Recombinant Schizosaccharomyces pombe Putative mannose 6-phosphate receptor-like protein C530.09c (SPBC530.09c)

What is the cellular localization of SPBC530.09c in S. pombe?

The SPBC530.09c protein, as a putative mannose 6-phosphate receptor-like protein, is primarily localized in the Golgi apparatus and endosomal compartments. To experimentally determine the precise localization, several methodologies can be employed:

Immunofluorescence microscopy using methanol fixation provides a reliable approach for protein localization. Cells should be fixed with cold methanol (-80°C) for 8 minutes, followed by antibody staining with anti-SPBC530.09c primary antibodies and fluorophore-conjugated secondary antibodies. Co-localization studies with known Golgi markers (e.g., GFP-tagged Gmf1p) can confirm the precise compartment localization .

For higher resolution analysis, immunogold electron microscopy can be employed. This technique requires spheroplasting S. pombe cells using 0.25 mg/ml Zymolyase 100T in SP buffer, followed by fixation, embedding, and immunogold labeling with specific antibodies against SPBC530.09c .

How does SPBC530.09c expression vary during the cell cycle?

SPBC530.09c expression patterns throughout the S. pombe cell cycle can be monitored using several approaches:

Synchronize cell cultures using either centrifugal elutriation or nitrogen starvation-based methods. For nitrogen starvation synchronization, grow cells to mid-log phase in EMM medium, then transfer to EMM-N (without nitrogen source) for 24 hours, followed by resuspension in complete medium .

Monitor cell cycle progression using DAPI staining and FACS analysis. Collect samples at 20-minute intervals following synchronization, fix cells with 70% ethanol, and stain with DAPI (4′,6-diamidino-2-phenylindole) at 1 μg/ml. This approach allows correlation of SPBC530.09c expression with specific cell cycle stages .

Extract RNA at each time point and perform qPCR using primers specific to SPBC530.09c. Expression data should be normalized to stable reference genes such as act1+ or cdc2+. Plot expression levels against the determined cell cycle stages to generate a comprehensive expression profile.

What glycosylation patterns are typical for SPBC530.09c?

S. pombe produces unique glycosylation patterns compared to other yeasts, which significantly impacts the properties of glycoproteins like SPBC530.09c:

S. pombe generates Man9GlcNAc2 N-glycans (rather than Man8GlcNAc2 seen in other yeasts) and incorporates galactose residues in addition to mannose in its glycan structures . To experimentally determine glycosylation patterns, perform EndoH treatment on purified SPBC530.09c and analyze the mobility shift using SDS-PAGE and Western blotting .

For detailed glycan structure analysis, purify SPBC530.09c using affinity chromatography, then employ mass spectrometry for glycan profiling. Tryptic digestion followed by LC-MS/MS can identify specific glycosylation sites and structures. The presence of galactose residues, which are unique to S. pombe among yeasts, can be confirmed through specific galactosidase treatments before mass spectrometry analysis .

What are the optimal conditions for recombinant expression of SPBC530.09c?

For successful recombinant expression of SPBC530.09c, consider the following methodological approach:

Clone the SPBC530.09c open reading frame into an appropriate S. pombe expression vector (such as pREP series) using high-fidelity PCR amplification with primers containing suitable restriction sites. For inducible expression, the nmt1 promoter system (available in three strength variants) provides excellent control, with thiamine repressing expression and its absence inducing expression .

For protein purification, add a C-terminal or N-terminal tag sequence (His6, FLAG, or GST) ensuring it doesn't interfere with protein folding or function. When using N-terminal tags, verify that the signal sequence for proper localization remains functional.

Transform the expression construct into an appropriate S. pombe strain (wild-type or specific mutant backgrounds depending on research questions). For optimal expression, culture cells in EMM medium at 30°C with appropriate selection markers .

Monitor expression levels using Western blotting with anti-tag antibodies or specific antibodies against SPBC530.09c. For membrane proteins like SPBC530.09c, optimize membrane preparation protocols using differential centrifugation methods to isolate appropriate cellular fractions.

How can genetic knockout or knockdown of SPBC530.09c be achieved?

For functional studies, genetic manipulation of SPBC530.09c can be performed using the following approaches:

For complete gene deletion (if viable), employ homologous recombination-based gene targeting. Design primers with 80-100 bp homology to regions flanking SPBC530.09c and amplify a selectable marker cassette (such as ura4+ or kanMX6). Transform the PCR product into a diploid S. pombe strain, select for marker integration, and confirm correct integration by PCR and sequencing. Induce sporulation and analyze haploid progeny to determine if SPBC530.09c is essential .

If SPBC530.09c proves essential (as many mannose-6-phosphate receptor proteins are), employ conditional expression systems. Replace the native promoter with the nmt81 (weak) or nmt41 (medium strength) thiamine-repressible promoter. This allows for controlled depletion of the protein by adding thiamine to the growth medium .

For partial knockdown, RNA interference can be employed using the ura4+ reporter gene and inverted repeat sequences of SPBC530.09c. This approach typically achieves 70-90% reduction in expression levels.

How can protein-protein interactions of SPBC530.09c be identified?

To elucidate the interaction network of SPBC530.09c, several complementary approaches can be employed:

Perform immunoprecipitation (IP) followed by mass spectrometry analysis. Express tagged versions of SPBC530.09c (FLAG, HA, or TAP-tag) in S. pombe, prepare cell lysates under gentle conditions (1% NP-40 or 0.5% digitonin) to preserve protein-protein interactions, and perform IP using appropriate antibodies or affinity resins. Identify co-precipitated proteins by LC-MS/MS analysis .

For validation of specific interactions, conduct co-immunoprecipitation experiments with candidate interacting proteins. Express differentially tagged versions of SPBC530.09c and potential interacting partners (e.g., FLAG-SPBC530.09c and HA-InteractingProteinX), perform IP with one tag, and detect the presence of the partner using antibodies against the other tag.

Yeast two-hybrid screening can identify direct protein interactions. Use SPBC530.09c (or specific domains) as bait against an S. pombe cDNA library to identify potential interacting partners. Confirm positive interactions through co-immunoprecipitation and functional studies.

How does SPBC530.09c contribute to cell wall integrity in S. pombe?

As a putative mannose-6-phosphate receptor-like protein, SPBC530.09c may play a role in trafficking enzymes involved in cell wall metabolism. Investigate its contribution using these approaches:

Analyze cell wall composition in SPBC530.09c-depleted or overexpressing strains. Prepare cell walls by mechanical disruption followed by detergent washing, and quantify cell wall components (β-1,3-glucan, β-1,6-glucan, α-glucan, and glycoproteins) using specific enzymes and colorimetric assays. Changes in cell wall composition can indicate functional roles in cell wall biogenesis or maintenance .

Perform phenotypic analysis using cell wall stress agents. Test sensitivity to compounds such as Calcofluor White (targets chitin), Congo Red (affects β-1,3-glucan), and enzymatic digestion (Zymolyase sensitivity). Increased sensitivity suggests compromised cell wall integrity .

Examine septum formation using microscopy techniques. Stain cells with Aniline Blue to visualize β-1,3-glucan, and assess septum morphology and integrity. Abnormal septum formation may indicate a role in cell division and cytokinesis .

What is the role of SPBC530.09c in protein glycosylation pathways?

To determine the role of SPBC530.09c in glycosylation pathways, implement the following experimental strategy:

Analyze N-glycan profiles in SPBC530.09c-depleted cells by isolating glycoproteins, releasing N-glycans using PNGase F, and analyzing glycan structures by HPLC or mass spectrometry. Compare profiles with wild-type cells to identify specific alterations in glycan structures .

Examine genetic interactions with known glycosylation pathway components. Construct double mutants between SPBC530.09c and genes involved in various aspects of glycosylation (such as och1+, which initiates outer chain elongation, or gms1+, the UDP-galactose transporter). Synthetic growth defects or epistatic relationships can reveal functional connections .

Perform pulse-chase analysis of model glycoproteins to track glycosylation kinetics. Express a reporter glycoprotein, pulse-label with radioactive amino acids, and chase with unlabeled medium. Analyze glycosylation status over time using glycosidase treatments and SDS-PAGE to detect defects in glycan processing or trafficking .

How does redox status affect SPBC530.09c function?

To investigate the relationship between redox conditions and SPBC530.09c function:

Express SPBC530.09c fused to redox-sensitive GFP (roGFP2) to monitor the redox environment of the protein in vivo. This fusion protein allows ratiometric measurements of oxidation states using fluorescence microscopy or plate reader assays. Calculate the oxidation degree using the formula: OxD = (R − Rred)/(Rox − Rred) × (Iλ2ox/Iλ2red) .

Expose cells to oxidative stress agents (H2O2, diamide) or reducing agents (DTT, β-mercaptoethanol) and monitor changes in SPBC530.09c localization, expression, and interaction partners. Compare these responses to wild-type cells to identify redox-dependent regulation mechanisms.

Identify potential redox-sensitive cysteine residues in SPBC530.09c through bioinformatic analysis and site-directed mutagenesis. Create cysteine-to-serine mutations at candidate sites and assess the impact on protein function, localization, and stability under different redox conditions.

What are the best approaches for purifying recombinant SPBC530.09c?

For successful purification of recombinant SPBC530.09c, consider the following methodology:

Express SPBC530.09c with an appropriate affinity tag (His6, GST, or MBP) in S. pombe. For membrane proteins like mannose-6-phosphate receptors, include a detergent screening step to identify optimal solubilization conditions. Test a panel of detergents including CHAPS, DDM, Triton X-100, and digitonin at various concentrations.

Prepare membrane fractions by differential centrifugation: lyse cells using glass beads, remove cell debris (1,000×g, 5 min), and collect membrane fractions by ultracentrifugation (100,000×g, 1 hour). Solubilize membranes with the optimal detergent and perform affinity chromatography using the appropriate resin .

For further purification, employ size exclusion chromatography to separate monomeric protein from aggregates or complexes. For higher purity requirements, add an ion exchange chromatography step based on the theoretical isoelectric point of SPBC530.09c.

Verify protein purity using SDS-PAGE with PAS-Silver staining to detect both protein and glycan components. Confirm identity using Western blotting with specific antibodies and mass spectrometry analysis .

How can the topology of SPBC530.09c in membranes be determined?

To elucidate the membrane topology of SPBC530.09c:

Perform proteinase K protection assays on isolated membrane fractions. Treat intact membrane vesicles with proteinase K with or without detergent (e.g., Triton X-100). Domains exposed to the cytosol will be digested in both conditions, while lumenal domains will only be accessible after detergent solubilization. Analyze the resulting fragments by Western blotting using antibodies against different regions of SPBC530.09c .

For a comprehensive topological map, create a series of SPBC530.09c constructs with reporter tags (such as GFP or epitope tags) inserted at different positions. Express these constructs in S. pombe and determine the localization of each tag using protease accessibility assays or immunofluorescence in selectively permeabilized cells.

Use chemical modification approaches with membrane-permeable and impermeable sulfhydryl reagents (e.g., NEM and MTSET) to probe the accessibility of cysteine residues. This approach requires generating cysteine-null versions of SPBC530.09c and then reintroducing individual cysteines at positions of interest.

What transcriptional analysis methods are most effective for studying SPBC530.09c regulation?

To investigate the transcriptional regulation of SPBC530.09c:

Perform RNA extraction using hot phenol method, which is particularly effective for S. pombe. Extract RNA from cells under various conditions (different growth phases, stress treatments, etc.), synthesize cDNA, and quantify SPBC530.09c expression using qPCR with appropriate reference genes for normalization .

For genome-wide analysis, conduct microarray hybridization or RNA-seq experiments to identify conditions that affect SPBC530.09c expression. This approach can reveal co-regulated genes and potential regulatory networks. Process biological triplicates for statistical reliability, and use appropriate software for data normalization and statistical analysis .

To identify transcription factors regulating SPBC530.09c, perform chromatin immunoprecipitation (ChIP) experiments. Use antibodies against candidate transcription factors, followed by qPCR with primers specific to the SPBC530.09c promoter region, or ChIP-seq for genome-wide binding analysis.

How can contradictory results in SPBC530.09c functional studies be reconciled?

When facing contradictory results in functional studies of SPBC530.09c:

Systematically analyze experimental differences that might explain discrepancies, including:

• Genetic background variations: Different S. pombe strains may contain genetic modifiers that influence SPBC530.09c function. Document strain lineages completely and sequence verify key strains.

• Expression level differences: Quantify protein expression levels using calibrated Western blotting. Over-expression or insufficient depletion can lead to contradictory results.

• Growth conditions: S. pombe phenotypes can be highly sensitive to media composition, temperature, and growth phase. Standardize conditions and report them comprehensively.

Perform epistasis analysis with known pathway components to place contradictory results in context. If SPBC530.09c appears to have different functions in different studies, it may operate in multiple pathways or its function may be context-dependent.

Design decisive experiments that can specifically distinguish between competing hypotheses. For example, if one study suggests SPBC530.09c functions in trafficking and another in biosynthesis, design cargo-specific trafficking assays and in vitro biosynthesis assays to directly test both possibilities.

What experimental controls are essential when studying SPBC530.09c glycosylation?

When investigating glycosylation aspects of SPBC530.09c, incorporate these essential controls:

Include glycosylation-deficient mutants as reference points. The Δoch1 S. pombe mutant, which lacks the α-1,6-mannosyltransferase that initiates hyperglycosylation, provides a valuable control for hypoglycosylated proteins. Similarly, include the UDP-galactose transporter mutant (Δgms1) to assess galactose-specific modifications .

Use enzymatic deglycosylation controls to confirm glycan-dependent effects. Treat samples with EndoH (which cleaves high-mannose N-glycans) or PNGase F (which removes most N-glycans) to distinguish between effects due to the protein backbone versus its glycan structures .

For each glycosylation analysis experiment, include standard glycoproteins with well-characterized glycan structures. For S. pombe-specific standards, acid phosphatase or cell surface galactomannan proteins provide reliable reference points for glycan profiling.

How might gene editing technologies enhance SPBC530.09c research?

CRISPR-Cas9 and related technologies offer significant advantages for SPBC530.09c research:

Implement CRISPR-Cas9 for precise genomic modifications, including:

• Introduction of point mutations to study specific functional residues without epitope tags that might interfere with function.

• Creation of fluorescent protein knock-ins at the endogenous locus to study native expression levels and localization.

• Generation of conditional degron systems for rapid protein depletion studies.

Design a CRISPR-based screening approach to identify genetic interactions. Construct a library of guide RNAs targeting non-essential S. pombe genes, introduce this library into SPBC530.09c mutant backgrounds, and use next-generation sequencing to identify synthetic lethal or suppressor interactions.

Establish base editing or prime editing systems in S. pombe to make precise nucleotide changes without double-strand breaks, allowing subtle modifications to regulatory regions or protein coding sequences of SPBC530.09c.

What comparative analyses with other organisms would advance SPBC530.09c research?

Comparative studies across species can provide valuable insights into SPBC530.09c function:

Perform phylogenetic analysis of mannose-6-phosphate receptor-like proteins across fungal species to identify conserved domains and species-specific adaptations. This can help distinguish core functions from specialized roles in S. pombe.

Conduct complementation studies by expressing SPBC530.09c homologs from other species (S. cerevisiae, Candida albicans, or mammalian cells) in S. pombe SPBC530.09c mutants. The degree of functional rescue can reveal evolutionary conservation of biochemical activities.

Create chimeric proteins containing domains from SPBC530.09c and its homologs to map functional regions. Express these in appropriate mutant backgrounds and assess their ability to rescue phenotypic defects.