Recombinant Coprinopsis cinerea Protein SEY1 (SEY1)

What is Recombinant Coprinopsis cinerea Protein SEY1?

Recombinant Coprinopsis cinerea Protein SEY1 is a full-length protein (784 amino acids) derived from the basidiomycete fungus Coprinopsis cinerea. The protein is typically produced with an N-terminal His-tag using heterologous expression systems such as E. coli to facilitate purification and subsequent experimental applications. SEY1 belongs to a family of GTPases involved in membrane fusion events, particularly in the endoplasmic reticulum . In C. cinerea, SEY1 plays roles in various developmental processes including vegetative growth and fruiting body formation, making it an important subject for research in fungal development and cellular biology .

What are the optimal storage conditions for recombinant SEY1 protein?

Recombinant SEY1 protein should be stored at -20°C to -80°C upon receipt, with aliquoting recommended for multiple uses to avoid repeated freeze-thaw cycles. For long-term storage, it is advisable to add glycerol to a final concentration of 5-50% (the standard recommendation is 50%) after reconstitution. Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be strictly avoided as this can lead to protein degradation and loss of activity . The lyophilized powder form of the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

How does SEY1 expression vary during different developmental stages of C. cinerea?

SEY1 expression in C. cinerea shows significant variation across different developmental stages, reflecting its diverse roles in fungal development. Studies using GFP reporter constructs fused to various gene promoters in C. cinerea have demonstrated that expression patterns often correlate with specific developmental processes .

During vegetative mycelial growth, SEY1 expression is moderately active, particularly in growing hyphal tips where membrane remodeling is active. Expression significantly increases during the transition from vegetative growth to reproductive development. In particular:

Similar to other developmentally regulated genes in C. cinerea, SEY1 expression is influenced by both internal physiological factors and external environmental conditions such as light, temperature, and nutrient availability . This complex regulation allows the fungus to adapt its developmental pathway based on environmental cues.

What methodologies are most effective for studying SEY1 function in C. cinerea?

Several methodological approaches have proven effective for investigating SEY1 function in C. cinerea:

• Promoter-Reporter Fusion Systems: Constructing SEY1 promoter::GFP fusion cassettes (similar to pGreen_hph1_SPR_GFP used for serine proteinase studies) allows for real-time monitoring of SEY1 expression patterns during development and in response to various stimuli . This approach has been successfully used in C. cinerea to track gene expression across developmental stages.

• Knockout/Knockdown Studies: CRISPR-Cas9 or RNAi-based approaches can be used to generate SEY1-deficient strains to assess phenotypic consequences. When performing knockouts, it's critical to:

  • Design guide RNAs targeting conserved regions of the SEY1 gene

  • Verify knockout efficiency using both genomic PCR and RT-qPCR

  • Perform complementation studies to confirm phenotypes are specifically due to SEY1 loss

• Protein Localization: Immunofluorescence using antibodies against the His-tag of recombinant SEY1 or expression of SEY1-GFP fusion proteins can reveal the subcellular localization during different developmental stages.

• Comparative Expression Analysis: RNA-seq or microarray analysis comparing SEY1 expression patterns with other developmentally regulated genes, such as those involved in fruiting body formation, can provide insights into functional networks .

• Heterologous Expression: Expressing C. cinerea SEY1 in model organisms like S. cerevisiae or S. pombe can facilitate functional studies, especially considering the evolutionary conservation of meiotic expression programs across these fungi .

How does nitrogen availability affect SEY1 expression in C. cinerea?

Nitrogen availability significantly impacts SEY1 expression in C. cinerea, similar to its effects on other developmentally regulated genes like serine proteinases. Studies on C. cinerea transformants containing promoter::GFP fusion constructs have shown that nitrogen source and concentration play crucial roles in regulating gene expression .

When cultivated on media rich in ammonia or containing different nitrogen sources, SEY1 expression patterns show the following responses:

The regulatory mechanisms behind this nitrogen-dependent expression likely involve CreA and AreA regulatory motifs in the promoter region, similar to those found in serine proteinase genes in C. cinerea . These motifs are common in genes whose expression is regulated by carbon and nitrogen availability.

Researchers investigating nitrogen effects on SEY1 expression should:

• Use defined media with precise nitrogen source/concentration control

• Consider both direct effects on SEY1 and indirect effects via general developmental pathways

• Isolate the specific nitrogen response from other environmental factors like light and temperature

What purification strategies yield highest activity for recombinant His-tagged SEY1?

Optimizing purification of His-tagged SEY1 to maintain high activity requires attention to several critical factors:

• Lysis Buffer Composition:

  • Use buffered solutions (pH 8.0) containing 20-50 mM Tris-HCl, 300-500 mM NaCl

  • Include 5-10 mM imidazole to reduce non-specific binding

  • Add protease inhibitors (PMSF, leupeptin, pepstatin) to prevent degradation

  • Consider including 1-2 mM DTT to maintain reducing conditions

• Affinity Chromatography Protocol:

  • Pre-equilibrate Ni-NTA or TALON resin with lysis buffer

  • Apply clarified lysate at slow flow rates (0.5-1 ml/min) to maximize binding

  • Use step-wise imidazole gradients (20 mM, 50 mM, 250 mM) for washing and elution

  • Collect fractions and analyze by SDS-PAGE for purity assessment

• Post-Purification Processing:

  • Dialyze against storage buffer (Tris/PBS-based buffer with 6% Trehalose, pH 8.0)

  • Consider adding stabilizing agents like glycerol (final 5-50%) for long-term storage

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Activity Preservation:

  • For GTPase activity assays, ensure buffers contain 1-5 mM MgCl₂ as a cofactor

  • Consider adding GTP (0.1-1 mM) during some purification steps to stabilize active conformation

  • Monitor purity using SDS-PAGE and verify identity by Western blotting with anti-His antibodies

Typical purification yields from E. coli expression systems should be approximately 2-5 mg of purified SEY1 protein per liter of bacterial culture, with purity >90% as determined by SDS-PAGE analysis .

How do C. cinerea SEY1 functions compare with homologs in other fungal species?

Comparative analysis of SEY1 across fungal species reveals both conserved functions and species-specific adaptations:

Particularly noteworthy is the finding that expression patterns of SEY1 in C. cinerea show stronger correlation with S. pombe than with S. cerevisiae, despite the vast evolutionary distance spanning over half a billion years . This suggests deep conservation of core meiotic expression programs in these fungi.

In comparative studies between C. cinerea and other basidiomycetes like A. bisporus, SEY1 appears to play roles in fruiting body development, with especially notable activity in young developing tissues where high protein turnover occurs during cell differentiation . This functional conservation suggests SEY1's fundamental importance in developmental processes across diverse fungal lineages.

What experimental approaches can address contradictions in SEY1 localization data?

Researchers sometimes encounter contradictory data regarding SEY1 localization, particularly between different developmental stages or experimental systems. To resolve such contradictions, the following experimental approaches are recommended:

• Multi-method Verification:

  • Combine immunolocalization, fluorescent protein tagging, and subcellular fractionation

  • Use multiple fixation methods (aldehyde-based, methanol, acetone) to rule out fixation artifacts

  • Compare N- and C-terminally tagged versions to identify potential interference from tags

• Developmental Time Course Analysis:

  • Perform high-resolution temporal sampling during developmental transitions

  • Use live-cell imaging with SEY1-GFP fusions to track dynamic localization changes

  • Correlate localization shifts with specific developmental events

• Co-localization Studies:

  • Use established organelle markers (ER, Golgi, mitochondria) to precisely define SEY1 location

  • Employ super-resolution microscopy techniques for improved spatial resolution

  • Quantify co-localization using statistical methods like Pearson's correlation coefficient

• Functional Domain Analysis:

  • Create truncated or domain-specific mutants to identify localization signals

  • Use site-directed mutagenesis to modify potential regulatory sites

  • Compare wild-type and mutant localization patterns under identical conditions

• Environmental Response Analysis:

  • Systematically vary culture conditions (temperature, light, nutrients) to determine if localization is condition-dependent

  • Investigate stress responses that might trigger relocalization

How can researchers design experiments to investigate SEY1's role in membrane dynamics?

Designing robust experiments to investigate SEY1's role in membrane dynamics requires multiple complementary approaches:

• In vitro Membrane Fusion Assays:

  • Prepare liposomes with fluorescent lipid markers (e.g., NBD-PE and Rh-PE)

  • Incorporate purified recombinant SEY1 into liposomes

  • Measure fusion using fluorescence resonance energy transfer (FRET)

  • Include appropriate controls: GTPase-dead mutants, GTP/GDP variations, divalent cation dependencies

• Membrane Dynamics in Living Cells:

  • Generate C. cinerea strains expressing fluorescent membrane markers

  • Perform SEY1 knockout or knockdown in these strains

  • Use live-cell imaging to track membrane dynamics during development

  • Measure parameters such as membrane fusion frequency, ER network morphology, and membrane fluidity

• Interaction Partner Identification:

  • Conduct pull-down assays using His-tagged SEY1 to identify binding partners

  • Perform yeast two-hybrid or split-GFP complementation assays to verify interactions

  • Use mass spectrometry to identify components of SEY1-containing complexes

  • Map interaction domains through truncation or site-directed mutagenesis

• Developmental Expression Correlation:

  • Track SEY1 expression across developmental stages using qRT-PCR

  • Correlate expression with specific membrane remodeling events

  • Compare with expression of other membrane dynamics proteins

  • Analyze promoter activity in different tissues using GFP reporter constructs

The experimental design should include appropriate controls, sufficient biological replicates (minimum n=3), and statistical analysis to ensure reproducibility and significance of results.

What are the critical controls needed for SEY1 functional studies in C. cinerea?

When conducting functional studies of SEY1 in C. cinerea, the following critical controls should be incorporated:

• For Gene Expression Studies:

  • Housekeeping gene controls (e.g., actin, GAPDH) for normalization

  • Expression profiles of known developmentally regulated genes for comparison

  • Empty vector controls for promoter reporter constructs

  • Multiple time points to capture expression dynamics

• For Protein Localization:

  • Unfused fluorescent protein controls to account for non-specific localization

  • Well-characterized organelle markers for co-localization studies

  • Both N- and C-terminal tags to identify potential interference

  • Fixed cell controls to compare with live-cell imaging

• For Knockout/Knockdown Studies:

  • Off-target control guides for CRISPR-Cas9 approaches

  • Complementation with wild-type SEY1 to confirm phenotype specificity

  • Heterozygous knockouts to identify dosage effects

  • Non-targeting RNAi controls for knockdown experiments

• For Biochemical Assays:

  • GTPase-dead mutants (typically S/T→N mutations in the G1 motif)

  • No-nucleotide and non-hydrolyzable GTP analog controls

  • Heat-inactivated protein controls

  • Buffer-only reactions to establish baselines

• For Developmental Studies:

  • Wild-type strains grown under identical conditions

  • Environmental condition controls (light/dark cycles, temperature, media composition)

  • Age-matched samples to control for developmental timing

  • Morphological markers to standardize developmental stages

Implementation of these controls ensures that observed effects can be confidently attributed to SEY1 function rather than experimental artifacts or secondary effects.

What are the optimal expression conditions for producing recombinant SEY1 in E. coli?

Optimizing expression conditions for recombinant SEY1 in E. coli requires careful consideration of multiple parameters:

• Strain Selection:

  • BL21(DE3) or derivatives like Rosetta(DE3) for enhanced expression of eukaryotic proteins

  • Arctic Express strains for expression at lower temperatures to improve folding

  • C41(DE3) or C43(DE3) for membrane-associated proteins like SEY1

• Vector and Tag Options:

  • pET series vectors with T7 promoter for high expression levels

  • N-terminal His-tag for purification, as demonstrated for the full-length SEY1

  • Consider larger solubility-enhancing tags (MBP, SUMO) if solubility is an issue

• Culture Conditions:

  • Medium: Rich media (LB, TB, 2xYT) for high cell density

  • Temperature: Initial growth at 37°C until OD600 ~0.6-0.8, then reduce to 16-18°C for induction

  • Induction: 0.1-0.5 mM IPTG, with lower concentrations often yielding better soluble protein

  • Duration: Extended expression (16-20 hours) at lower temperatures often improves yield

• Optimized Protocol:

• Troubleshooting Low Yields:

  • Check for codon bias and consider codon-optimized synthetic genes

  • Test different media formulations, including auto-induction media

  • Supplement with 0.5-1% glucose to reduce basal expression before induction

  • Add 5-10% glycerol to stabilize protein during expression

Following these optimized conditions typically yields 2-5 mg of purified SEY1 protein per liter of bacterial culture, with purity greater than 90% as determined by SDS-PAGE analysis .

How can researchers effectively analyze SEY1 expression patterns across different C. cinerea tissues?

Effective analysis of SEY1 expression patterns across C. cinerea tissues requires a multi-faceted approach:

• Tissue-Specific RNA Extraction Protocol:

  • Harvest distinct tissues (mycelium, primordia, stipe, cap, gill) at defined developmental stages

  • Flash-freeze samples in liquid nitrogen and pulverize while frozen

  • Extract RNA using fungal-optimized protocols (e.g., TRIzol with additional phenol extractions)

  • Verify RNA quality using both spectrophotometric methods (A260/A280 ratios) and gel electrophoresis

• Quantitative Expression Analysis:

  • Perform RT-qPCR using SEY1-specific primers with efficiency between 90-110%

  • Normalize expression to multiple reference genes validated for stability in C. cinerea

  • Calculate relative expression using the 2^(-ΔΔCt) method

  • Present data with appropriate statistical analysis (ANOVA with post-hoc tests)

• Spatial Expression Mapping:

  • Generate C. cinerea transformants with SEY1 promoter::GFP fusion constructs

  • Perform confocal microscopy on different tissues at various developmental stages

  • Use counterstaining (e.g., DAPI for nuclei, FM4-64 for membranes) to provide cellular context

  • Quantify fluorescence intensity across different tissue regions

• Single-Cell Resolution Approaches:

  • Apply laser capture microdissection to isolate specific cell types

  • Perform RNA-seq on isolated populations

  • Use fluorescence-activated cell sorting on protoplasts from SEY1-GFP strains

  • Consider single-cell RNA-seq for highest resolution of expression heterogeneity

• Correlation with Developmental Markers:

  • Compare SEY1 expression with established markers for different developmental stages

  • Analyze under varied environmental conditions known to affect development

  • Create expression heat maps to visualize patterns across tissues and developmental time

This comprehensive approach provides both quantitative data on expression levels and qualitative information about spatial distribution, offering a complete picture of SEY1 expression dynamics throughout C. cinerea development.

What are the emerging techniques that will advance SEY1 research in basidiomycetes?

Several cutting-edge techniques are poised to significantly advance SEY1 research in C. cinerea and other basidiomycetes:

• CRISPR-Cas9 Genome Editing: The adaptation of CRISPR systems for efficient editing in basidiomycetes will enable precise manipulation of SEY1, including introduction of point mutations, domain deletions, and fluorescent protein fusions at endogenous loci.

• Single-Molecule Tracking: Using photoactivatable fluorescent proteins fused to SEY1 will allow tracking of individual molecules in living cells, revealing dynamic behaviors and interaction kinetics that are masked in bulk measurements.

• Cryo-Electron Microscopy: Structural studies of SEY1 and its complexes at near-atomic resolution will provide insights into the mechanism of GTPase activity and membrane interactions, potentially revealing fungal-specific features that could be targeted for antifungal development.

• Spatial Transcriptomics: Emerging methods for in situ sequencing will enable mapping of SEY1 expression with unprecedented spatial resolution in intact fruiting bodies and mycelia, correlating expression with specific morphological features.

• Optogenetics: Development of light-controlled SEY1 variants will allow temporal and spatial control of protein function, enabling precise dissection of SEY1's role in specific developmental processes.

These emerging technologies will help resolve current contradictions in the literature and provide deeper insights into the multifaceted roles of SEY1 in fungal development and membrane dynamics.

How does evolutionary conservation of SEY1 inform functional studies across fungal species?

The evolutionary conservation of SEY1 across diverse fungal lineages provides valuable insights for functional studies:

• Comparative Genomics Approach: Analysis reveals that SEY1 shows varying degrees of conservation across fungi, with particularly strong homology observed between basidiomycetes like C. cinerea and A. bisporus. Interestingly, the expression pattern of SEY1 in C. cinerea correlates more strongly with S. pombe than with S. cerevisiae, despite the vast evolutionary distance of over half a billion years . This suggests fundamental conservation of core meiotic expression programs across these diverse fungi.

• Function-Structure Relationships: Highly conserved domains likely represent functional cores essential for basic SEY1 activity, while divergent regions may indicate species-specific adaptations. Targeted mutagenesis of conserved versus variable regions can help delineate universal versus specialized functions.

• Cross-Species Complementation: The ability of SEY1 from one species to rescue phenotypes in another provides strong evidence for functional conservation. Such experiments between C. cinerea and model organisms like S. cerevisiae can expedite functional characterization.

• Developmental Context Conservation: The observation that SEY1 expression is associated with similar developmental processes across multiple basidiomycete species suggests conserved regulatory networks. These patterns can guide experimental design when studying novel basidiomycete species.

• Translational Relevance: Conservation patterns between model fungi like C. cinerea and pathogenic fungi highlight potential targets for antifungal development, where sufficient divergence from human homologs exists.