Recombinant Schizosaccharomyces pombe Phosphatidylinositol N-acetylglucosaminyltransferase subunit gpi15 (gpi15)

What is gpi15 and what is its functional role in Schizosaccharomyces pombe?

The gpi15 protein in Schizosaccharomyces pombe is a critical subunit of the Phosphatidylinositol N-acetylglucosaminyltransferase (GPI-GnT) complex, which catalyzes the first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis. This process is essential for attaching specific proteins to the cell membrane.

The functional characterization of gpi15 has been established through homology studies with Saccharomyces cerevisiae, where it plays a crucial role in the formation of N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). The S. pombe gpi15+ gene was cloned by complementation of the S. cerevisiae gpi1 mutant, which exhibits temperature-sensitive defects in growth and GPI membrane anchoring of proteins .

Experimental disruption of gpi1+ in S. pombe is lethal, with haploid Δgpi1+::his7+ spores being able to germinate but proceeding through no more than three rounds of cell division. Many cells cease growth as binucleate, septate cells with thickened septa, suggesting that GPI synthesis is not only essential for viability but also specifically required for proper completion of cytokinesis .

What expression systems and conditions are optimal for producing recombinant S. pombe gpi15?

For optimal expression of recombinant S. pombe gpi15:

• Host System: E. coli is the preferred expression system for recombinant production, as demonstrated in multiple studies .

• Expression Construct: The full-length coding sequence (1-160 amino acids) should be cloned into an expression vector with an N-terminal His tag to facilitate purification .

• Culture Conditions: After transformation, cultures should be grown in standard E. coli media (such as LB) with appropriate antibiotics based on the selection marker in the expression vector.

• Storage Considerations:

  • Store the lyophilized protein at -20°C/-80°C upon receipt

  • Aliquot to prevent repeated freeze-thaw cycles

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) for long-term storage

  • Working aliquots can be stored at 4°C for up to one week

The expression of the native protein in S. pombe can be studied using the pat1-114 temperature-sensitive mutation system, which allows for the induction of synchronous meiosis and provides a controlled environment for studying protein expression and function during the cell cycle .

How does S. pombe gpi15 compare to its homologs in other species?

S. pombe gpi15 shares functional homology with its counterparts in other species, particularly:

• Saccharomyces cerevisiae: The S. pombe gpi1+ gene encodes a protein with 29% identity to amino acids 87-609 of the S. cerevisiae Gpi1 protein. Despite limited sequence homology, it functions as a true ortholog, as demonstrated by its ability to restore [³H]inositol-labelling of protein and in vitro GlcNAc-PI synthetic activity to both S. cerevisiae gpi1 and gpi1::URA3 cells .

• Candida albicans: Studies have shown that CaGpi15 is functionally homologous to S. cerevisiae Gpi15. CaGPI15 acts as a master activator of CaGPI2 and CaGPI19 in the GPI-GnT complex. Mutations in CaGPI15 result in azole sensitivity and reduced filamentation capacity, highlighting its regulatory role in the GPI biosynthetic pathway .

• Human homolog: S. pombe gpi15 is homologous to the human PIGH protein (Phosphatidylinositol N-acetylglucosaminyltransferase subunit H), which similarly participates in the first step of GPI anchor biosynthesis .

This cross-species functional conservation underscores the evolutionary importance of the GPI biosynthetic pathway and positions S. pombe as a valuable model organism for studying processes relevant to human cell biology.

What methodologies are most effective for studying the function of gpi15 in S. pombe?

For investigating gpi15 function in S. pombe, several complementary approaches are recommended:

• Conditional expression systems: Since gpi15 is essential, use of temperature-sensitive mutants or tetracycline-regulatable promoters allows for controlled depletion of the protein while observing cellular consequences.

• Biochemical assays: Western blot analysis and enzyme assays can be used to detect gpi15 expression and activity, similar to methods used for analyzing PHB synthase expression in recombinant S. pombe strains .

• Fluorescence microscopy: GFP-tagging of gpi15 can reveal its subcellular localization during different stages of the cell cycle and under various stress conditions.

• Co-immunoprecipitation: To identify interaction partners within the GPI-GnT complex and potentially other cellular components.

These methodologies have been successfully applied to study other aspects of S. pombe biology and can be adapted for investigating gpi15 specifically.

How can researchers design experiments to investigate gpi15's role in GPI anchor assembly?

To investigate gpi15's specific role in GPI anchor assembly, researchers should consider the following experimental approaches:

• In vitro GPI biosynthesis assays: Similar to those used for S. cerevisiae, measuring the formation of GlcNAc-PI. Using membrane preparations from wild-type and gpi15-depleted cells, researchers can compare the efficiency of the first step in GPI anchor assembly .

• Complementation studies: Express mutant versions of gpi15 in gpi15-deficient backgrounds to identify critical residues for function. This approach was successfully used to demonstrate that S. pombe gpi1+ can complement S. cerevisiae gpi1 mutants .

• Pulse-chase experiments: Use [³H]inositol labeling to track GPI biosynthesis and transfer to proteins in cells with varying levels of gpi15 expression.

• Structure-function analysis: Generate a series of truncated or point-mutated gpi15 constructs to identify domains critical for:

  • Interaction with other GPI-GnT subunits

  • Binding to phosphatidylinositol

  • Transfer of N-acetylglucosamine

• Quantitative analysis of GPI-anchored proteins: Use proteomics approaches to identify and quantify changes in the GPI-anchored proteome in response to altered gpi15 levels.

• Genetic interaction screens: Identify genetic enhancers or suppressors of gpi15 conditional mutants to place gpi15 in broader cellular pathways.

These experimental designs would provide complementary information about the specific biochemical and cellular functions of gpi15 in the GPI anchor assembly process.

What techniques can be used to study protein-protein interactions of gpi15 within the GPI-GnT complex?

To investigate protein-protein interactions involving gpi15 within the GPI-GnT complex, researchers can employ these techniques:

• Yeast Two-Hybrid (Y2H) screening: Though challenging for membrane proteins, modified Y2H systems can be used to screen for protein interactions involving the soluble domains of gpi15.

• Co-immunoprecipitation (Co-IP): Using epitope-tagged gpi15, researchers can identify interacting partners through pull-down experiments followed by mass spectrometry:

  • Express His-tagged gpi15 in S. pombe

  • Prepare cell lysates under conditions that preserve protein complexes

  • Perform affinity purification using Ni-NTA resin

  • Analyze co-precipitating proteins by mass spectrometry

• Bimolecular Fluorescence Complementation (BiFC): By fusing gpi15 and potential interacting partners to complementary fragments of a fluorescent protein, interactions can be visualized in living cells.

• Proximity-dependent biotin identification (BioID): Fusion of gpi15 to a biotin ligase allows for labeling of proximal proteins, which can then be purified and identified.

• Förster Resonance Energy Transfer (FRET): Using fluorescently tagged proteins to detect nanometer-scale interactions between gpi15 and other GPI-GnT components.

• Cross-linking Mass Spectrometry (XL-MS): Chemical cross-linking followed by mass spectrometry can capture transient interactions and provide structural information about the complex.

These techniques can reveal not only the composition of the GPI-GnT complex in S. pombe but also the specific domains of gpi15 involved in protein-protein interactions and the dynamics of these interactions during different cellular states.

How does gpi15 deficiency specifically affect cell division and cytokinesis in S. pombe?

The effects of gpi15 deficiency on cell division and cytokinesis in S. pombe can be studied through:

• Time-lapse microscopy: Tracking cell cycle progression in gpi15-depleted cells allows for precise identification of the stage at which division fails. Previous studies show that Δgpi1+::his7+ spores cease growth after no more than three rounds of cell division, often as binucleate cells with thickened septa .

• Septum formation analysis: Calcofluor white staining or electron microscopy can reveal detailed structural abnormalities in the septum when gpi15 is depleted. The observation of thickened septa suggests a specific role for GPI-anchored proteins in septum maturation or cell separation .

• Cell wall composition analysis: Since many GPI-anchored proteins are ultimately incorporated into the cell wall, biochemical analysis of cell wall composition in gpi15-deficient cells can identify specific defects.

• Examination of GPI-anchored septum-specific proteins: Identify which GPI-anchored proteins involved in septation are mislocalized or dysfunctional when gpi15 is compromised.

• Genetic interaction studies: Combining conditional gpi15 alleles with mutations in known septation genes can reveal functional relationships within the septation pathway.

Methodologically, these approaches should be implemented using temperature-sensitive mutants or inducible depletion systems, as complete loss of gpi15 is lethal. The S. pombe pat1-114 system offers controlled conditions for analyzing these phenotypes in a synchronous population .

How can contradictory data regarding gpi15 function be reconciled in experimental design?

When facing contradictory data about gpi15 function, researchers should implement the following methodological approaches:

• Standardize experimental conditions: Many contradictions arise from variations in:

  • Strain backgrounds

  • Growth conditions

  • Protein expression levels

  • Assay methodologies

  Researchers should establish standardized protocols similar to those used for inducing synchronous meiosis in S. pombe, where precise media compositions and temperature shifts are critical .

• Use multiple complementary techniques: For example, when studying protein-protein interactions, combine biochemical approaches (Co-IP, in vitro binding assays) with in vivo techniques (FRET, BiFC) to validate findings.

• Control for protein expression levels: Use quantitative Western blotting to ensure that phenotypes attributed to specific mutations aren't simply due to altered protein expression levels.

• Consider genetic background effects: Test phenotypes in multiple strain backgrounds or create isogenic strains that differ only in the allele of interest.

• Utilize domain-specific mutations: Instead of complete gene knockouts, which can lead to pleiotropic effects, use targeted mutations in specific functional domains to dissect protein function.

• Incorporate time-course analyses: Single time-point measurements may miss dynamic aspects of gpi15 function, particularly during cell cycle progression.

• Cross-validate results across species: Compare findings in S. pombe with those in S. cerevisiae and C. albicans to identify conserved versus species-specific aspects of gpi15 function .

By implementing these methodological controls, researchers can better reconcile contradictory data and develop a more comprehensive understanding of gpi15 function.

What methodologies are appropriate for analyzing gpi15 function during meiosis in S. pombe?

For studying gpi15 function during meiosis in S. pombe, researchers should consider these methodological approaches:

• Chromatin immunoprecipitation (ChIP): To determine if gpi15 or related factors associate with specific DNA regions during meiosis.

• DNA recombination analysis: While S. pombe is an excellent model for studying meiotic recombination , the specific connection to gpi15 would need to be established through genetic approaches such as:

  • Quantifying recombination frequencies in gpi15 mutant backgrounds

  • Analyzing formation and resolution of recombination intermediates

  • Examining crossover/non-crossover ratios

• Protein expression profiling: Western blot analysis at different meiotic time points can reveal dynamic changes in gpi15 expression and post-translational modifications throughout meiosis.

• Fluorescence microscopy: GFP-tagged gpi15 can be visualized during the characteristic "horsetail" movement of chromosomes in meiotic prophase, a stage unique to S. pombe .

These approaches leverage S. pombe's advantages as a model organism for meiotic studies while focusing specifically on gpi15's potential functions during this specialized cell division process.

How can researchers design genetic screens to identify synthetic interactions with gpi15?

To identify genetic interactions with gpi15, researchers can employ these methodological approaches:

• Synthetic Genetic Array (SGA) analysis with a conditional gpi15 allele:

  • Create a query strain with a temperature-sensitive or auxin-inducible degron-tagged gpi15

  • Cross this strain systematically with the S. pombe deletion library

  • Identify combinations that show enhanced growth defects (synthetic sickness) or lethality

• Transposon mutagenesis screens:

  • Generate random insertions in a strain with a conditional gpi15 allele

  • Screen for colonies with growth defects under semi-permissive conditions

  • Identify insertion sites that enhance gpi15 phenotypes

• Multicopy suppressor screens:

  • Transform a conditional gpi15 mutant with an S. pombe genomic library

  • Select for clones that rescue growth defects under restrictive conditions

  • Identify genes that, when overexpressed, compensate for gpi15 dysfunction

• Chemical-genetic screens:

  • Expose conditional gpi15 mutants to a library of compounds

  • Identify chemicals that specifically enhance gpi15 phenotypes

  • Use these compounds as tools to identify pathways connected to gpi15 function

• CRISPR interference (CRISPRi) screens:

  • Create a strain with dCas9-based repression of gpi15

  • Introduce a genome-wide sgRNA library

  • Identify sgRNAs that modify the gpi15 repression phenotype

For all these approaches, special attention should be paid to genes involved in GPI biosynthesis, cell wall integrity, and cytokinesis pathways, as these are likely to interact with gpi15 based on its known functions .

What approaches can be used to investigate regulatory networks controlling gpi15 expression?

To study the regulatory networks controlling gpi15 expression, researchers should consider:

• Promoter analysis:

  • Identify transcription factor binding sites in the gpi15 promoter

  • Create reporter constructs with wild-type and mutated binding sites

  • Measure reporter expression under different conditions to identify regulatory elements

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Identify transcription factors that bind to the gpi15 promoter

  • Compare binding patterns under different growth conditions or stress responses

• RNA sequencing (RNA-seq):

  • Compare gpi15 expression levels across different conditions, developmental stages, or cell cycle phases

  • Identify co-regulated genes that may be part of the same regulatory network

• Epigenetic analysis:

  • Examine histone modifications at the gpi15 locus

  • Based on findings in Candida albicans, investigate H3 acetylation patterns at the gpi15 promoter, possibly mediated by Rtt109

• Cross-species analysis:

  • Compare regulatory mechanisms between S. pombe, S. cerevisiae, and C. albicans

  • In C. albicans, CaGPI15 has been shown to function as a master activator of CaGPI2 and CaGPI19, while also being reciprocally regulated by these genes

• Quantitative trait locus (QTL) mapping:

  • Use S. pombe natural variants to identify genetic loci that influence gpi15 expression levels

  • S. pombe exhibits genetic diversity that can be leveraged for such studies

These approaches would provide complementary data about the regulatory mechanisms controlling gpi15 expression and how they integrate with broader cellular processes.

How can the essentiality of gpi15 be experimentally addressed in S. pombe research?

To experimentally address the essentiality of gpi15 while still enabling its functional study, researchers can employ:

• Conditional expression systems:

  • Tetracycline-repressible promoters to control gpi15 expression levels

  • Temperature-sensitive alleles that maintain function at permissive temperatures

  • Auxin-inducible degron (AID) tags for rapid protein depletion

• Partial loss-of-function alleles:

  • Generate a library of point mutations or truncations

  • Screen for hypomorphic alleles that maintain viability but show specific defects

  • Use these alleles to study specific aspects of gpi15 function

• Domain-specific complementation:

  • Express individual domains of gpi15 in a conditional mutant background

  • Determine which domains are sufficient for specific aspects of gpi15 function

• Heterologous complementation:

  • Test whether gpi15 homologs from other species can complement S. pombe gpi15 deficiency

  • Previous studies have shown that S. pombe gpi1+ can complement S. cerevisiae gpi1 mutants

• Bypass suppressor screening:

  • Identify mutations that allow cells to survive complete loss of gpi15

  • These suppressors may reveal alternative pathways or compensatory mechanisms

• Mosaic analysis:

  • Create genetic mosaics where only some cells lack gpi15

  • Study cell-autonomous versus non-cell-autonomous effects of gpi15 deficiency

These approaches allow researchers to circumvent the lethality associated with complete gpi15 loss while still investigating its functions in cellular processes such as GPI anchor biosynthesis and cytokinesis.

What technical considerations are important when expressing and purifying recombinant S. pombe gpi15 for structural studies?

For successful expression and purification of recombinant S. pombe gpi15 for structural studies, researchers should consider:

• Expression system optimization:

  • While E. coli is commonly used , consider eukaryotic expression systems (e.g., insect cells) for proper folding

  • Optimize codon usage for the expression host

  • Test multiple affinity tags (His, GST, MBP) for improved solubility and yield

• Membrane protein-specific considerations:

  • Include detergents or amphipols to maintain solubility and native conformation

  • Try fusion partners known to enhance membrane protein solubility

  • Consider co-expression with interacting partners from the GPI-GnT complex

• Purification protocol:

  • Use affinity chromatography with His-tag binding to Ni-NTA resin as the initial step

  • Follow with size exclusion chromatography to separate monomeric protein from aggregates

  • For structural studies, achieve >90% purity as assessed by SDS-PAGE

• Storage and stability:

  • Store in Tris/PBS-based buffer with 6% trehalose, pH 8.0

  • Add 5-50% glycerol for long-term storage at -20°C/-80°C

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

• Protein quality assessment:

  • Verify protein identity by mass spectrometry

  • Assess structural integrity by circular dichroism or thermal shift assays

  • Check for aggregation by dynamic light scattering

• Reconstitution for structural studies:

  • For crystallization attempts, concentrate to 5-15 mg/mL

  • For cryo-electron microscopy, ensure sample homogeneity

  • For NMR studies, prepare isotopically labeled protein