Recombinant Schizosaccharomyces pombe Uncharacterized RING finger protein C57A7.09 (SPAC57A7.09)

What is known about the genomic location and basic characteristics of SPAC57A7.09?

SPAC57A7.09 is an uncharacterized protein encoded by the fission yeast S. pombe. The protein is located on chromosome I within the SPAC57A7 locus, which contains several other uncharacterized proteins with potential regulatory functions. The protein contains a RING finger domain, suggesting involvement in ubiquitin-mediated protein degradation pathways. Given its genomic proximity to other uncharacterized proteins (e.g., SPAC57A7.07c, SPAC57A7.15c), it may participate in coordinated cellular processes, although experimental validation is required to confirm these relationships.

What structural and functional predictions can be made about SPAC57A7.09 based on its RING finger domain?

The presence of a RING finger domain in SPAC57A7.09 strongly suggests ubiquitin ligase activity. RING finger-containing proteins typically bind ubiquitin-conjugating enzymes (E2s) and facilitate E2-dependent ubiquitination . The RING domain likely contains metal-coordinating residues that bind zinc ions, which are critical for maintaining the protein's structural integrity and functional activity. Mutations of these cation-coordinating residues or chelation of zinc would likely abolish any ubiquitination activity . Comparative analysis with other RING finger proteins such as AO7, BRCA1, and Siah-1 suggests SPAC57A7.09 may function as an E3 ubiquitin ligase, potentially targeting specific proteins for proteasomal degradation .

How does S. pombe compare to other model organisms for studying uncharacterized proteins?

S. pombe offers several advantages for studying uncharacterized proteins like SPAC57A7.09. With a generation time of approximately 108 minutes and well-defined cell cycle phases (G1: 38 min, S: 17 min, G2: 78 min, M: 17 min), S. pombe provides a relatively rapid experimental timeline . The fission yeast has a GC content of 36% and a comparatively small genome, simplifying genetic manipulations . Additionally, S. pombe maintains eukaryotic post-translational modification capabilities while having less complex cellular organization than mammalian cells. The mean protein content per dividing cell is approximately 12 pg, and cell volumes range from 129μm³ at 25°C to 149μm³ at 35°C, providing sufficient material for biochemical analysis from reasonable culture volumes .

What are the recommended expression systems for recombinant production of SPAC57A7.09?

For optimal expression of recombinant SPAC57A7.09, two main systems should be considered:

• Homologous expression in S. pombe:

  • Advantages: Native post-translational modifications, proper protein folding

  • Methodology: Integration into the leu1 locus using vectors such as pREP1 or pCAD1

  • Regulation: Inducible expression using the nmt1 promoter under glucose regulation

• Heterologous expression in E. coli:

  • Advantages: Higher yield, simplified purification

  • Considerations: May lack proper post-translational modifications

  • Methodology: Expression with fusion tags (His6, GST) to facilitate purification

The choice depends on experimental goals: functional studies may benefit from the homologous system, while structural studies might prioritize higher yields from E. coli expression.

What purification strategy would yield the highest purity for SPAC57A7.09?

A multi-step purification strategy is recommended for obtaining high-purity SPAC57A7.09:

Critical parameters to monitor include buffer conditions (pH 7.0-8.0 recommended for RING finger proteins), salt concentration (150-300 mM NaCl to prevent aggregation), and addition of reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain the integrity of zinc-coordinating cysteine residues .

How can proper folding of the RING finger domain be verified?

Verifying the proper folding of the RING finger domain is critical since this structure is essential for potential ubiquitin ligase activity. Multiple complementary techniques are recommended:

• Circular Dichroism (CD) Spectroscopy:

  • Measures secondary structure elements characteristic of folded RING domains

  • Expected features: Peaks at 208 nm and 222 nm indicating α-helical content

• Zinc Content Analysis:

  • Inductively coupled plasma mass spectrometry (ICP-MS) to quantify zinc ions

  • Expected stoichiometry: Two zinc ions per RING finger domain

• Functional Binding Assays:

  • Pull-down assays with known E2 enzymes (UbcH5 family members)

  • Expected outcome: Specific binding to E2s indicating properly folded RING domain

• Thermal Shift Assays:

  • Measure protein stability in presence/absence of zinc

  • Expected result: Higher melting temperature in zinc-containing buffer

An integrated approach combining these methods provides the most reliable assessment of RING domain integrity.

What experimental design would best characterize the potential E3 ligase activity of SPAC57A7.09?

A comprehensive experimental approach to characterize potential E3 ligase activity would include:

• In vitro ubiquitination assays:

  • Components: Purified SPAC57A7.09, E1 (Uba1), E2 (UbcH5 family), ubiquitin, ATP

  • Controls: Reactions lacking individual components; mutated RING domain variant

  • Detection: Anti-ubiquitin western blotting to detect polyubiquitin chains

  • Expected result: RING-dependent formation of polyubiquitin chains

• E2 binding assays:

  • Yeast two-hybrid screening with UbcH5 family members as bait

  • Co-immunoprecipitation of tagged SPAC57A7.09 with endogenous E2s

  • Surface plasmon resonance to determine binding kinetics

• Substrate identification:

  • BioID proximity labeling in S. pombe cells expressing SPAC57A7.09-BirA fusion

  • Comparative proteomics before/after SPAC57A7.09 overexpression

  • Validation of putative substrates through in vitro ubiquitination assays

The integration of these techniques would establish whether SPAC57A7.09 functions as an E3 ligase and identify its potential substrates and biological roles.

How should site-directed mutagenesis be implemented to confirm the importance of the RING domain?

Strategic site-directed mutagenesis targeting conserved residues in the RING domain is essential for confirming its functional importance:

• Target residues for mutation:

  • Cysteine and histidine residues that coordinate zinc ions

  • Based on alignment with characterized RING domains (e.g., from AO7)

  • Recommended substitutions: Cys→Ala and His→Ala to disrupt zinc coordination

• Mutation protocol:

  • PCR-based site-directed mutagenesis using overlap extension

  • Verification by DNA sequencing before expression

• Functional comparison:

  • Express wild-type and mutant proteins under identical conditions

  • Compare E2 binding capacity via co-immunoprecipitation

  • Assess ubiquitination activity in vitro and in vivo

  • Expected outcome: Mutations should abolish E2 binding and ubiquitination

• Structural analysis:

  • CD spectroscopy to assess structural changes

  • Zinc content analysis to confirm loss of metal coordination

This systematic mutational approach provides strong evidence for the mechanism of SPAC57A7.09 function and confirms the essential role of the RING domain in its activity.

What advanced approaches can resolve contradictory functional data for SPAC57A7.09?

When faced with contradictory functional data, a multi-faceted approach using knowledge-driven techniques is recommended:

• Bayesian signal processing and machine learning:

  • Implement uncertainty-aware and physics-informed models

  • Develop robust optimization protocols that account for experimental variability

  • Design experiments that optimally reduce performance loss in predictive models

• Condition-dependent functional analysis:

  • Test protein activity under various cellular stresses (oxidative, thermal, nutrient)

  • Evaluate activity across different cell cycle phases

  • Examine function in different genetic backgrounds (e.g., E2 deletion strains)

• Cross-validation with orthogonal techniques:

  • Combine biochemical, genetic, and computational approaches

  • Implement CRISPR-based gene editing to create genomic mutations

  • Perform epistasis analysis with known ubiquitination pathway components

• Single-molecule techniques:

  • Use FRET to monitor conformational changes during E2 binding

  • Apply super-resolution microscopy to track protein localization during cell cycle

This integrated approach acknowledges the complexity of protein function and allows resolution of seemingly contradictory data through systematic investigation.

What genetic manipulation strategies are most effective for studying SPAC57A7.09 in S. pombe?

Several genetic approaches can be employed for studying SPAC57A7.09 in S. pombe:

• Gene deletion/replacement:

  • Homologous recombination targeting the SPAC57A7.09 locus

  • Replacement with selectable markers (e.g., kanMX6, hphMX6)

  • Assessment of phenotypic consequences across growth conditions

• Fluorescent tagging for localization studies:

  • C-terminal tagging with GFP or mCherry

  • Visualization throughout cell cycle and in response to stressors

  • Co-localization with known ubiquitination machinery components

• Promoter replacement for controlled expression:

  • Substitution of native promoter with nmt1 (thiamine-repressible)

  • Creation of expression gradients through promoter variants (nmt1, nmt41, nmt81)

  • Assessment of dosage-dependent phenotypes

• CRISPR-Cas9 genome editing:

  • Precise introduction of point mutations in the RING domain

  • Creation of conditional alleles (temperature-sensitive mutations)

  • Integration of degron tags for rapid protein depletion

These approaches leverage the genetic tractability of S. pombe and provide complementary insights into SPAC57A7.09 function.

How does the cell cycle of S. pombe influence experimental design for studying SPAC57A7.09?

The well-characterized cell cycle of S. pombe significantly impacts experimental design for studying SPAC57A7.09:

For optimal synchronization, consider:

• Centrifugal elutriation to select G2 cells based on size

• Nitrogen starvation to arrest in G1

• Hydroxyurea treatment to synchronize at G1/S boundary

• cdc25-22 temperature-sensitive mutants for G2 arrest

Sampling intervals should account for the 108-minute generation time, with more frequent sampling during rapid transitions between phases .

How can quantitative proteomics be applied to study the global impact of SPAC57A7.09 activity?

Quantitative proteomics offers powerful approaches to elucidate the global impact of SPAC57A7.09:

• SILAC (Stable Isotope Labeling with Amino acids in Cell culture):

  • Culture wild-type and SPAC57A7.09Δ strains with heavy/light lysine and arginine

  • Compare proteome changes using LC-MS/MS

  • Identify proteins with altered abundance, suggesting potential substrates

• Ubiquitin remnant profiling:

  • Enrich for ubiquitinated peptides using K-ε-GG antibodies

  • Compare ubiquitinome between wild-type and SPAC57A7.09Δ strains

  • Map ubiquitination sites on potential substrates

• Protein turnover analysis:

  • Pulse-chase experiments with SILAC or dynamic SILAC

  • Calculate protein half-lives in presence/absence of SPAC57A7.09

  • Identify proteins with SPAC57A7.09-dependent degradation rates

• Absolute quantification:

  • Determine copy numbers of relevant proteins using SRM/MRM

  • Expected stoichiometry: Based on known E3 ligases, SPAC57A7.09 abundance may be 500-2000 molecules per cell, compared to ~150,000 ribosomes in S. pombe

These approaches provide a systems-level understanding of SPAC57A7.09 function within the cellular proteostasis network.

What computational approaches can predict potential substrates of SPAC57A7.09?

Several computational strategies can be employed to predict potential substrates:

• Sequence-based motif analysis:

  • Identify degrons (recognition motifs) in the S. pombe proteome

  • Analyze compositional bias in known E3 ligase substrates

  • Apply machine learning algorithms trained on verified ubiquitination substrates

• Structural modeling and docking:

  • Generate homology models of SPAC57A7.09 RING domain based on characterized RING structures

  • Perform molecular docking with E2~Ub conjugates

  • Simulate substrate binding using flexible protein-protein docking

• Protein interaction network analysis:

  • Mine existing protein interaction databases for SPAC57A7.09 associations

  • Apply graph theory algorithms to identify hub proteins within the same functional modules

  • Implement Bayesian approaches for uncertainty-aware prediction of interactions

• Evolutionary conservation analysis:

  • Compare orthologous sequences across yeast species

  • Identify co-evolving protein pairs as potential E3-substrate relationships

  • Apply knowledge-driven learning algorithms to integrate multiple data types

These computational predictions should be validated experimentally, creating an iterative cycle of prediction and verification.

What are the common challenges in RING finger protein purification and how can they be addressed?

Several challenges are commonly encountered when purifying RING finger proteins like SPAC57A7.09:

Monitoring zinc content throughout purification is critical, as loss of zinc often correlates with loss of activity in RING finger proteins . A multi-parameter optimization approach using Bayesian experimental design can efficiently identify optimal conditions .

How can contradictory results between in vitro and in vivo ubiquitination assays be reconciled?

Contradictions between in vitro and in vivo results are common in ubiquitination studies and require systematic troubleshooting:

• Physiological relevance of in vitro conditions:

  • Adjust E2:E3 ratios to reflect cellular concentrations

  • Include potential cofactors or scaffold proteins

  • Test varying concentrations of substrates

  • Include cellular fractions to supply missing components

• Cellular context considerations:

  • Examine cell cycle-dependent activity (G1: 38 min, S: 17 min, G2: 78 min, M: 17 min)

  • Test activity under various stress conditions

  • Consider compartmentalization and localization effects

  • Evaluate post-translational modifications of SPAC57A7.09 itself

• Technical approaches to bridge the gap:

  • Semi-in vitro assays using cell extracts

  • Reconstitution experiments with purified cellular complexes

  • Single-cell analysis to capture population heterogeneity

  • Development of biosensors to monitor activity in real-time

• Computational integration:

  • Apply Bayesian frameworks to reconcile disparate datasets

  • Develop physics-informed machine learning models

  • Design experiments specifically to address contradictions

This systematic approach acknowledges the complexity of ubiquitination networks and works toward a unified understanding of SPAC57A7.09 function across experimental contexts.