Recombinant Schizosaccharomyces pombe Uncharacterized membrane protein C18H10.18c (SPBC18H10.18c)

What are the recommended storage and handling conditions for recombinant SPBC18H10.18c?

The optimal storage of recombinant SPBC18H10.18c protein requires careful attention to temperature and buffer conditions. The lyophilized protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use scenarios . For working with the protein, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended .

To maintain stability, the addition of 5-50% glycerol (final concentration) before aliquoting for long-term storage at -20°C/-80°C is advised, with 50% being the default concentration . The protein is supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . It's important to note that repeated freeze-thaw cycles should be avoided, and working aliquots can be stored at 4°C for up to one week . Prior to opening, a brief centrifugation of the vial is recommended to bring contents to the bottom .

How can I verify the expression and purification quality of recombinant SPBC18H10.18c?

Quality assessment of recombinant SPBC18H10.18c should employ multiple analytical techniques. Start with SDS-PAGE analysis to confirm the expected molecular weight (the full-length protein is 242 amino acids plus the His-tag) . A purified preparation should show >90% purity as determined by SDS-PAGE .

For further verification, Western blot analysis using an anti-His antibody can confirm the presence of the tagged protein. If studying potential glycosylation, researchers should consider EndoH treatment followed by mobility shift analysis on SDS-PAGE . Mass spectrometry provides the most definitive confirmation of protein identity and can detect any post-translational modifications or proteolytic processing.

For functional verification, membrane localization studies using fractionation techniques like sucrose density gradient centrifugation would be appropriate, particularly since SPBC18H10.18c is annotated as a membrane protein .

What is the subcellular localization of SPBC18H10.18c in S. pombe?

While specific localization data for SPBC18H10.18c is limited in the provided references, appropriate methodological approaches for determining subcellular localization include both fluorescent tagging and immunolabeling techniques. Based on methodologies described for related membrane proteins, researchers should consider:

• Fluorescent protein tagging: C- or N-terminal tagging with fluorescent proteins like GFP, though it's important to verify that tagging doesn't disrupt protein localization or function .

• Immunolabeling with specific antibodies when fluorescent tagging proves challenging .

• Cellular fractionation via sucrose density gradient centrifugation to separate membrane compartments, followed by Western blot analysis to track the protein's distribution .

Given that SPBC18H10.18c is annotated as a membrane protein, researchers should be particularly attentive to potential artifacts in localization studies and consider multiple complementary approaches for verification.

What are the potential functional roles of SPBC18H10.18c based on homology and structural features?

Though SPBC18H10.18c remains uncharacterized, computational comparative analyses can provide valuable functional insights. While direct information about SPBC18H10.18c's function isn't explicitly stated in the provided references, the methodological approach should include:

• Homology analysis: Compare SPBC18H10.18c sequence with characterized proteins across species using BLAST and multiple sequence alignment tools. Special attention should be paid to conserved domains and motifs that might suggest function.

• Structural prediction: Use programs like Phyre2 or I-TASSER to generate 3D structural models, which can reveal functional domains not obvious from sequence analysis alone.

• Examination of membrane topology: Determine the number and orientation of transmembrane domains using prediction algorithms like TMHMM or TOPCONS, as this can suggest potential functions (transport, signaling, anchoring, etc.).

The search results mention that proteins like Sup11p in S. pombe show homology to Saccharomyces cerevisiae Kre9, which is involved in β-1,6-glucan synthesis . While no direct connection between SPBC18H10.18c and Sup11p is established in the provided information, this suggests a methodological approach of investigating whether SPBC18H10.18c might have similar functions in cell wall biosynthesis or remodeling.

How can gene deletion or conditional expression systems be used to study SPBC18H10.18c function?

For investigating SPBC18H10.18c function through genetic manipulation, researchers should consider whether the gene is essential, as this will determine the appropriate experimental strategy. Based on methodologies described for related proteins:

• Essentiality testing: Create a heterozygous deletion in a diploid strain, induce sporulation, and analyze tetrads to determine if haploid deletion mutants are viable.

• For essential genes: Employ conditional expression systems like the nmt (no message in thiamine) promoter series (nmt1, nmt41, nmt81) that offer different levels of repression in the presence of thiamine . The nmt81 promoter, being the weakest, allows for partial depletion that can reveal phenotypes while maintaining viability.

• For non-essential genes: Create complete deletion strains and characterize phenotypes related to growth, morphology, stress resistance, and cell wall integrity.

• For all approaches: Employ comprehensive phenotypic analysis including:

  • Growth rate measurements under various conditions

  • Microscopic examination of cell morphology and septum formation

  • Cell wall composition analysis

  • Stress response evaluation (osmotic, temperature, cell wall-perturbing agents)

The research on Sup11p demonstrates how depletion of an essential protein can reveal its function in processes like cell wall formation and septum assembly , providing a methodological template for SPBC18H10.18c functional studies.

What methods are appropriate for studying protein-protein interactions of SPBC18H10.18c?

For investigating interaction partners of SPBC18H10.18c, a multi-faceted approach is required that accounts for its membrane localization:

• Proximity-based labeling: BioID or APEX2 fusion proteins can identify proximal proteins in living cells, which is particularly valuable for membrane proteins where traditional yeast two-hybrid systems are challenging.

• Co-immunoprecipitation with membrane-appropriate detergents: Use mild non-ionic detergents (like digitonin or DDM) for solubilization followed by pull-down with anti-His antibodies and mass spectrometry identification of co-precipitating proteins.

• Genetic interaction screening: Synthetic genetic array (SGA) analysis can reveal functional relationships by identifying genes whose deletion enhances or suppresses phenotypes of SPBC18H10.18c mutants.

• Split-ubiquitin membrane yeast two-hybrid: A specialized two-hybrid system designed for membrane proteins.

• Crosslinking mass spectrometry: In vivo or in vitro crosslinking followed by MS analysis can capture transient interactions.

Analysis of the resulting data should prioritize reproducibility across methods and biological significance of interactions, with validation through reciprocal co-IP or functional studies.

How might SPBC18H10.18c be involved in cell wall biogenesis or remodeling?

While direct evidence for SPBC18H10.18c's involvement in cell wall processes is not specifically mentioned in the provided references, a comprehensive experimental approach to investigate this possibility would include:

• Cell wall composition analysis: Compare β-1,3-glucan, β-1,6-glucan, and mannan content between wild-type and SPBC18H10.18c-depleted cells using specific enzymes and antibodies.

• Cell wall stress tests: Examine sensitivity to cell wall-perturbing agents (Calcofluor White, Congo Red) and cell wall digestive enzymes in mutant strains.

• Genetic interaction studies: Test for genetic interactions with known cell wall synthesis genes, particularly those involved in β-1,6-glucan biosynthesis.

• Transcriptome analysis: Examine changes in expression of cell wall-related genes upon SPBC18H10.18c depletion, similar to the approach used for Sup11p that revealed regulation of several cell wall glucan modifying enzymes .

• Microscopic examination: Analyze septum formation and cell morphology using transmission electron microscopy and fluorescent staining of cell wall components.

Based on the research on Sup11p, which showed that its depletion led to the absence of β-1,6-glucan from the cell wall and severe morphological defects with malformation of the septum , similar approaches could reveal whether SPBC18H10.18c has related functions.

What transcriptomic changes occur upon depletion of SPBC18H10.18c?

A methodologically sound approach to investigating transcriptomic changes upon SPBC18H10.18c depletion would involve:

• Establishing a conditional expression system (preferably using the nmt81 promoter for moderate repression) to create a SPBC18H10.18c knock-down strain.

• Time-course sampling during protein depletion to capture primary and secondary transcriptional responses.

• RNA extraction followed by either microarray hybridization or RNA-seq analysis, with appropriate biological and technical replicates.

• Bioinformatic analysis including:

  • Differential expression analysis

  • Gene Ontology enrichment

  • Pathway analysis

  • Comparison with transcriptional responses to other perturbations

• Validation of key findings by RT-qPCR and functional studies.

The approach used for Sup11p, which identified significant regulation of cell wall glucan modifying enzymes , provides a methodological template. Researchers should pay particular attention to genes involved in membrane processes, protein trafficking, and cell wall biogenesis given SPBC18H10.18c's membrane localization.

How can post-translational modifications of SPBC18H10.18c be studied?

Investigation of post-translational modifications (PTMs) of SPBC18H10.18c requires a comprehensive analytical approach:

• Mass spectrometry-based PTM mapping: Purify the protein using tandem affinity purification tags, perform in-gel or in-solution digestion with multiple proteases, and analyze using high-resolution MS/MS with PTM-specific enrichment strategies.

• Glycosylation analysis: Given the potential for both N- and O-glycosylation in S. pombe proteins, researchers should:

  • Test for N-glycosylation using EndoH treatment and mobility shift analysis

  • Examine O-mannosylation through metabolic labeling with [14C]mannose

  • Consider PNGase F treatment for N-glycan release and subsequent glycan profiling

• Phosphorylation studies: Use phospho-specific antibodies or phosphoproteomic approaches with TiO2 enrichment.

• Site-directed mutagenesis: Create mutants of predicted modification sites to assess their functional significance.

The research on Sup11p showed that it can be hypo-mannosylated when expressed in an O-mannosylation mutant background, and that it can be N-glycosylated on an unusual N-X-A sequon when the S/T-rich O-mannosylation sites are unavailable . This suggests that careful analysis of glycosylation patterns in different genetic backgrounds would be particularly valuable for membrane proteins like SPBC18H10.18c.

What approaches are suitable for analyzing membrane topology of SPBC18H10.18c?

Determining the membrane topology of SPBC18H10.18c requires multiple complementary experimental approaches:

• Protease protection assays: Perform proteinase K digestion of intact spheroplasts, microsomes, or whole cells, followed by Western blot analysis using antibodies against different regions of the protein or epitope tags placed at various positions.

• Reporter fusion proteins: Create fusions with reporters like GFP, alkaline phosphatase (PhoA), or β-galactosidase at different positions in the protein sequence. The activity or fluorescence of these reporters depends on their cellular localization (cytoplasmic vs. luminal).

• Substituted cysteine accessibility method (SCAM): Introduce cysteines at various positions followed by labeling with membrane-impermeable sulfhydryl reagents to determine exposed regions.

• Fluorescence protease protection (FPP) assay: Tag the protein with fluorescent proteins at different termini and monitor fluorescence after selective plasma membrane permeabilization and protease addition.

• Cryo-electron microscopy: For high-resolution structural determination, though this requires substantial amounts of purified protein.

The methodology described for subcellular localization studies of Sup11p, which includes both fluorescent tagging and immunolabeling approaches , provides useful guidance for designing experiments to analyze membrane protein topology.

How can the effect of SPBC18H10.18c on β-1,6-glucan synthesis be evaluated?

To evaluate whether SPBC18H10.18c plays a role in β-1,6-glucan synthesis, similar to what was found for Sup11p , researchers should employ the following methodological approaches:

• Quantitative analysis of β-1,6-glucan content: Compare levels between wild-type cells and those with depleted or overexpressed SPBC18H10.18c using:

  • Enzymatic digestion with specific β-1,6-glucanases followed by quantification of released glucose

  • Immunolabeling with antibodies specific to β-1,6-glucan epitopes

  • Binding assays with β-1,6-glucan-specific probes

• Genetic interaction studies:

  • Test for genetic interactions with known β-1,6-glucan synthesis genes

  • Examine potential redundancy or synthetic relationships with β-1,6-glucanase family members

• Cell wall fractionation:

  • Isolate cell walls and analyze polysaccharide composition through fractionation and specific enzyme digestions

  • Examine changes in association of cell wall proteins with the wall matrix

• Microscopic examination:

  • Analyze septum formation using transmission electron microscopy

  • Use fluorescent probes to visualize β-1,6-glucan distribution in wild-type and mutant cells

The research on Sup11p demonstrated that its depletion led to the absence of β-1,6-glucan from the cell wall , providing a methodological framework for similar studies with SPBC18H10.18c.

What integrated approaches would most effectively characterize SPBC18H10.18c function?

A comprehensive characterization of SPBC18H10.18c function would benefit from an integrated multi-omics approach combining:

• Structural biology: Cryo-EM or X-ray crystallography of purified protein to determine 3D structure.

• Transcriptomics: RNA-seq analysis of gene expression changes upon protein depletion or overexpression.

• Proteomics: Identification of interaction partners through IP-MS and analysis of post-translational modifications.

• Metabolomics: Assessment of changes in cell wall precursors and other relevant metabolites.

• Genetic screens: Systematic analysis of genetic interactions to place the protein in functional networks.

• Comparative genomics: Analysis of conservation and variation across fungal species to infer evolutionary constraints and functional importance.

Integration of these datasets through systems biology approaches would provide a holistic view of the protein's function. Particular attention should be paid to potential roles in cell wall biogenesis, given the significant insights gained from studies of related proteins like Sup11p .