Recombinant Schizosaccharomyces pombe Uncharacterized membrane protein C757.15 (SPCC757.15)

What is the basic structure and properties of SPCC757.15?

SPCC757.15 is an uncharacterized membrane protein from the fission yeast Schizosaccharomyces pombe with a full length of 69 amino acids . It is classified as a membrane protein, suggesting it contains hydrophobic regions that anchor it within cellular membranes. The recombinant form of this protein can be produced with a histidine tag to facilitate purification and experimental manipulation . While considered "uncharacterized," preliminary functional annotation suggests it may be associated with mitochondrial cytochrome c oxidase assembly processes with a confidence score of 0.64 .

What expression systems are recommended for producing recombinant SPCC757.15?

E. coli has been successfully used as an expression host for recombinant SPCC757.15 protein production . When designing an expression system for this membrane protein, researchers should consider:

• Vector selection: Choose vectors with strong, inducible promoters like T7 or tac promoters for controlled expression

• Fusion tags: His-tagging has been validated for this protein and facilitates purification via metal affinity chromatography

• Expression conditions: Optimize temperature, induction time, and inducer concentration to maximize yield while maintaining proper folding

• Membrane protein considerations: Lower expression temperatures (15-25°C) often improve proper folding of membrane proteins

• Solubilization strategies: Test various detergents to effectively extract the protein from membranes while maintaining native conformation

How can researchers verify the identity and purity of recombinant SPCC757.15?

A multi-faceted approach to verification is recommended:

• SDS-PAGE analysis: Confirms molecular weight (expected ~7-8 kDa plus tag size)

• Western blotting: Using anti-His antibodies for tagged protein detection

• Mass spectrometry: For accurate mass determination and sequence verification

• N-terminal sequencing: To confirm the intact N-terminus

• Circular dichroism: To assess secondary structure elements characteristic of membrane proteins

• Size-exclusion chromatography: To evaluate homogeneity and oligomeric state

What experimental approaches are recommended for determining the function of SPCC757.15?

Given the current bioinformatic prediction linking SPCC757.15 to mitochondrial cytochrome c oxidase assembly , several complementary approaches are recommended:

• Gene knockout/knockdown studies: Generate SPCC757.15 deletion strains in S. pombe and characterize phenotypic effects, particularly focusing on:

  • Mitochondrial morphology and function

  • Cytochrome c oxidase activity assays

  • Growth under respiratory versus fermentative conditions

  • Stress response, particularly oxidative stress

• Localization studies: Employ fluorescent protein tagging (GFP/mCherry) to determine subcellular localization, with special attention to mitochondrial colocalization

• Protein-protein interaction studies:

  • Co-immunoprecipitation with known components of cytochrome c oxidase assembly machinery

  • Yeast two-hybrid screening

  • Proximity labeling approaches (BioID/APEX)

  • Cross-linking mass spectrometry

• Functional complementation: Test whether SPCC757.15 can rescue phenotypes in strains lacking other known cytochrome c oxidase assembly factors

How should researchers design experiments to validate the predicted role of SPCC757.15 in mitochondrial cytochrome c oxidase assembly?

A systematic experimental design approach should include:

• Baseline characterization:

  • Measure cytochrome c oxidase activity in wild-type S. pombe

  • Assess mitochondrial respiration rates

  • Quantify ATP production

• Comparative analysis:

  • Generate SPCC757.15 deletion strain

  • Compare cytochrome c oxidase activity, respiration, and ATP production between wild-type and deletion strains

  • Perform growth assays under different carbon sources (glucose vs. glycerol/ethanol)

• Rescue experiments:

  • Reintroduce SPCC757.15 to knockout strain under native or inducible promoter

  • Test whether phenotypes are rescued

  • Include control with mutated versions of SPCC757.15 to identify critical residues/domains

• Biochemical interaction studies:

  • Purify SPCC757.15 and test direct binding to cytochrome c oxidase subunits or assembly factors

  • Perform in vitro reconstitution assays

• Control for confounding variables:

  • Use multiple deletion clones to rule out off-target effects

  • Include controls for general mitochondrial function

  • Test specificity by examining other respiratory chain complexes

What computational approaches are most effective for predicting SPCC757.15 function?

Based on recent functional annotation research, multi-faceted bioinformatic approaches yield the most reliable predictions:

• Sequence similarity analysis: PANNZER2 has been successfully used for functional annotation of uncharacterized S. pombe proteins, including SPCC757.15 . This tool:

  • Conducts sequence similarity searches against UniProtKB database

  • Filters sequence neighborhoods based on multiple criteria

  • Provides GO annotations and free text descriptions

  • Assigns confidence scores to predictions (SPCC757.15 received a 0.64 score for mitochondrial cytochrome c oxidase assembly)

• Structural prediction:

  • AlphaFold2 or RoseTTAFold for 3D structure prediction

  • Membrane topology prediction using TMHMM or Phobius

  • Secondary structure prediction using JPred or PSIPRED

• Evolutionary analysis:

  • Identification of conserved domains or motifs

  • Phylogenetic profiling to identify proteins with similar evolutionary patterns

  • Comparative genomics across yeast species

• Network-based approaches:

  • Functional association networks (STRING database)

  • Co-expression analysis across multiple conditions

  • Genomic context analysis (gene neighborhood)

How can researchers integrate experimental and computational data to improve functional predictions for SPCC757.15?

An iterative approach combining wet-lab experiments with computational refinement is recommended:

• Initial bioinformatic predictions:

  • Begin with sequence-based analysis (as performed with PANNZER2)

  • Generate hypotheses about potential functions (e.g., mitochondrial cytochrome c oxidase assembly)

• Experimental validation:

  • Design targeted experiments to test predictions

  • Generate new data (localization, interaction partners, phenotypic effects)

• Refinement of predictions:

  • Update models with new experimental data

  • Employ machine learning approaches that incorporate diverse data types

  • Use Bayesian frameworks to update confidence in functional predictions

• Iterative validation:

  • Design subsequent experiments based on refined predictions

  • Focus on areas of uncertainty or contradiction

• Data integration platforms:

  • Use platforms that can integrate multiple data types (e.g., InterMine, KBase)

  • Develop custom pipelines for S. pombe uncharacterized proteins

How does SPCC757.15 expression change in response to metformin treatment, and what are the implications for aging research?

Recent research has investigated the expression patterns of uncharacterized proteins, including SPCC757.15, in response to metformin treatment in S. pombe . The findings suggest:

• Expression changes:

  • SPCC757.15 was identified among differentially expressed genes in metformin-treated S. pombe cells

  • This expression pattern was observed in both standard (3% glucose) and overnutrition (5% glucose) conditions

• Functional context:

  • The predicted association with mitochondrial function aligns with known metformin mechanisms involving mitochondrial activity

  • Changes in expression may reflect adaptive responses to metabolic alterations induced by metformin

• Research implications:

  • SPCC757.15 represents a potential new target for aging research

  • Its involvement in mitochondrial processes connects to the mitochondrial theory of aging

  • Understanding its precise role could illuminate mechanisms of metformin's life-extending effects

• Experimental approaches:

  • Time-course analysis of expression changes following metformin treatment

  • Comparison of wild-type versus SPCC757.15 deletion strains in longevity assays

  • Investigation of interactions with known aging-related pathways (TOR, AMPK, sirtuins)

What experimental design considerations are important when studying SPCC757.15 in the context of aging research?

Aging research requires careful experimental design to account for numerous variables and complex phenotypes:

• Strain selection and validation:

  • Use well-characterized S. pombe strains with consistent genetic backgrounds

  • Generate multiple independent SPCC757.15 deletion or overexpression strains

  • Validate genetic modifications through sequencing and expression analysis

• Lifespan assay design:

  • Measure both chronological and replicative lifespan

  • Use appropriate media (SD medium has been validated for chronological lifespan experiments in S. pombe)

  • Include proper controls (wild-type, known long-lived and short-lived mutants)

• Variables to control:

  • Growth conditions (temperature, media composition, culture density)

  • Cell cycle stage and synchronization

  • Metabolic state (respiratory vs. fermentative)

  • Stress factors (oxidative, nutrient availability)

• Key measurements:

  • Lifespan (chronological and replicative)

  • Mitochondrial function (membrane potential, respiration rate)

  • ROS production and oxidative damage

  • ATP levels and metabolic profiles

  • Protein aggregation and proteostasis markers

• Statistical considerations:

  • Appropriate sample sizes for detecting expected effect sizes

  • Multiple biological and technical replicates

  • Control for batch effects

  • Longitudinal measurements to capture temporal dynamics

How does SPCC757.15 compare to other uncharacterized membrane proteins in S. pombe?

A comparative analysis approach can provide valuable context:

• Structural comparison:

  • Size comparison: SPCC757.15 is relatively small at 69 amino acids

  • Transmembrane domain prediction and comparison

  • Presence of conserved motifs or domains

• Expression pattern analysis:

  • Under standard growth conditions

  • In response to stressors (oxidative stress, nutrient deprivation)

  • During different cell cycle phases

  • In response to metformin treatment

• Co-expression network analysis:

  • Identify other uncharacterized proteins with similar expression patterns

  • Construct functional association networks

  • Identify clusters of co-regulated uncharacterized proteins

• Evolutionary conservation:

  • Presence or absence of orthologs in related species

  • Conservation patterns across fungal lineages

  • Comparison to similar proteins in model organisms with better-characterized proteomes

What methodological approaches are effective for conducting functional screens of multiple uncharacterized proteins including SPCC757.15?

High-throughput functional screening approaches include:

• Parallel phenotypic analysis:

  • Generate a library of deletion strains for multiple uncharacterized proteins

  • Perform growth assays under various conditions (temperature, carbon source, stress)

  • Conduct high-content imaging for morphological phenotypes

  • Use flow cytometry for cell cycle and viability analysis

• Pooled functional genomics:

  • CRISPR-based screens in S. pombe

  • Barcoded deletion libraries with next-generation sequencing readout

  • Synthetic genetic array (SGA) analysis to identify genetic interactions

• Protein localization screening:

  • Systematic GFP tagging

  • High-throughput fluorescence microscopy

  • Automated image analysis and classification

• Biochemical approaches:

  • Affinity purification-mass spectrometry for multiple targets

  • Protein microarrays for interaction screening

  • Activity-based protein profiling

• Data integration and analysis:

  • Machine learning approaches to classify proteins based on multiple data types

  • Network-based function prediction

  • Clustering algorithms to identify functional groups

What are the challenges in working with small membrane proteins like SPCC757.15, and how can they be addressed?

Working with small membrane proteins (SPCC757.15 is 69 amino acids ) presents several technical challenges:

• Expression and purification challenges:

  • Low yield due to toxicity or improper folding

  • Aggregation during overexpression

  • Difficulty maintaining native conformation during solubilization

  Solutions:

  • Use speciality expression hosts (C41/C43 E. coli strains designed for membrane proteins)

  • Fusion with solubility-enhancing tags (MBP, SUMO) in addition to His-tag

  • Screen multiple detergents for optimal solubilization

  • Consider cell-free expression systems

• Structural characterization difficulties:

  • Small size makes electron microscopy challenging

  • Crystallization of membrane proteins is notoriously difficult

  • Insufficient protein material for NMR

  Solutions:

  • Computational structure prediction (AlphaFold2)

  • Circular dichroism for secondary structure elements

  • Fusion with crystallization chaperones

  • Solid-state NMR approaches

• Functional assay limitations:

  • Redundancy may mask phenotypes in deletion strains

  • Small size limits domains for protein-protein interactions

  • Difficulty distinguishing direct vs. indirect effects

  Solutions:

  • Combine deletion with related genes to overcome redundancy

  • Use overexpression in addition to deletion studies

  • Employ sensitive reporters for subtle phenotypes

  • Develop in vitro reconstitution systems

What quality control measures should be implemented when working with recombinant SPCC757.15?

A comprehensive quality control strategy should include:

• Pre-experimental validation:

  • Sequence verification of expression constructs

  • Optimization of expression conditions with small-scale tests

  • Detergent screening for optimal solubilization

• Purification quality assessment:

  • Multi-step chromatography (IMAC, size exclusion, ion exchange)

  • SDS-PAGE with appropriate gel systems for small proteins

  • Mass spectrometry confirmation of intact mass

  • Western blotting with anti-His antibodies

• Functional integrity assessment:

  • Secondary structure analysis via circular dichroism

  • Thermal stability assays

  • Reconstitution into liposomes or nanodiscs

  • Binding assays with predicted interaction partners

• Storage stability:

  • Optimization of buffer conditions

  • Testing of various stabilizing additives

  • Freeze-thaw stability analysis

  • Long-term activity retention measurement

• Reproducibility measures:

  • Multiple biological replicates

  • Independent protein preparations

  • Lot-to-lot consistency checks

  • Detailed record-keeping of all procedures

What are the most promising research directions for further characterizing SPCC757.15?

Based on current knowledge, several research avenues appear particularly promising:

• Detailed mitochondrial function studies:

  • Investigation of specific aspects of cytochrome c oxidase assembly potentially involving SPCC757.15

  • Analysis of mitochondrial morphology and dynamics in deletion/overexpression strains

  • Examination of SPCC757.15 roles in mitochondrial stress responses

• Aging and longevity connections:

  • Expansion of metformin-related studies to include other lifespan-extending interventions

  • Analysis of SPCC757.15 expression changes during chronological and replicative aging

  • Investigation of potential connections to conserved longevity pathways

• Structural biology approaches:

  • Determination of 3D structure using cryo-EM, NMR, or crystallography

  • Structure-function studies to identify critical domains or residues

  • Membrane topology and integration studies

• Systems biology integration:

  • Positioning SPCC757.15 within broader functional networks

  • Multi-omics approaches to understand regulatory contexts

  • Computational modeling of potential functional roles

• Translational relevance:

  • Investigation of human orthologs or functional analogs

  • Connections to mitochondrial disorders in humans

  • Potential relevance to metabolic diseases

How can researchers effectively design experiments to resolve contradictory data about SPCC757.15 function?

When facing contradictory results, a systematic approach is necessary:

• Critical evaluation of existing data:

  • Assess methodological differences between studies

  • Examine genetic background variations

  • Consider environmental and experimental conditions

  • Evaluate statistical robustness of conflicting findings

• Triangulation approach:

  • Deploy multiple orthogonal techniques to assess the same function

  • Combine genetic, biochemical, and computational approaches

  • Use both gain-of-function and loss-of-function strategies

• Conditional analyses:

  • Test function under various environmental conditions

  • Investigate cell cycle or development stage-specific roles

  • Examine genetic background dependencies

• Collaboration strategies:

  • Establish collaborative validation studies between labs with conflicting results

  • Standardize protocols and reagents

  • Perform blinded analyses when appropriate

• Mechanistic dissection:

  • Move beyond correlative studies to direct mechanistic tests

  • Develop reconstituted systems to test direct biochemical functions

  • Generate structure-guided mutations to test specific hypotheses