Recombinant Schizosaccharomyces pombe Uncharacterized RING finger membrane protein C15C4.06c (SPBC15C4.06c, SPBC21H7.01c)

Basic Research Questions

• What is SPBC15C4.06c and what structural characteristics does it possess?

  SPBC15C4.06c (also known as SPBC21H7.01C) is an uncharacterized RING finger membrane protein in Schizosaccharomyces pombe with a full length of 556 amino acids . As a RING finger protein, it contains a cysteine-rich domain that characteristically binds two zinc ions, typically with a 'cross-brace' topology . The RING finger domain consists of an α-helix and three short-stranded β-sheets arranged close to the Zn²⁺ ions, which help stabilize the protein structure . This structural arrangement is crucial for facilitating protein-protein interactions and potentially mediating ubiquitination processes.

  Protein Characteristics:

  Feature	Description
  Protein Name	Uncharacterized RING finger membrane protein C15C4.06C
  Alternative IDs	SPBC15C4.06c, SPBC21H7.01C
  Source Organism	Schizosaccharomyces pombe
  Length	556 amino acids
  Domain	RING finger (cysteine-rich)
  Cellular Location	Membrane-associated
  Key Structural Feature	Zinc-binding motif

• Why is Schizosaccharomyces pombe a valuable model organism for studying proteins like SPBC15C4.06c?

  S. pombe serves as an excellent model organism for several reasons:

  • It possesses only three chromosomes, making genetic manipulations and analyses more straightforward

  • Cells maintain their shape by growing exclusively through cell tips and divide by medial fission to produce two daughter cells of equal size, making it valuable for cell cycle studies

  • Its genome contains approximately 4,970 protein-coding genes, providing a manageable yet comprehensive eukaryotic system

  • It exhibits efficient homologous recombination, facilitating precise genome editing

  • Many fundamental cellular processes discovered in S. pombe are conserved in higher eukaryotes, including humans

  The relevance of S. pombe was recognized when Paul Nurse, a fission yeast researcher, received the 2001 Nobel Prize in Physiology or Medicine for work on cell cycle regulation .

• What expression systems are recommended for producing recombinant SPBC15C4.06c?

  Several expression systems can be utilized to produce recombinant SPBC15C4.06c, each with distinct advantages:

  Expression System	Advantages	Recommended Tags	Notes
  E. coli	Rapid growth, high yield	His, MBP, GST	Successfully used for full-length protein
  Yeast (S. cerevisiae)	Post-translational modifications	His, FLAG	Better for membrane proteins
  Insect cells (Sf9, Sf21)	Complex folding capability	His, FLAG, GFP	Suitable for structural studies
  Mammalian cells	Native-like processing	His, FLAG, GFP	Best for functional studies

  For membrane proteins like SPBC15C4.06c, expression in eukaryotic systems often provides better results due to proper membrane insertion machinery and post-translational modifications .

• What genetic manipulation techniques are effective in S. pombe for studying SPBC15C4.06c?

  S. pombe offers several effective genetic manipulation techniques:

  • Homologous recombination: The primary method for gene targeting in S. pombe, exploiting the innate homology-targeted repair mechanism

  • Stable integration vectors (SIVs): These produce non-repetitive, stable genomic loci and integrate predominantly as single copies

  • PCR-based gene targeting: Allows precise deletion, tagging, or modification of genes using homologous flanking sequences

  • Random spore analysis: Enables examination of large numbers of spores when studying recombination frequencies or constructing strains

  • Tetrad analysis: Crucial for studying recombination when not all spore classes are viable

  For determining the function of SPBC15C4.06c, gene deletion followed by phenotypic analysis would be the initial approach, potentially supplemented with complementation studies using tagged versions of the protein.

Advanced Research Questions

• How should I design experiments to characterize the function of an uncharacterized protein like SPBC15C4.06c?

  A comprehensive experimental design for characterizing SPBC15C4.06c should include:

  1. Genetic approaches:

     • Generate deletion mutants through homologous recombination

     • Create conditional mutants if deletion is lethal

     • Perform phenotypic analyses under various stress conditions

  2. Localization studies:

     • Generate fluorescently tagged versions of the protein

     • Perform co-localization with known cellular compartment markers

     • Examine localization changes during the cell cycle or stress conditions

  3. Interaction studies:

     • Perform immunoprecipitation coupled with mass spectrometry

     • Conduct yeast two-hybrid screens

     • Validate interactions through co-immunoprecipitation

  4. Functional assays:

     • Test for E3 ubiquitin ligase activity (given the RING domain)

     • Examine effects on protein stability of potential targets

     • Assess DNA damage responses and replication stress sensitivity

  5. Transcriptomic and proteomic analyses:

     • Perform RNA-seq or PRO-seq to identify transcriptional changes in mutants

     • Use ChIP-seq if the protein potentially associates with chromatin

     • Conduct proteome-wide analyses to identify globally affected pathways

  When designing these experiments, follow proper experimental design principles: define variables clearly, create testable hypotheses, include appropriate controls, and plan for statistical analyses .

• What methods can effectively identify protein-protein interactions for membrane-bound RING finger proteins like SPBC15C4.06c?

  Identifying interaction partners for membrane-bound RING finger proteins presents unique challenges. Recommended methodologies include:

  1. Proximity-based labeling methods:

     • BioID: Fusion of SPBC15C4.06c with a biotin ligase to biotinylate neighboring proteins

     • APEX2: Peroxidase-based labeling of proximal proteins

  2. Modified immunoprecipitation approaches:

     • Crosslinking prior to membrane solubilization

     • Sequential detergent extraction to maintain interactions

     • Use of specialized detergents that maintain membrane protein interactions

  3. Split reporter systems:

     • Split-ubiquitin yeast two-hybrid specifically designed for membrane proteins

     • Bimolecular fluorescence complementation (BiFC) in live cells

  4. Mass spectrometry-based approaches:

     • SILAC combined with immunoprecipitation for quantitative interaction analysis

     • Label-free quantitative proteomics comparing wild-type and deletion strains

  For membrane proteins specifically, careful optimization of detergent conditions during extraction is crucial to maintain native protein interactions while effectively solubilizing the membrane fraction.

• How can I analyze contradictory experimental data regarding SPBC15C4.06c?

  Contradictions in experimental data often arise from differences in experimental conditions, genetic backgrounds, or technical approaches. To reconcile contradictory findings:

  1. Systematic context analysis:

     • Document all experimental variables (strain background, growth conditions, experimental methods)

     • Determine if contradictions are complete (mutually exclusive) or partial (differing degrees)

     • Categorize contradictions as arising from technical, biological, or contextual differences

  2. Structured classification of contradiction patterns:

     • Apply the (α, β, θ) notation system: where α is the number of interdependent items, β is the number of contradictory dependencies, and θ is the minimal number of required Boolean rules

     • Analyze multidimensional interdependencies using Boolean minimization techniques

  3. Experimental validation:

     • Design experiments that directly test contradictory results under identical conditions

     • Perform replicate experiments with strict control of variables

     • Consider collaborations with labs reporting contradictory results

  4. Meta-analysis approaches:

     • Implement computational methods to detect underlying patterns across contradictory datasets

     • Utilize Bayesian analysis to account for varying degrees of certainty in different results

  When reporting, clearly state all experimental conditions and acknowledge limitations, as incomplete context (different species, temporal contexts, or environmental conditions) is a common source of apparent contradictions in biological research .

• What are the implications of the RING finger domain for SPBC15C4.06c's potential cellular functions?

  The presence of a RING finger domain in SPBC15C4.06c suggests several potential functions:

  1. Ubiquitination pathway involvement:

     • Likely functions as an E3 ubiquitin ligase, facilitating the transfer of ubiquitin from E2 enzymes to specific substrates

     • May regulate protein degradation through the ubiquitin-proteasome system

     • Could be involved in non-degradative ubiquitination (e.g., signaling, localization changes)

  2. Additional biochemical functions associated with RING proteins:

     • Potential role in transcriptional and translational regulation

     • Possible involvement in neddylation (the attachment of NEDD8 to proteins)

     • May participate in apoptosis and cell cycle regulation

  3. Potential cellular processes:

     • DNA damage response and repair pathways

     • Cell cycle checkpoint regulation

     • Stress response pathways

     • Membrane protein trafficking and quality control

  The specificity of RING E3 ligases is typically determined by their substrate recognition domains, suggesting that identifying interacting proteins would provide significant insights into SPBC15C4.06c's biological function.

• How can I design genetic screens to identify genes that interact with SPBC15C4.06c?

  Genetic interaction screens can provide valuable insights into the functional pathways involving SPBC15C4.06c. Recommended approaches include:

  1. Synthetic Genetic Array (SGA) methodology:

     • Generate a query strain with SPBC15C4.06c deletion or mutation

     • Cross with an array of deletion or mutation strains

     • Analyze growth phenotypes to identify synthetic lethal or synthetic sick interactions

     • Quantify interaction strength through colony size measurement and statistical analysis

  2. Suppressor screens:

     • If SPBC15C4.06c deletion shows a strong phenotype, screen for suppressors

     • Use random mutagenesis or overexpression libraries to identify genes that rescue the phenotype

     • Sequence suppressors to identify the genetic basis of rescue

  3. Specialized condition screens:

     • Test genetic interactions under stress conditions (DNA damage, temperature, nutrient limitation)

     • Examine drug sensitivity profiles (e.g., test with MPA as done for other genes)

     • Analyze genetic interactions in specific genetic backgrounds (e.g., heterochromatin formation mutants)

  4. High-throughput phenotypic analysis:

     • Combine genetic perturbations with automated microscopy to assess multiple phenotypes

     • Use fluorescent reporters to monitor specific cellular processes

  These approaches should be followed by validation of key interactions through detailed phenotypic analysis, biochemical studies, and potentially structural studies to understand the molecular basis of the interactions.

• What experimental techniques can determine if SPBC15C4.06c is involved in chromatin regulation or DNA replication?

  Given that some RING finger proteins participate in chromatin regulation and DNA replication processes, several approaches can determine SPBC15C4.06c's potential role:

  1. Chromatin immunoprecipitation (ChIP) approaches:

     • Perform ChIP-seq to map SPBC15C4.06c binding sites across the genome

     • Analyze co-localization with replication origins, transcription start sites, or specific chromatin marks

     • Conduct sequential ChIP to identify co-occupancy with known replication or chromatin factors

  2. Replication-specific assays:

     • Test sensitivity to replication stress (hydroxyurea, MMS)

     • Examine replication fork progression using DNA combing or BrdU incorporation

     • Investigate potential involvement in replication fork barriers using the RTS1 system

  3. Chromatin modification analysis:

     • Assess global levels of histone modifications in deletion mutants (e.g., H3K9 methylation)

     • Examine localization of chromatin marks at specific genomic regions

     • Test for genetic interactions with chromatin modifiers

  4. Transcriptional analysis:

     • Perform PRO-seq to analyze nascent transcription in mutants

     • Map RNA polymerase II occupancy using ChIP-seq

     • Identify differentially expressed genes through RNA-seq

  Importantly, research on other proteins has shown that RING finger proteins can affect subtelomeric H3K9 methylation , suggesting potential roles in heterochromatin maintenance that should be investigated for SPBC15C4.06c.