Recombinant Schizosaccharomyces pombe Probable phospholipase C1020.13c (SPCC1020.13c, SPCC14G10.05), partial

What is the functional role of phospholipase C1020.13c in S. pombe?

Phospholipase C1020.13c (SPCC1020.13c, SPCC14G10.05) in S. pombe is involved in lipid-mediated signal transduction and membrane dynamics. While specific research on C1020.13c is limited, studies on phospholipases in S. pombe have demonstrated their role in hydrolyzing phospholipids, particularly phosphatidylcholine, to generate second messengers involved in cellular processes. Phospholipases in S. pombe are involved in cytoskeletal reorganization, secretion, and membrane trafficking similar to their counterparts in other eukaryotic cells . When working with this enzyme, researchers should be aware that its activity may increase during specific cellular events such as mating and sporulation, as observed with related phospholipases in this organism .

How do I express recombinant S. pombe phospholipase in a laboratory setting?

For successful expression of recombinant S. pombe phospholipase, utilize a genetic transformation system based on a dominant marker with targets for multiple integration of an expression cassette into the genome. The expression system should be designed considering that S. pombe has been used successfully for heterologous protein expression due to its eukaryotic post-translational modification capabilities . When expressing phospholipases specifically, enzyme activity can be determined spectrophotometrically at 412 nm using crude extracts of the transgenic strain compared against the plasmid-free strain. Typical expression systems have yielded enzyme activities with Vmax values of approximately 175.7 ± 12 nmol/min/mg for recombinant proteins in S. pombe, significantly higher than the baseline activity in non-transgenic strains (2.7 ± 0.02 nmol/min/mg) .

What are the key structural domains in S. pombe phospholipases?

S. pombe phospholipases contain several conserved domains critical for their function. Based on studies of related phospholipases, these enzymes typically feature:

• Four core catalytic domains (I-IV) containing regions required for activity (PLDc)

• Catalytic domains II and IV with a highly conserved HxK(x)4D (HKD) motif essential for function

• N-terminal lipid-binding motifs including PX (Phox) and PH (Pleckstrin homology) domains

• A polybasic motif located between catalytic domains II and III

The HKD motif is particularly important, as mutational studies have demonstrated that altering the lysine residue within this motif can completely eliminate enzyme activity. In S. pombe phospholipases, the polybasic domain often contains a glycine residue instead of the proline found in most other species, which may contribute to unique regulatory properties .

How can I measure phospholipase C1020.13c activity in vitro?

For accurate measurement of phospholipase C1020.13c activity in vitro, employ a fluorescence-based assay using fluorescently labeled phospholipid analogs. This methodology allows for sensitive detection of enzymatic activity:

• Prepare whole-cell extracts from S. pombe cultures expressing the recombinant phospholipase

• Incubate extracts with fluorescently labeled phosphatidylcholine analog as substrate

• Separate reaction products using thin-layer chromatography

• Visualize and quantify hydrolysis products with a Fluorimager

The transphosphatidylation reaction, characteristic of phospholipases, can be assessed by adding different alcohols to the reaction mixture. The production of fluorescently labeled phosphatidylalcohol provides confirmation of phospholipase activity. When testing activity under various conditions, appropriate controls should include samples from both mating types (h+ and h-) of S. pombe, as both have demonstrated phospholipase activity in previous studies .

What are the optimal conditions for kinetic analysis of S. pombe phospholipases?

For optimal kinetic analysis of S. pombe phospholipases, use spectrophotometric assays at 412 nm with varying substrate concentrations. Plot the activities using Lineweaver-Burk plot models to determine key kinetic parameters. Based on studies with related enzymes in transgenic S. pombe strains, expect Km values in the range of 6.45 ± 2 mM and Vmax values around 175.7 ± 12 nmol/min/mg, compared to wild-type values of approximately 0.33 ± 0.05 mM and 2.7 ± 0.02 nmol/min/mg respectively .

When investigating regulatory factors, note that unlike some related enzymes, S. pombe phospholipases may show distinct responses to potential modulators. For example, phospholipase activity in S. pombe has been shown to be unaffected by phosphatidylinositol 4,5-bisphosphate (PIP2) but slightly stimulated by oleate . This differs from phospholipases in other yeast species, highlighting the importance of species-specific characterization.

What verification methods confirm successful cloning and expression of recombinant phospholipase C1020.13c?

To verify successful cloning and expression of recombinant phospholipase C1020.13c in S. pombe, employ a multi-faceted approach:

• Molecular verification:

  • PCR amplification with gene-specific primers

  • Restriction enzyme digestion pattern analysis

  • DNA sequencing to confirm correct gene insertion

• Expression verification:

  • Western blotting with antibodies specific to the phospholipase or epitope tags

  • Activity assays comparing transformed vs. non-transformed strains

  • Spectrophotometric measurement of enzyme activity

• Structural verification:

  • Infrared spectroscopy

  • NMR spectroscopy (both 1H and 13C)

The enzyme activity assays should demonstrate significantly higher activity in transformed strains compared to controls. Previous studies with recombinant proteins in S. pombe have shown approximately 65-fold increase in enzyme activity compared to wild-type strains .

How does the regulation of phospholipase C1020.13c differ from related enzymes in other yeast species?

The regulation of phospholipase activity in S. pombe exhibits notable differences from related enzymes in other yeast species. A significant distinction is its response to phosphatidylinositol regulators. While phospholipases in most organisms, including Saccharomyces cerevisiae and Candida albicans, are typically stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2), the S. pombe phospholipase activity remains unaffected by PIP2 .

This regulatory difference can be attributed to structural variations in the polybasic domain. Specifically, S. pombe phospholipases contain a glycine residue in place of a highly conserved proline found in over 300 other phospholipase sequences. This proline-to-glycine substitution appears unique to Schizosaccharomyces species and likely contributes to the distinctive regulatory properties .

A comparative analysis of regulatory responses shows:

These differences highlight the importance of species-specific characterization despite high sequence homology between the enzymes .

What experimental approaches can resolve contradictory findings regarding phospholipase substrate specificity?

To resolve contradictory findings regarding phospholipase substrate specificity in S. pombe, implement a multi-method approach:

• Comprehensive substrate panel testing:

  • Examine activity against various phospholipid substrates including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine

  • Test both natural and synthetic substrates with varying fatty acid compositions

  • Include fluorescently labeled analogs for enhanced detection sensitivity

• Transphosphatidylation assays:

  • Compare transphosphatidylation efficiency with straight-chain and branched-chain alcohols

  • This distinctive property of S. pombe phospholipases (ability to use branched-chain alcohols) can help differentiate from other phospholipases

• Domain-specific mutagenesis:

  • Generate targeted mutations in key domains, particularly the HKD motif and the polybasic region

  • Assess how these mutations affect substrate specificity

  • Compare results with similar mutations in related enzymes from other species

• Structural analysis:

  • Use X-ray crystallography or cryo-EM to determine the three-dimensional structure of the active site

  • Perform molecular docking simulations with various substrates

When analyzing contradictory data, consider that enzyme activity may vary significantly based on cell cycle stage and physiological conditions, as phospholipase activity in S. pombe has been shown to increase during mating and sporulation .

How can I design experiments to investigate the role of phospholipase C1020.13c in S. pombe cell wall integrity?

To investigate the role of phospholipase C1020.13c in S. pombe cell wall integrity, design experiments that combine genetic manipulation with cell wall stress assays:

• Gene deletion/overexpression approach:

  • Generate phospholipase C1020.13c deletion strains using homologous recombination

  • Create overexpression strains using inducible promoters

  • Develop point mutation variants targeting key catalytic residues to create enzymatically inactive forms

• Cell wall stress assays:

  • Expose strains to cell wall stressors such as micafungin, calcofluor white, and Congo red

  • Test for synergistic effects with other compounds known to affect cell wall integrity, such as FK506, which has demonstrated synergistic effects with micafungin in wild-type cells

  • Monitor growth inhibition patterns under various stress conditions

• Molecular phenotyping:

  • Perform transcriptomic analysis to identify genes differentially expressed in response to phospholipase manipulation

  • Analyze the composition of cell wall components in wild-type vs. mutant strains

  • Conduct phospholipidomic profiling to determine changes in membrane lipid composition

• Microscopy and cell biology:

  • Use fluorescent markers to track cell wall synthesis and integrity

  • Employ electron microscopy to examine ultrastructural changes in cell wall architecture

When designing these experiments, include appropriate controls and ensure replication across different genetic backgrounds. Consider that phospholipase activity may interact with other signaling pathways involved in cell wall integrity, requiring a systems biology approach for comprehensive understanding .

Why might expression levels of recombinant phospholipase C1020.13c vary between experiments?

Variation in expression levels of recombinant phospholipase C1020.13c between experiments can stem from multiple factors:

• Integration site variability:

  • The genomic location of integration can significantly impact expression levels

  • Multiple integration sites may yield higher expression but potentially greater variability

  • Solution: Use targeted integration approaches to ensure consistent genomic positioning

• Culture condition inconsistencies:

  • Growth phase differences at harvest time can alter expression levels

  • Media composition variations can affect protein expression

  • Temperature fluctuations can impact protein folding and stability

  • Solution: Standardize culture conditions with precise timing and monitoring

• Protein stability issues:

  • Phospholipases may exhibit variable stability in different buffer conditions

  • Post-translational modifications can affect enzyme half-life

  • Solution: Include protease inhibitors in extraction buffers and optimize storage conditions

• Assay variability:

  • Spectrophotometric measurements may vary based on sample preparation

  • Substrate quality between batches can affect activity measurements

  • Solution: Include internal standards and perform technical replicates

To minimize these variations, establish standardized protocols with clearly defined parameters at each step from transformation to protein extraction and activity measurements. When comparing expression levels between experiments, calculate relative values against consistent controls rather than absolute measurements .

What are the most common pitfalls in analyzing phospholipase activity and how can they be avoided?

Common pitfalls in analyzing phospholipase activity and their solutions include:

• Substrate accessibility issues:

  • Pitfall: Poor substrate solubility or micelle formation can limit enzyme access

  • Solution: Optimize substrate preparation using appropriate detergents or carrier lipids; ensure consistent substrate presentation across experiments

• Interfering enzymatic activities:

  • Pitfall: Crude extracts may contain multiple phospholipases or other lipid-modifying enzymes

  • Solution: Include specific inhibitors for other lipid-modifying enzymes; consider partial purification steps; use specific assays that can distinguish between different phospholipase activities

• Inadequate controls:

  • Pitfall: Failing to account for non-enzymatic hydrolysis or background activity

  • Solution: Include heat-inactivated enzyme controls and samples from non-transformed strains as baseline measurements

• Misinterpretation of kinetic data:

  • Pitfall: Assuming Michaelis-Menten kinetics without verification

  • Solution: Test multiple kinetic models and substrate concentration ranges; be aware that phospholipases often exhibit complex kinetic behaviors including substrate inhibition

• Ignoring physiological context:

  • Pitfall: Focusing solely on in vitro activity without considering cellular context

  • Solution: Correlate in vitro measurements with physiological responses such as changes during cell cycle or response to stressors

When working specifically with S. pombe phospholipases, remember that unlike some other yeast phospholipases, they may be unresponsive to PIP2 while showing stimulation by oleate . This distinct regulatory profile should be considered when designing control experiments and interpreting results.

How can I optimize transphosphatidylation reactions for studying S. pombe phospholipase C1020.13c?

To optimize transphosphatidylation reactions for studying S. pombe phospholipase C1020.13c:

• Alcohol selection and concentration:

  • Test a range of primary alcohols (methanol, ethanol, 1-butanol) at concentrations between 1-5%

  • Include branched-chain alcohols (isopropanol, 2-butanol) which S. pombe phospholipases can uniquely utilize

  • Determine optimal alcohol concentration that maximizes transphosphatidylation while minimizing enzyme inhibition

• Reaction conditions optimization:

  • Buffer composition: Test buffers with pH range 6.0-8.0

  • Ionic strength: Vary NaCl concentration from 50-200 mM

  • Temperature: Typically 30°C is optimal for S. pombe enzymes, but test range from 25-37°C

  • Time course: Monitor reaction progress at intervals to determine optimal incubation time

• Product detection and quantification:

  • Use thin-layer chromatography with appropriate standards

  • For higher sensitivity, employ fluorescently labeled phosphatidylcholine

  • Visualize products with a Fluorimager for best detection limits

• Controls and validation:

  • Include samples without alcohol to measure hydrolysis activity

  • Run parallel reactions with well-characterized phospholipases as positive controls

  • Confirm product identity using mass spectrometry when possible

The unique ability of S. pombe phospholipases to use branched-chain alcohols in transphosphatidylation reactions provides an excellent tool for distinguishing their activity from other phospholipases and can be leveraged for more specific assays .

What emerging technologies could advance our understanding of phospholipase C1020.13c function?

Several emerging technologies hold promise for advancing our understanding of phospholipase C1020.13c function in S. pombe:

• CRISPR-Cas9 gene editing:

  • Create precise mutations in key domains to study structure-function relationships

  • Generate conditional knockout systems to study essential functions

  • Implement CRISPRi/CRISPRa for tunable gene expression modulation

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize enzyme localization at nanoscale

  • FRET-based biosensors to monitor phospholipase activity in real-time in living cells

  • Correlative light and electron microscopy to link enzyme localization with ultrastructural features

• Single-cell technologies:

  • Single-cell transcriptomics to identify cell-to-cell variability in phospholipase expression

  • Single-cell proteomics to study post-translational modifications

  • Microfluidic approaches to analyze individual cell responses to stressors

• Structural biology advancements:

  • Cryo-EM to determine high-resolution structures of phospholipase-substrate complexes

  • Hydrogen-deuterium exchange mass spectrometry to study protein dynamics

  • AlphaFold2 and related AI tools to predict structural changes caused by mutations

• Systems biology approaches:

  • Multi-omics integration to place phospholipase function in broader cellular context

  • Mathematical modeling of lipid metabolism networks

  • Synthetic biology approaches to build minimal systems for studying phospholipase function

These technologies can help address key questions about phospholipase function, particularly regarding its regulation by factors such as oleate and its independence from PIP2 regulation, which distinguishes it from phospholipases in other yeast species .

How might phospholipase C1020.13c interact with other signaling pathways in S. pombe?

Phospholipase C1020.13c likely interacts with multiple signaling pathways in S. pombe through its enzymatic products and protein-protein interactions:

• Cell wall integrity pathway:

  • Phospholipid-derived second messengers may regulate cell wall synthesis enzymes

  • Potential interaction with the FK506-sensitive pathway that synergizes with cell wall-targeting compounds

  • Hypothesized crosstalk with MAP kinase cascades that respond to cell wall stress

• Mating and sporulation pathways:

  • Increased phospholipase activity during mating and sporulation suggests functional relevance

  • Potential regulation of membrane fusion events during conjugation

  • Possible roles in spore wall formation through lipid metabolism modulation

• Stress response networks:

  • Phosphatidic acid production may contribute to membrane remodeling during stress

  • Integration with TOR signaling through lipid-derived second messengers

  • Potential regulation by stress-activated protein kinases

• Membrane trafficking pathways:

  • Regulation of vesicle formation and fusion events essential for polarized growth

  • Influence on lipid raft composition affecting protein sorting and signaling

  • Potential impact on secretory pathways through modification of membrane properties

A comprehensive understanding of these interactions will require systematic experimental approaches combining genetic manipulation with phenotypic and biochemical analyses. Investigation of genetic interactions through synthetic genetic arrays could reveal functional connections with other signaling components. Additionally, phosphoproteomic analysis following phospholipase manipulation could identify downstream effectors within these pathways .

What are the implications of phospholipase C1020.13c research for broader understanding of eukaryotic cell biology?

Research on phospholipase C1020.13c in S. pombe has several important implications for our broader understanding of eukaryotic cell biology:

• Evolutionary conservation and divergence of lipid signaling:

  • The unique regulatory features of S. pombe phospholipases, such as PIP2 independence, provide insights into how lipid signaling pathways have evolved across eukaryotes

  • Comparative studies between S. pombe, S. cerevisiae, and C. albicans phospholipases reveal how highly conserved proteins can acquire different regulatory mechanisms

• Model for specialized membrane dynamics:

  • S. pombe's rod-shaped morphology requires distinct membrane remodeling processes

  • Understanding phospholipase's role in this context illuminates fundamental principles of membrane organization in polarized cells

• Insights into phospholipid metabolism across domains of life:

  • The ability of S. pombe phospholipases to perform transphosphatidylation with branched-chain alcohols suggests unique catalytic properties

  • This distinctive feature may reveal previously unrecognized catalytic mechanisms relevant to phospholipases across species

• Translational potential:

  • Mechanisms governing phospholipase regulation in S. pombe may inform development of specific inhibitors with potential applications in medicine and biotechnology

  • The unique glycine substitution in the polybasic domain could serve as a target for species-specific enzyme modulation

• Methodological advances:

  • Techniques developed for studying phospholipases in S. pombe can be adapted for other challenging enzymes

  • The model system offers opportunities to study membrane-associated enzymes in a genetically tractable organism

By leveraging S. pombe as a model system, researchers can address fundamental questions about phospholipid metabolism while developing approaches applicable across eukaryotic biology. The distinctive features of S. pombe phospholipases serve as valuable comparison points for understanding both conserved and divergent aspects of lipid signaling .