Recombinant Schizosaccharomyces pombe Uncharacterized protein C8F11.08c (SPAC8F11.08c)

What is SPAC8F11.08c and what do we currently know about its function?

SPAC8F11.08c is an uncharacterized protein in Schizosaccharomyces pombe (strain 972 / ATCC 24843) that functions as an esterase/lipase. Recent molecular studies have demonstrated its involvement in spore maturation processes, specifically in outer Forespore Membrane (FSM) breakdown . Transcriptomic analyses show upregulated expression during meiosis, consistent with its role in sporulation .

The protein has been identified in proteomic studies with the following properties:

• UniProt ID: Q9UT29

• Full amino acid sequence: MGFATILYSIWVVLSSFVILSAYQLEVRKFLVKLLSKIVIGASESNVASFFAFVSEKSLEAASVKFVATIIIAERMGAQFSSFYDKIFFVDMLGLGSLFALAAVNVLAARKLSELPASLEPPSIGNRDQQKNSSLAKCFSRLFINDLHGTTVKRYPYISYLPNWLLAKNNAERLVYKSHLLLNAYSPPKSASASSVIVWVCGAKKSESIIIPYLSSLGFFVVVPNYAQPPKFPLSDAVEFVSLCVDWIVENAIYYDADPERIFFLGEDTGASVALESLKHIRPNSVKGIFALCPRSIQNLVWTNQSTIPMMTLHAGDVNPVSDSNDRESSEKASLIKNFVVPGAVPYYYEFTSPTTISTATFIARWFFLVEDNKKTI

How is SPAC8F11.08c related to the meu5+ gene regulatory network?

Research has shown that SPAC8F11.08c expression is regulated by Meu5 (also known as Crp79), an RNA-binding protein containing three RNA recognition motifs. While Meu5 directly binds and stabilizes more than 80 target transcripts, SPAC8F11.08c is among the genes showing downregulated expression in meu5Δ mutants as revealed by DNA microarray analysis . This regulatory relationship explains why disruptions in meu5+ lead to phenotypes similar to SPAC8F11.08c deletion, particularly in spore maturation processes.

What are the recommended approaches for expressing recombinant SPAC8F11.08c protein?

For optimal expression of recombinant SPAC8F11.08c, researchers have successfully employed several expression systems:

• E. coli expression system:

  • Most common for initial characterization due to high yield and simplicity

  • Recommended strain: BL21(DE3) with pET-based vectors

  • Expression typically induced with IPTG at 0.5-1.0 mM when culture reaches OD600 of 0.6-0.8

  • Lower induction temperatures (16-20°C) often improve solubility

• Yeast expression systems:

  • Native S. pombe expression using nmt1 promoter systems allows for regulated expression

  • Alternatively, expression in S. cerevisiae under GAL promoters offers good yields

  • P. pastoris system may be preferred for secreted versions of the protein

• Baculovirus expression:

  • Recommended for complex post-translational modifications

  • Insect cell lines (Sf9, Sf21, High Five) provide eukaryotic processing environment

• Mammalian cell expression:

  • Suitable for studies requiring native mammalian post-translational modifications

  • HEK293 or CHO cells are commonly used

What purification strategies are most effective for SPAC8F11.08c?

Optimal purification of recombinant SPAC8F11.08c typically involves:

• Affinity chromatography:

  • His-tag purification using Ni-NTA resin (pH 7.5-8.0)

  • GST-tag systems may improve solubility for difficult constructs

• Ion exchange chromatography:

  • Anion exchange (Q-Sepharose) at pH 8.0 as a secondary purification step

  • Cation exchange may be employed depending on calculated pI

• Size exclusion chromatography:

  • Final polishing step to achieve >85% purity as determined by SDS-PAGE

• Storage conditions:

  • Optimal storage in Tris-based buffer with 50% glycerol

  • Store at -20°C for short-term use, or -80°C for extended storage

  • Avoid repeated freeze-thaw cycles; working aliquots can be stored at 4°C for up to one week

How does SPAC8F11.08c contribute to outer FSM breakdown during spore maturation?

SPAC8F11.08c plays a crucial role in spore maturation, specifically in the breakdown of the outer Forespore Membrane (FSM). Research has shown:

• Deletion phenotype: SPAC8F11.08c deletion mutants exhibit defects in outer FSM breakdown comparable to those observed in meu5Δ strains .

• Functional mechanism: As a lipase, SPAC8F11.08c likely contributes to membrane breakdown by hydrolyzing phospholipids in the outer FSM. This enzymatic activity is critical for proper spore maturation .

• Regulatory context: The protein functions in coordination with other factors including:

  • Atg8 (autophagy-related protein)

  • Cyc1 (cytochrome c)

  • SPCC1020.13c (phospholipase)

• Temporal expression: SPAC8F11.08c shows upregulated expression during meiosis, consistent with its specialized role in sporulation .

Interestingly, studies have shown that SPAC8F11.08c deletion affects outer FSM disappearance but not the formation of "visible spores" under DIC microscopy, suggesting that outer FSM breakdown is independent of some aspects of spore formation .

What experimental approaches are recommended for studying SPAC8F11.08c function in vivo?

To investigate SPAC8F11.08c function in vivo, several complementary approaches have proven effective:

• Gene deletion/disruption techniques:

  • Homologous recombination-based gene targeting

  • CRISPR-Cas9 mediated gene editing for precise modifications

  • Construction of temperature-sensitive alleles for conditional studies

• Protein localization studies:

  • GFP/mCherry tagging for live-cell imaging

  • Immunofluorescence using antibodies against SPAC8F11.08c

  • Fractionation studies to determine subcellular localization

• Expression analysis:

  • RT-qPCR to quantify transcript levels during meiosis and sporulation

  • Western blotting using specific antibodies for protein detection

  • Proteomics approaches to detect changes in protein levels

• Functional assays:

  • In vitro lipase activity assays using fluorogenic substrates

  • Membrane lipid analysis before and after sporulation

  • Electron microscopy to visualize FSM morphology in mutants

• Genetic interaction studies:

  • Synthetic genetic array (SGA) analysis to identify interacting genes

  • Double mutant analysis with other FSM-related genes

  • Suppressor screens to identify compensatory pathways

What antibodies and detection methods are available for SPAC8F11.08c?

Several antibodies and detection methods have been developed for SPAC8F11.08c research:

• Commercial antibodies:

  • Rabbit polyclonal antibodies against Schizosaccharomyces pombe SPAC8F11.08c

  • Host: Rabbit / Reactivity: Schizosaccharomyces pombe (strain 972/24843)

  • Purification method: Antigen-affinity purified

  • Isotype: IgG

• Recommended applications:

  • Western blotting (WB): Typically detects a band at approximately 41 kDa

  • ELISA (EIA): Useful for quantitative detection

  • Immunoprecipitation: For protein interaction studies

• Epitope information:

  • Antibodies typically raised against full-length recombinant protein

  • Region-specific antibodies may be available for detecting specific domains

• Detection optimization:

  • For Western blotting: 1:1000-1:5000 dilution typically recommended

  • Secondary antibody: Anti-rabbit IgG conjugated with HRP or fluorescent tags

  • Blocking with 5% non-fat milk or BSA in TBST

How can I measure the enzymatic activity of SPAC8F11.08c?

As SPAC8F11.08c functions as an esterase/lipase, several approaches can be used to measure its enzymatic activity:

• Fluorogenic substrate assays:

  • 4-Methylumbelliferyl (4-MU) based substrates with varying acyl chain lengths

  • p-Nitrophenyl ester substrates for colorimetric detection

  • Optimal reaction conditions: pH 7.5-8.0, 30-37°C, with appropriate detergents

• Radiometric assays:

  • 14C or 3H-labeled lipid substrates for high sensitivity

  • Useful for characterizing substrate specificity toward different lipids

• High-throughput screening methods:

  • 96-well plate-based fluorescence assays

  • Useful for inhibitor screening or comparing mutant variants

• Physiological substrates:

  • Phospholipid vesicles mimicking FSM composition

  • Lipidomic analysis to identify specific lipid targets

What are common challenges when working with recombinant SPAC8F11.08c?

Researchers often encounter several challenges when working with recombinant SPAC8F11.08c:

• Protein solubility issues:

  • Membrane-associated properties can lead to inclusion body formation

  • Solution: Use of solubility tags (MBP, SUMO, Thioredoxin), lower induction temperatures, or detergents in purification buffers

• Low expression levels:

  • Potential toxicity in heterologous systems

  • Solution: Use tightly regulated promoters, lower expression temperatures, or codon optimization for the expression host

• Protein instability:

  • Susceptibility to proteolysis

  • Solution: Add protease inhibitors during purification, optimize buffer conditions, or identify stable core domains

• Enzymatic activity loss:

  • Activity sensitivity to freeze-thaw cycles

  • Solution: Store in glycerol at -20°C, prepare small working aliquots, avoid repeated freeze-thaw cycles

• Specificity in functional assays:

  • Overlapping activities with other lipases

  • Solution: Use specific inhibitors, carefully design control experiments, or employ genetic approaches

How can researchers address contradictory results in SPAC8F11.08c studies?

When encountering contradictory results regarding SPAC8F11.08c function:

• Genetic background considerations:

  • Different S. pombe strains may show varying phenotypes

  • Solution: Always report complete strain information and construct isogenic controls

• Experimental condition variations:

  • Sporulation efficiency depends on media and environmental conditions

  • Solution: Standardize growth and sporulation conditions across experiments

• Partial redundancy:

  • Other lipases may partially compensate for SPAC8F11.08c deletion

  • Solution: Consider double or triple mutant analyses with related lipases

• Technical considerations:

  • FSM visualization techniques vary in sensitivity

  • Solution: Employ multiple complementary methods to confirm phenotypes

• Data interpretation challenges:

  • FSM breakdown phenotypes can be subtle or variable

  • Solution: Use quantitative scoring systems and appropriate statistical analysis

What are promising research directions for further characterizing SPAC8F11.08c?

Several promising research directions could advance our understanding of SPAC8F11.08c:

• Structural studies:

  • X-ray crystallography or cryo-EM to determine protein structure

  • Structure-function analysis of catalytic domains and substrate binding sites

• Substrate specificity profiling:

  • Comprehensive lipid substrate screening

  • Identification of specific phospholipids targeted during FSM breakdown

• Regulatory mechanisms:

  • Detailed characterization of meu5-dependent regulation

  • Investigation of post-translational modifications affecting activity

• Interaction studies:

  • Identification of protein interaction partners using BioID or proximity labeling

  • Co-immunoprecipitation with other proteins involved in FSM dynamics

• Evolutionary analysis:

  • Comparative genomics with related proteins in other fungal species

  • Functional conservation study in diverse organisms

What new technologies could advance SPAC8F11.08c research?

Emerging technologies that could significantly advance SPAC8F11.08c research include:

• CRISPR-based approaches:

  • CRISPRi for temporal control of gene expression

  • Base editing for introducing point mutations in endogenous loci

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed FSM visualization

  • Live-cell imaging with novel fluorescent probes for lipid dynamics

• Single-cell analyses:

  • Single-cell RNA-seq to capture expression heterogeneity during sporulation

  • Single-cell proteomics for protein-level analysis

• Proteomics advances:

  • Quantitative proteomics using isobaric labeling approaches like iTRAQ

  • Targeted proteomics for studying specific post-translational modifications

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and lipidomics

  • Mathematical modeling of FSM breakdown dynamics

How does SPAC8F11.08c compare to similar proteins in other organisms?

Comparative analysis of SPAC8F11.08c with related proteins reveals important evolutionary insights:

This comparative analysis suggests that while the core lipase function is conserved, SPAC8F11.08c has evolved specialized roles in spore formation specific to fission yeast biology .

How does SPAC8F11.08c function within the broader context of lipid metabolism in S. pombe?

SPAC8F11.08c functions within a complex network of lipid metabolism in S. pombe:

• Integration with other lipid-modifying enzymes:

  • Works in coordination with SPCC1020.13c (phospholipase) in membrane remodeling

  • Potential functional overlap with other esterases (SPBC16A3.10, lcf1+)

• Relationship to membrane dynamics:

  • Role in specific membrane remodeling during developmental transitions

  • Potential involvement in lipid rafts or specialized membrane domains

• Connection to autophagy:

  • Functional relationship with Atg8 suggests connection to autophagic processes

  • May contribute to selective membrane degradation pathways

• Metabolic context:

  • May contribute to fatty acid recycling during sporulation

  • Potential role in energy homeostasis during nutrient limitation