Use multiple peptide epitopes from different regions, validate with knockout controls, consider nanobody development, implement epitope tagging strategies

• Phenotype subtlety in knockout strains:

  • Challenge: No obvious phenotype in standard conditions

  • Solution: Perform growth under various stress conditions, create double mutants with functionally related genes, use high-sensitivity assays, examine competitive fitness over multiple generations

• Protein-protein interaction detection difficulties:

  • Challenge: Transient or weak interactions

  • Solution: Implement crosslinking strategies, use proximity labeling approaches (BioID, APEX), optimize buffer conditions, employ more sensitive detection methods

• Functional redundancy masking phenotypes:

  • Challenge: Compensatory mechanisms hiding functional defects

  • Solution: Generate multiple gene knockouts, use conditional degron systems for acute depletion, employ overexpression strategies, analyze genetic interaction profiles

• Reproducibility issues in chromatin studies:

  • Challenge: High variability in chromatin assays

  • Solution: Standardize growth conditions, synchronize cells, use internal controls, increase biological replicates, implement spike-in normalization strategies

These approaches parallel successful strategies used to overcome similar challenges in studying other uncharacterized S. pombe proteins, particularly those involved in chromatin regulation like the heterochromatin assembly factors .

How should researchers design experiments to distinguish between direct and indirect effects of SPAC23H4.13c manipulation?

To distinguish between direct and indirect effects of SPAC23H4.13c manipulation:

• Temporal resolution approaches:

  • Auxin-inducible degron (AID) system for rapid protein depletion

  • Temperature-sensitive alleles for conditional inactivation

  • Chemical genetics using engineered sensitivity to small molecules

  • Time-course analyses to establish order of events

• Spatial resolution strategies:

  • Protein tethering to specific genomic loci using CRISPR-dCas9

  • Forced localization to different cellular compartments

  • Domain-specific mutations to separate distinct functions

  • Chimeric proteins to test sufficiency of specific domains

• Biochemical directness testing:

  • In vitro reconstitution with purified components

  • Direct binding assays with potential interaction partners or substrates

  • Enzymatic activity assays with recombinant proteins

  • Crosslinking mass spectrometry to identify direct contacts

• Genetic approaches:

  • Separation-of-function mutations

  • Epistasis analysis with upstream and downstream factors

  • Suppressor screens to identify pathway components

  • Targeted rescue experiments with specific pathway components

• Multi-omics integration:

  • Combined analysis of immediate transcriptome, proteome, and chromatin changes

  • Network analysis to distinguish primary from secondary effects

  • Mathematical modeling of direct regulatory connections

  • Perturbation time-series to establish causality

This comprehensive approach would establish causal relationships and distinguish direct molecular functions from secondary cellular responses, similar to strategies used in functional characterization of other chromatin-associated proteins in S. pombe .

What are the most promising research directions for further characterizing SPAC23H4.13c?

The most promising research directions for characterizing SPAC23H4.13c include:

• Integration with known heterochromatin pathways:

  • Detailed investigation of genetic and physical interactions with established heterochromatin assembly factors like pip1/rbx1, pob3, and other proteins identified in search results

  • Exploration of potential roles in silencing mechanisms similar to those affected by hst4+ mutation

  • Assessment of relationships with histone modification pathways

• Condition-specific function analysis:

  • Examination of SPAC23H4.13c roles under various stress conditions

  • Investigation of cell cycle-dependent functions and regulation

  • Analysis of meiosis-specific roles given the importance of chromatin regulation in sexual development in S. pombe

• Multi-omics characterization:

  • Integrated analysis of transcriptome, proteome, and chromatin structure changes in knockout strains

  • Identification of direct binding sites through ChIP-seq or CUT&RUN approaches

  • Metabolomic profiling if enzymatic activity is predicted

• Structural biology approaches:

  • Determination of three-dimensional structure through X-ray crystallography or cryo-EM

  • Structure-function relationship studies through targeted mutagenesis

  • Protein dynamics analysis through hydrogen-deuterium exchange mass spectrometry

• Translational relevance exploration:

  • Comparative analysis with human orthologs if identified

  • Investigation of roles in genome stability relevant to disease models

  • Potential as a target for antifungal development if essential functions are discovered

These directions would build upon the foundational knowledge of S. pombe chromatin biology established through studies of related proteins and pathways .

How can researchers contribute to community resources for studying SPAC23H4.13c and related uncharacterized proteins?

Researchers can contribute to community resources for studying SPAC23H4.13c through:

• Data deposition and standardization:

  • Submit validated strains to repositories like the Yeast Genetic Resource Center

  • Deposit plasmids in AddGene with detailed protocols

  • Share raw data in appropriate databases (GEO for genomics, PRIDE for proteomics)

  • Implement standardized nomenclature and experimental conditions

• Method development and optimization:

  • Establish optimized protocols for SPAC23H4.13c purification and analysis

  • Develop specific antibodies or reporter constructs

  • Create specialized assay systems for functional characterization

  • Design CRISPR guide RNA libraries targeting uncharacterized genes

• Database and resource contribution:

  • Update PomBase entries with experimental findings

  • Contribute to GO term annotations based on experimental evidence

  • Participate in community curation efforts

  • Provide feedback on computational prediction accuracy

• Collaborative initiatives:

  • Establish consortium approaches for systematic characterization

  • Develop shared phenotyping platforms with standardized conditions

  • Create integrated data visualization tools

  • Implement open science approaches for real-time data sharing

• Educational resource development:

  • Create detailed protocols and troubleshooting guides

  • Develop training materials for new researchers

  • Establish mentor networks for technique transfer

  • Organize focused workshops on uncharacterized protein characterization

These contributions would accelerate research progress not only on SPAC23H4.13c but also on the broader challenge of functionally annotating the uncharacterized proteome of S. pombe, building upon successful community approaches used for other model organisms and protein families .