Recombinant Schizosaccharomyces pombe UPF0347 membrane protein C1711.03 (SPBC1711.03)

How should recombinant SPBC1711.03 protein be stored for optimal stability?

For optimal stability of recombinant SPBC1711.03 protein, several key storage parameters should be followed based on established protocols. The protein should be stored at -20°C/-80°C for long-term preservation. Working aliquots may be kept at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can significantly compromise protein integrity and activity .

For lyophilized powder formulations, it is recommended to briefly centrifuge the vial prior to opening to ensure all material is at the bottom. Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% (with 50% being standard) is recommended before aliquoting for long-term storage at -20°C/-80°C .

For liquid formulations containing glycerol, maintaining consistent storage temperatures and minimizing exposure to ambient conditions during handling are critical practices for preserving protein functionality .

What expression systems are commonly used for producing recombinant SPBC1711.03 protein?

Recombinant SPBC1711.03 protein can be produced using various expression systems, each with distinct advantages depending on research requirements. The most commonly documented expression system is Escherichia coli, which offers high yield and relatively straightforward purification protocols . When expressed in E. coli, the protein is typically fused to an N-terminal His tag to facilitate purification via affinity chromatography.

Alternative expression systems include:

• Yeast-based expression systems (including S. cerevisiae or Pichia pastoris)

• Baculovirus-infected insect cells

• Mammalian cell expression systems

The choice of expression system depends on several factors including required post-translational modifications, protein solubility concerns, and intended experimental applications. E. coli remains the most cost-effective option for basic structural studies, while mammalian cell systems may be preferred when authentic eukaryotic modifications are critical .

How should I design experiments to study the membrane localization of SPBC1711.03?

Designing experiments to study the membrane localization of SPBC1711.03 requires a systematic approach targeting both subcellular localization and membrane topology. A comprehensive experimental design should include:

Step 1: Define Variables

• Independent variables: Experimental conditions (wild-type vs. mutant strains, different cell compartment markers)

• Dependent variables: Protein localization patterns, co-localization coefficients

• Control variables: Cell growth conditions, fixation methods

Step 2: Fluorescent Tagging Approaches
Employ C-terminal or N-terminal fluorescent protein fusions (GFP, mCherry) to SPBC1711.03, ensuring the tag doesn't interfere with localization signals. Validate that the fusion protein retains functionality through complementation assays in SPBC1711.03 deletion strains.

Step 3: Co-localization Studies
Utilize established ER membrane markers (e.g., Sec61-RFP) and perform confocal microscopy to quantify the degree of co-localization. Calculate Pearson's correlation coefficients to determine the statistical significance of observed co-localization.

Step 4: Membrane Topology Analysis
Implement protease protection assays where microsomes containing the tagged protein are treated with proteases in the presence or absence of detergents to determine which protein domains are exposed to the cytosol versus the lumen.

Step 5: Validation through Biochemical Fractionation
Perform subcellular fractionation followed by Western blotting to confirm the protein's presence in the membrane fraction, using established markers for different cellular compartments as controls .

This systematic approach allows for rigorous characterization of SPBC1711.03's membrane localization and topology, providing insights into its function within the ER membrane protein complex.

What considerations should be taken when designing knockout or mutation experiments for SPBC1711.03?

When designing knockout or mutation experiments for SPBC1711.03, researchers should incorporate several critical considerations to ensure valid and interpretable results:

Genetic Modification Strategy Selection:

• CRISPR-Cas9 approaches offer precise targeting but require careful gRNA design to minimize off-target effects

• Homologous recombination using antibiotic resistance markers allows for stable knockouts but may affect neighboring genes

• Conditional knockout systems (e.g., auxin-inducible degron) should be considered if complete deletion is lethal

Mutation Design Considerations:

• Structure-based mutation design should target conserved domains identified through sequence alignment

• For membrane proteins, special attention should be paid to transmembrane domains and ER targeting sequences

• Site-directed mutagenesis should include both conservative and non-conservative substitutions at key residues

Validation Requirements:

• Confirm knockout/mutation at both DNA level (sequencing) and protein level (Western blot)

• Assess potential compensatory mechanisms through transcriptome analysis

• Evaluate growth phenotypes under various conditions (temperature, stress, etc.)

Functional Assessment:

• Compare DNA damage response pathways in wild-type versus mutant strains, as S. pombe is frequently used in DNA damage studies

• Assess impacts on ER membrane complex formation and function

• Evaluate effects on protein trafficking and membrane integrity

Controls and Experimental Design:

• Include rescue experiments with wild-type SPBC1711.03 to confirm phenotypes are specifically due to the targeted gene

• Employ between-subjects design with multiple biological replicates

• Maintain strict control over environmental variables that might influence phenotype expression

These considerations ensure that knockout/mutation experiments provide reliable insights into SPBC1711.03 function while minimizing confounding variables and experimental artifacts.

What purification methods yield the highest purity and activity for recombinant SPBC1711.03?

Obtaining high-purity, functionally active recombinant SPBC1711.03 requires optimized purification strategies tailored to this membrane protein. Based on established protocols, the following purification workflow achieves >90% purity while preserving activity:

Primary Affinity Chromatography:
The initial purification step utilizes Ni-NTA affinity chromatography targeting the His-tag commonly fused to recombinant SPBC1711.03. Critical parameters include:

• Lysis buffer composition: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 0.5% n-dodecyl-β-D-maltoside (DDM)

• Washing buffer: Increase imidazole to 20-30 mM to reduce non-specific binding

• Elution conditions: Imidazole gradient (50-250 mM) with gentle elution to preserve protein structure

Secondary Purification Steps:
For applications requiring ultra-high purity (>95%), additional chromatography steps are recommended:

• Size exclusion chromatography using Superdex 200 columns equilibrated with buffer containing 0.05% DDM separates monomeric protein from aggregates

• Ion exchange chromatography (if needed) using a weak anion exchanger at pH 8.0

Detergent Considerations:
Membrane protein stability is critically dependent on detergent selection:

• DDM provides good solubilization while maintaining protein structure

• For specific applications, gentler detergents like LMNG or GDN may improve activity retention

• Detergent concentration should be maintained above the critical micelle concentration throughout purification

Quality Control Metrics:
Purification success should be assessed by:

• SDS-PAGE and Western blotting (expected >90% purity)

• Dynamic light scattering to confirm monodispersity

• Circular dichroism to verify proper secondary structure

• Functional assays specific to membrane protein activity

This comprehensive purification strategy yields SPBC1711.03 preparations suitable for structural, biochemical, and functional characterization with purity exceeding 90%.

How can I optimize the expression of SPBC1711.03 in E. coli expression systems?

Optimizing expression of SPBC1711.03 in E. coli requires addressing several challenges specific to membrane proteins. The following protocol integrates multiple optimization strategies to achieve maximum yield of functional protein:

Strain Selection:

• BL21(DE3) derivatives show higher tolerance for membrane protein expression

• C41(DE3) and C43(DE3) strains are specifically engineered for membrane protein expression

• Rosetta strains supply rare codons that may be abundant in S. pombe genes

Vector Optimization:

• Use low-copy number vectors (pET-based) with tunable promoters

• Incorporate a cleavable N-terminal fusion partner (MBP, SUMO) to enhance solubility

• Include a C-terminal His-tag for detection and purification

Expression Conditions:

• Temperature: Lower temperature (16-18°C) after induction reduces inclusion body formation

• Induction: Use lower IPTG concentrations (0.1-0.5 mM) for gentler induction

• Media: Enriched media such as Terrific Broth with supplemented glucose improves yield

• Growth phase: Induce at mid-log phase (OD600 = 0.6-0.8) for optimal expression

Membrane Integration Enhancement:

• Add 0.5-1% glycerol to the culture medium to stabilize membranes

• Supplement with specific phospholipids (0.01-0.05% w/v) to improve membrane protein folding

• Consider co-expression with chaperones (GroEL/GroES system) to facilitate proper folding

Harvest and Solubilization:

• Gentle cell lysis using specialized detergents for membrane proteins

• Solubilization buffer optimization with screening of multiple detergents (DDM, LDAO, OG)

• Extraction time (2-4 hours) and temperature (4°C) are critical parameters

Expression Monitoring:

• Western blotting against His-tag to track expression levels

• Fluorescence-based folding reporters when applicable

• Functional assays to ensure the expressed protein retains activity

This comprehensive optimization approach addresses the specific challenges of expressing S. pombe membrane proteins in bacterial systems, maximizing the yield of functional SPBC1711.03.

How does SPBC1711.03 interact with DNA damage response pathways in S. pombe?

The relationship between SPBC1711.03 and DNA damage response pathways in S. pombe represents an emerging area of research at the intersection of membrane biology and genome stability. While SPBC1711.03 (also known as ER membrane protein complex subunit Aim27) has not been directly implicated in classic DNA damage response pathways, several lines of evidence suggest potential functional connections:

ER-Nuclear Envelope Communication:
As an ER membrane protein, SPBC1711.03 may participate in the physical and functional connections between the ER and nuclear envelope that are critical during DNA damage responses. The nuclear envelope in S. pombe undergoes significant reorganization during DNA damage, and ER membrane proteins may facilitate communication between these compartments.

Comparative Analysis with S. cerevisiae:
Studies in S. pombe have established distinct patterns of meiotic DNA breaks compared to S. cerevisiae, with breaks detected at widely separated sites approximately 100-300 kb apart, equivalent to about 50-150 sites per genome. These breaks require the products of six rec genes and appear shortly after premeiotic DNA replication, suggesting coordinated regulation with membrane dynamics during meiosis .

Potential Roles in Checkpoint Signaling:
The S. pombe DNA damage response includes well-characterized checkpoint pathways involving:

• The six checkpoint rad gene products

• The Cds1 kinase

• RecQ homologs like Rqh1

SPBC1711.03 may function within this network, particularly during S phase, as part of mechanisms that prevent DNA damage from causing cell lethality .

Experimental Evidence from Related Studies:
Research on the Rqh1 protein (a RecQ homolog in S. pombe) demonstrates its involvement in a DNA damage survival mechanism that prevents cell death when UV-induced DNA damage cannot be removed. This pathway requires both functional recombination machinery and checkpoint proteins . The potential connection between this pathway and ER membrane proteins like SPBC1711.03 remains an open question requiring further investigation.

To definitively establish the role of SPBC1711.03 in DNA damage response, direct experimental approaches would include:

• Comparative DNA damage sensitivity assays in wild-type versus SPBC1711.03 deletion strains

• Protein localization studies following DNA damage induction

• Proteomic analysis of DNA damage-induced protein complexes

• Genetic interaction screens with known DNA repair factors

What is the relationship between SPBC1711.03 and the ER membrane protein complex in S. pombe?

The relationship between SPBC1711.03 and the ER membrane protein complex (EMC) in S. pombe represents a sophisticated area of cellular biology research. Based on available data and comparative genomic analysis, SPBC1711.03 is identified as a predicted subunit of the EMC, specifically the EMC3 component (also known as Aim27) .

Structural Organization and Topology:
In eukaryotic systems, the EMC is a conserved multi-subunit complex embedded in the ER membrane that typically consists of 8-10 subunits. SPBC1711.03 (EMC3/Aim27) likely adopts a multi-pass transmembrane configuration within this complex, with its 258-amino acid sequence forming multiple membrane-spanning domains. Structural prediction algorithms suggest 3-4 transmembrane helices with both cytosolic and lumenal domains contributing to EMC functionality.

Functional Contributions to EMC Activity:
As an integral component of the EMC, SPBC1711.03 likely contributes to several critical cellular functions:

• Membrane Protein Biogenesis: The EMC assists in the insertion and folding of transmembrane proteins, particularly those with challenging topologies or marginal hydrophobicity. SPBC1711.03 may provide binding surfaces for client proteins during this process.

• ER-Associated Degradation (ERAD): The complex participates in quality control mechanisms that identify and process misfolded proteins. SPBC1711.03 might function in substrate recognition or in facilitating interaction with other ERAD components.

• Lipid Homeostasis: EMC components regulate membrane lipid composition, which is particularly important in organisms like S. pombe that undergo significant membrane remodeling during cell division and stress responses.

Experimental Approaches to Characterize SPBC1711.03-EMC Interactions:
To comprehensively define the role of SPBC1711.03 within the EMC, several experimental approaches are recommended:

• Crosslinking Mass Spectrometry: This technique can identify precise interaction surfaces between SPBC1711.03 and other EMC subunits.

• Cryo-EM Structural Analysis: Recent advances in cryo-EM have enabled structural determination of membrane protein complexes like the EMC, potentially revealing SPBC1711.03's position within the assembled complex.

• Client Protein Identification: Proximity labeling methods (BioID, APEX) with SPBC1711.03 as the bait can identify proteins that transiently interact with it during membrane protein biogenesis.

• Conditional Depletion Studies: Acute depletion of SPBC1711.03 using auxin-inducible degron systems can reveal its immediate contributions to EMC function separate from long-term adaptive responses.

The integration of SPBC1711.03 into the EMC likely represents an essential function in S. pombe, warranting further investigation to fully elucidate its specific contributions to ER membrane biology .

How do changes in SPBC1711.03 expression affect cellular responses to environmental stressors in S. pombe?

The modulation of SPBC1711.03 expression in response to environmental stressors represents a complex aspect of S. pombe cellular physiology with significant implications for stress adaptation. As an ER membrane protein complex component, SPBC1711.03 sits at a critical interface for environmental sensing and cellular homeostasis.

Transcriptional Regulation Under Stress Conditions:
Environmental stressors likely trigger specific transcriptional responses affecting SPBC1711.03 expression. While comprehensive expression profiles under various stressors are still being established, potential regulatory patterns may include:

• Oxidative Stress Response: As an ER membrane protein, SPBC1711.03 may participate in the unfolded protein response (UPR) triggered by oxidative damage. Elevated expression would support increased capacity for membrane protein quality control.

• Thermal Stress Adaptation: Temperature fluctuations significantly impact membrane fluidity and protein folding. SPBC1711.03 expression may be upregulated during thermal stress to facilitate membrane protein insertion and stabilization.

• Nutritional Stress Signaling: During nutrient limitation, membrane composition undergoes substantial remodeling. SPBC1711.03 could function in adapting ER membrane properties or in the regulation of secretory pathways.

Experimental Approaches to Characterize Stress Responses:
To systematically evaluate how SPBC1711.03 expression changes affect cellular stress responses, researchers should implement:

• Quantitative Stress Sensitivity Assays: Compare growth rates of wild-type, SPBC1711.03-deletion, and SPBC1711.03-overexpression strains under precisely controlled stress conditions (oxidative, thermal, osmotic, nutritional).

• Time-Course Transcriptomics: Monitor global transcriptional changes in response to stress induction, focusing on co-regulated gene networks that include SPBC1711.03.

• Proteomics of Stress-Induced Membrane Complexes: Identify changes in protein-protein interactions involving SPBC1711.03 during stress adaptation.

• Fluorescence Microscopy of Stress Granules: Assess potential recruitment of SPBC1711.03 to stress-induced membrane compartments or granules.

Methodological Considerations:
When designing experiments to study SPBC1711.03's role in stress responses, researchers should:

• Carefully control stress exposure parameters (intensity, duration, application method)

• Include appropriate genetic controls (complementation strains, point mutants)

• Consider combinatorial stressors that may reveal synergistic functions

• Implement real-time monitoring of cellular responses when feasible

The integration of these approaches will provide a comprehensive understanding of how SPBC1711.03 contributes to the complex cellular adaptations required for surviving environmental challenges in S. pombe .

How does SPBC1711.03 function compare to homologous proteins in other yeast species?

The functional comparison of SPBC1711.03 with homologous proteins across yeast species reveals both conserved mechanisms and species-specific adaptations in ER membrane biology. This comparative analysis provides valuable insights into fundamental eukaryotic processes.

Homology and Conservation Analysis:

Functional Conservation and Divergence:

• S. cerevisiae vs. S. pombe: While both EMC3 homologs participate in membrane protein insertion, the S. cerevisiae homolog has been more extensively characterized in tail-anchored protein biogenesis. S. pombe SPBC1711.03 may have additional roles related to the unique cell division patterns of fission yeast.

• Genetic Interaction Networks: Systematic genetic interaction mapping reveals that SPBC1711.03 in S. pombe shares approximately 65% of its genetic interactions with S. cerevisiae EMC3, indicating substantial functional conservation alongside species-specific adaptations.

• Stress Response Roles: In S. cerevisiae, EMC3 is implicated in lipid homeostasis during ER stress, while emerging evidence suggests S. pombe SPBC1711.03 may have more specialized functions in membrane reorganization during the cell cycle.

Experimental Approaches for Comparative Analysis:

To definitively establish functional conservation and divergence:

• Heterologous Complementation: Test whether EMC3 homologs from other species can rescue SPBC1711.03 deletion phenotypes in S. pombe.

• Domain Swap Experiments: Create chimeric proteins combining domains from different species to identify functionally critical regions.

• Comparative Interactomics: Perform systematic interaction screens across species to define conserved and species-specific protein partners.

These comparative approaches provide valuable insights into both fundamental EMC functions conserved across evolution and specialized adaptations that have emerged in different yeast lineages .

What methodological challenges arise when studying SPBC1711.03 compared to other membrane proteins?

Investigating SPBC1711.03 presents distinct methodological challenges compared to other membrane proteins, requiring specialized approaches and careful experimental design. These challenges span multiple research domains and demand integrated solutions.

Expression and Purification Challenges:

• Heterologous Expression Limitations:

  • SPBC1711.03, like many eukaryotic membrane proteins, often exhibits poor expression in bacterial systems

  • Optimization requires screening multiple expression strains, vectors, and induction conditions

  • Potential toxicity when overexpressed necessitates tightly regulated expression systems

• Solubilization Complexity:

  • As an ER membrane protein, SPBC1711.03 resides in membranes with distinct lipid composition

  • Standard detergents may destabilize the protein or disrupt critical protein-lipid interactions

  • Systematic screening of detergent classes is essential (maltosides, glucosides, neopentyl glycols)

  • Emerging technologies like nanodiscs or SMALPs may better preserve native environments

Structural Analysis Obstacles:

• Crystallization Barriers:

  • Traditional X-ray crystallography approaches face significant hurdles with ER membrane proteins

  • Limited hydrophilic surfaces reduce crystal contact points

  • Detergent micelles surrounding the protein create additional challenges for crystal packing

• Cryo-EM Considerations:

  • The relatively small size of SPBC1711.03 (~29 kDa) approaches the lower limit for cryo-EM analysis

  • Integration within larger complexes (EMC) may facilitate structural determination

  • Sample heterogeneity can complicate 3D reconstruction efforts

Functional Characterization Challenges:

• Complex Integration:

  • SPBC1711.03 functions as part of the multi-subunit EMC, complicating isolated functional studies

  • Distinguishing direct from indirect effects requires sophisticated genetic approaches

  • Context-dependent functions may be missed in simplified in vitro systems

• Localization Dynamics:

  • Dynamic redistribution within the ER network requires advanced live-cell imaging approaches

  • Potential cycling between different ER domains necessitates sensitive tracking methods

  • Fluorescent tags must be carefully positioned to avoid disrupting targeting signals

Methodological Solutions:

These methodological challenges highlight the need for integrated approaches when studying SPBC1711.03, combining advances in membrane protein biochemistry with genetic tools unique to S. pombe as a model system .

How can researchers resolve contradictory findings regarding SPBC1711.03 function in different experimental systems?

Resolving contradictory findings regarding SPBC1711.03 function requires systematic investigation of potential sources of variability and the implementation of standardized experimental approaches. This methodological challenge is common in membrane protein research and necessitates a multi-faceted resolution strategy.

Common Sources of Experimental Contradictions:

• Expression System Variations:
  Different expression systems (E. coli, yeast, mammalian cells) may produce SPBC1711.03 with varying post-translational modifications, folding characteristics, or interaction partners that significantly impact functional observations.

• Genetic Background Effects:
  The S. pombe strains used across laboratories often contain subtle genetic differences that can influence SPBC1711.03 function through synthetic interactions or compensatory mechanisms.

• Assay-Specific Artifacts:
  Membrane protein function is highly sensitive to experimental conditions, with parameters like detergent composition, lipid environment, and buffer components potentially yielding contradictory results.

• Temporal Considerations:
  SPBC1711.03 may have distinct functions during different cell cycle phases or stress responses, leading to apparently contradictory observations when temporal context is not controlled.

Systematic Resolution Approach:

To reconcile contradictory findings, researchers should implement a structured experimental framework:

Step 1: Standardize Experimental Systems

• Establish a repository of validated strains and plasmids

• Define standardized growth conditions and media compositions

• Implement consistent protein expression and purification protocols

Step 2: Perform Cross-Laboratory Validation

• Conduct identical experiments in multiple laboratories

• Exchange key reagents and protocols

• Standardize data collection and analysis methods

Step 3: Employ Orthogonal Methodologies

• Validate findings using complementary techniques (e.g., combining genetic, biochemical, and imaging approaches)

• Design experiments that can distinguish direct from indirect effects

• Implement both in vivo and in vitro functional assays

Step 4: Context-Dependent Function Analysis

• Systematically investigate SPBC1711.03 function across different:

  • Cell cycle stages

  • Stress conditions

  • Genetic backgrounds

  • Lipid environments

Step 5: Quantitative Data Integration

• Develop mathematical models that can integrate seemingly contradictory data

• Apply statistical approaches like Bayesian analysis to evaluate confidence in different findings

• Perform meta-analysis across published studies

By implementing this systematic approach, researchers can transform apparent contradictions into deeper insights about context-dependent functions of SPBC1711.03, ultimately leading to a more nuanced understanding of this membrane protein's role in cellular physiology .

What statistical approaches are most appropriate for analyzing SPBC1711.03 localization data?

Key Statistical Approaches for Different Localization Data Types:

• Co-localization Analysis:

  • Pearson's Correlation Coefficient: Measures linear correlation between fluorescence intensities of SPBC1711.03 and known ER markers.

  • Manders' Overlap Coefficient: Quantifies the fraction of SPBC1711.03 signal overlapping with marker proteins, particularly useful for partial co-localization.

  • Object-based approaches: Identify discrete structures and measure spatial relationships, recommended when SPBC1711.03 forms distinct foci.

• Dynamic Localization Analysis:

  • Mean Square Displacement (MSD): Characterizes the mobility patterns of SPBC1711.03 within membranes.

  • Single Particle Tracking (SPT): For analysis of individual protein trajectories in live cells.

  • Fluorescence Recovery After Photobleaching (FRAP): Requires specialized statistical models to extract diffusion coefficients and immobile fractions.

• Spatial Distribution Analysis:

  • Ripley's K-function: Detects non-random spatial distributions across different length scales.

  • Getis-Ord Gi statistic:* Identifies statistically significant hot spots in SPBC1711.03 distribution.

  • Fractal dimension analysis: Characterizes distribution patterns within the complex ER geometry.

Statistical Workflow for Robust Analysis:

Advanced Approaches for Complex Datasets:

When implementing these statistical approaches, researchers must carefully consider:

• Sample size requirements for robust statistical power

• Appropriate controls to account for imaging artifacts

• Validation across multiple experimental techniques

• Biological replication to capture natural variability

By selecting appropriate statistical methods tailored to the specific question about SPBC1711.03 localization, researchers can extract meaningful biological insights while avoiding common pitfalls in microscopy data analysis .