Recombinant Schizosaccharomyces pombe Putative uncharacterized protein C36.13 (SPBC36.13)

What is the primary structure and basic properties of SPBC36.13?

SPBC36.13 is a small protein consisting of 90 amino acids, with the following sequence:
MKRKTKISKMINRITFYFPLPTKKKTEIFFLSFAKQLFEKALLLFIPLSSFDSFFFVYFPASIKEITHYVAWRNAIQKRIRVLHHNKVIV

The protein has a UniProt ID of G2TRS4 and is officially classified as a putative uncharacterized protein, indicating its function has not been experimentally validated. Its small size (90 aa) suggests it may function as a regulatory protein rather than an enzyme with catalytic domains . While commercially available as a recombinant protein with an N-terminal His-tag expressed in E. coli, researchers should be aware that its native conformation and post-translational modifications in S. pombe may differ .

How does SPBC36.13 compare to other uncharacterized proteins in the S. pombe proteome?

SPBC36.13 is part of the approximately 70% of the S. pombe theoretical proteome that remains incompletely characterized. Proteomic profiling studies have detected only about 30% of the predicted fission yeast proteins, suggesting that many proteins like SPBC36.13 may be expressed at low levels or under specific conditions not routinely captured in standard proteomic analyses .

In multidimensional prefractionation and LC ESI MS/MS studies, researchers have identified 1,465 proteins representing approximately 29.5% of the predicted fission yeast proteome. This identification rate was found to be consistent across various molecular weights, isoelectric points, and gene ontology attributes . When designing studies on SPBC36.13, researchers should consider that uncharacterized proteins were underrepresented in these analyses compared to proteins with known functions.

What computational predictions exist regarding SPBC36.13 function?

While direct experimental data is limited, computational approaches can provide initial hypotheses about SPBC36.13 function. The protein's amino acid sequence contains multiple lysine residues (K) and arginine residues (R), particularly concentrated in the N-terminal region, suggesting possible DNA/RNA binding capabilities or nuclear localization signals. Researchers should employ multiple prediction algorithms for:

• Secondary structure prediction

• Functional domain identification

• Subcellular localization prediction

• Ortholog identification in related species

These computational analyses should be considered starting points for experimental validation rather than definitive functional assignments.

What are the key considerations when designing experiments to characterize SPBC36.13?

When designing experiments to characterize SPBC36.13, researchers should follow established principles of experimental design:

• Define independent and dependent variables: When studying SPBC36.13, carefully select measurable outcomes (dependent variables) that might reveal function, such as growth rates, stress responses, or molecular interactions .

• Control variables: Identify and standardize all factors that could influence results, including growth conditions, strain background, and expression levels .

• Statistical power: Ensure sufficient biological and technical replicates to detect potentially subtle phenotypes associated with SPBC36.13 manipulation .

• Validation methods: Plan for multiple orthogonal approaches to validate findings, especially important for previously uncharacterized proteins where functional hypotheses may be speculative .

A particularly effective approach may involve both gain-of-function (overexpression) and loss-of-function (deletion or depletion) experiments to assess SPBC36.13's role under various conditions.

How should recombinant SPBC36.13 be handled to maintain its stability and activity?

Based on the commercial protein specifications, researchers should follow these guidelines to maintain stability and potential activity of recombinant SPBC36.13:

• Initial handling: Briefly centrifuge the vial before opening to bring contents to the bottom .

• Reconstitution: Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Storage preparation: Add glycerol to a final concentration of 5-50% (50% is recommended) and aliquot for long-term storage .

• Temperature considerations: Store working aliquots at 4°C for up to one week; for longer storage, maintain at -20°C/-80°C. Avoid repeated freeze-thaw cycles as they may compromise protein integrity .

• Buffer compatibility: The protein is supplied in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which should be considered when designing experimental buffers to maintain stability .

What methods are most appropriate for studying potential protein-protein interactions of SPBC36.13?

For investigating potential protein-protein interactions of SPBC36.13, consider these methodological approaches:

• Affinity purification coupled with mass spectrometry (AP-MS): This approach has been successfully used to characterize protein complexes in S. pombe, as demonstrated in studies of histone deacetylase complexes . Using tagged versions of SPBC36.13 (similar to the "nts1-FLAG::3 kanMX6" approach described for other proteins), researchers can identify interaction partners .

• Yeast two-hybrid (Y2H) screening: While not mentioned in the provided search results, Y2H remains a valuable method for detecting binary protein interactions and could reveal direct binding partners of SPBC36.13.

• Co-immunoprecipitation verification: Following identification of candidate interactors, co-IP experiments using antibodies against the tagged SPBC36.13 can verify interactions under various physiological conditions.

• Proximity-based labeling: Methods such as BioID or APEX could provide insights into the proximal protein environment of SPBC36.13 in living cells.

When designing these experiments, researchers should consider both stringent and more permissive conditions, as SPBC36.13 may form stable complexes or participate in transient interactions that are more difficult to capture.

How can contradictory data about SPBC36.13 function be reconciled in research publications?

When faced with contradictory findings regarding SPBC36.13 function, researchers should apply a systematic approach to reconciliation:

• Context-dependent interpretation: Similar to how the Gospel writers include apparent contradictions for deeper literary points, scientific "contradictions" about SPBC36.13 may reflect context-dependent functions . For example, a protein may show different behaviors under different experimental conditions.

• Terminology clarification: Ensure that seeming contradictions don't stem from different uses of the same term, as illustrated by the example of "world" having different meanings in different biblical contexts .

• Resolution through deeper analysis: Contradictions at a superficial level often encourage deeper thinking about the underlying biology . For SPBC36.13, contradictory findings might reveal condition-specific roles or multiple functions.

• Experimental design evaluation: Assess whether differences in experimental design, such as tagging strategies, expression levels, or strain backgrounds could explain apparently contradictory results .

A practical approach is to create a comprehensive comparison table of contradictory findings, noting all experimental variables that differ between studies, which often reveals the source of apparent contradictions.

What is the relationship between SPBC36.13 mRNA levels and protein abundance, and how might this impact functional studies?

Understanding the relationship between SPBC36.13 mRNA and protein levels is crucial for interpreting functional studies:

• General correlation patterns: Proteomic and transcriptomic profiling of S. pombe has revealed that mRNA-protein correlations vary significantly depending on protein function. The correlation is stronger for proteins involved in signaling and metabolic processes but more discordant for components of protein complexes .

• Clustered expression patterns: Components of protein complexes often cluster in groups with similar mRNA-protein ratios . If SPBC36.13 functions as part of a complex, its expression might correlate with its interaction partners rather than showing a direct 1:1 relationship with its own mRNA.

• Cross-species considerations: When comparing SPBC36.13 with potential homologs in other yeasts like S. cerevisiae, researchers should note that despite the organisms' relatedness, there is "considerable divergence in gene expression patterns" at both mRNA and protein levels .

• Methodological implications: For functional studies, this means that mRNA quantification alone may not reliably predict SPBC36.13 protein levels. Using direct protein detection methods like western blotting or mass spectrometry is recommended alongside transcriptional analysis.

How might SPBC36.13 relate to histone deacetylase complexes in S. pombe?

While direct evidence linking SPBC36.13 to histone deacetylase (HDAC) complexes is not presented in the search results, this represents an important research question given the significance of HDAC complexes in S. pombe:

• HDAC complex diversity: S. pombe contains multiple HDAC complexes, including the recently characterized Clr6 HDAC complex I′′, which includes novel proteins Nts1, Mug165, and Png3 . Researchers should investigate whether SPBC36.13 might be associated with this or other HDAC complexes.

• Experimental approach: Following methodologies used to characterize the Clr6 HDAC complex, researchers could construct strains with tagged SPBC36.13 (similar to "nts1-FLAG::3 kanMX6" strains) and perform co-immunoprecipitation experiments to detect potential associations with known HDAC components .

• Functional testing: If associations are found, functional studies similar to those using strains like "sds3-FLAG::3 kanMX6 nts1::natMX6 mug165::hphMX6 png3::natMX6" could reveal whether SPBC36.13 affects HDAC complex assembly or function .

• Reporter assays: Systems like "tf2-2(LTR-lacZ):ura4+" could be employed to assess whether SPBC36.13 influences transcriptional silencing, a common function of HDAC complexes .

These approaches would help determine whether SPBC36.13 plays a role in chromatin regulation through HDAC complexes, an important aspect of gene expression control in S. pombe.

What proteomic approaches are most effective for detecting and quantifying low-abundance proteins like SPBC36.13?

Detecting low-abundance proteins like SPBC36.13 requires specialized proteomic approaches:

• Multidimensional prefractionation: The extensive prefractionation scheme described in search result significantly improves detection of low-abundance proteins. This approach includes:

  • Preparative isoelectric focusing on immobilized pH gradients

  • Liquid-phase format separation

  • One-dimensional gel electrophoresis

  • Strong ion-exchange chromatography

  • Analysis of fractions by LC ESI MS/MS

• On-line 2D LC ESI MS/MS (MudPIT): This method, applied to total fission yeast lysate after in-solution digestion into tryptic peptides, provides complementary coverage to the prefractionation approaches .

• Statistical modeling for quantification: Normalizing spectral counts to the number of predicted tryptic peptides enables label-free quantification, which has successfully quantified 1,465 proteins in S. pombe .

• Coverage considerations: Researchers should note that current methods have detected only ~30% of the theoretical fission yeast proteome, with uncharacterized proteins and transmembrane proteins being underrepresented . Specialized approaches may be necessary for comprehensive detection.

How should researchers integrate transcriptomic and proteomic data when studying SPBC36.13?

Effective integration of transcriptomic and proteomic data requires consideration of several factors:

• Correlation patterns by functional category: The mRNA-protein correlation varies by functional category, with stronger correlations for proteins involved in signaling and metabolic processes, but more discordant patterns for components of protein complexes . When analyzing SPBC36.13, consider which functional category it might belong to.

• Self-organizing map (SOM) clustering: This approach has revealed coordinate but not always concordant expression of components of functional pathways and protein complexes in fission and budding yeast . Applying SOM clustering to datasets including SPBC36.13 may reveal its functional associations.

• Cross-species comparative analysis: Comparisons between S. pombe and S. cerevisiae have shown "considerable divergence in gene expression patterns" despite their relatedness . If studying SPBC36.13 homologs across species, expect potential differences in expression patterns.

• Integration methodology:

  • Begin with independent analysis of each data type

  • Apply correlation analysis to identify concordant and discordant patterns

  • Use pathway enrichment analysis to contextualize findings

  • Apply machine learning approaches to identify patterns not apparent through simple correlation

This integrated approach provides a more complete understanding of SPBC36.13's expression and functional context than either data type alone.

What are the most promising future directions for research on SPBC36.13?

Based on the limited characterization of SPBC36.13 to date, several promising research directions emerge:

• Functional genomics screens: Systematic assessment of phenotypes associated with SPBC36.13 deletion or overexpression under various stress conditions may reveal condition-specific functions.

• Protein complex characterization: Given that many S. pombe proteins function as parts of complexes, identifying SPBC36.13's interaction partners could provide significant functional insights.

• Structural biology approaches: Determining the three-dimensional structure of SPBC36.13 could reveal functional domains and potential interaction surfaces not apparent from sequence analysis alone.

• Comparative genomics: Identifying and characterizing potential orthologs in related species could provide evolutionary context and functional clues.

• Integration with emerging technologies: Approaches like Crispr-Cas9 genome editing, proximity labeling proteomics, and single-cell analysis could reveal new aspects of SPBC36.13 function.

These directions should be pursued with careful experimental design principles, ensuring appropriate controls and statistical power to detect potentially subtle phenotypes associated with this uncharacterized protein.

How can researchers best contribute to the community knowledge about uncharacterized proteins like SPBC36.13?

To advance collective understanding of uncharacterized proteins like SPBC36.13, researchers should:

• Adopt standardized reporting formats: Ensure all experimental conditions, strain constructions, and methodological details are comprehensively reported to enable reproduction.

• Deposit raw data in public repositories: Submit mass spectrometry data, RNA-seq results, and other primary data to appropriate databases.

• Contribute to community resources: Update database annotations as new information becomes available, and participate in community curation efforts.

• Address contradictory findings directly: When new results contradict published work, directly address and attempt to reconcile differences rather than ignoring them .

• Share negative results: Information about conditions under which SPBC36.13 shows no phenotype or interactions can be as valuable as positive findings for guiding future research.