Recombinant Schizosaccharomyces pombe Vacuolar protein sorting-associated protein 28 homolog (vps28)

What cellular functions does VPS28 perform in S. pombe?

VPS28 plays critical roles in cellular trafficking pathways as a component of the ESCRT-I complex. Its primary functions include:

• Participation in multivesicular body (MVB) formation

• Regulation of extracellular vesicle (EV) secretion

• Involvement in protein sorting mechanisms within the endosomal system

• Potential roles in cellular communication, particularly in neurons

Research indicates that VPS28 knockdown significantly impacts MVB formation, as evidenced by the altered size and distribution of MVB markers like HGS. Transmission electron microscopy analysis shows reduced numbers and density of MVBs in cells with VPS28 deficiency . These functions are conserved across species, with research in zebrafish demonstrating similar effects.

How do I design expression systems for recombinant S. pombe VPS28?

When designing expression systems for recombinant S. pombe VPS28, researchers should consider:

• Vector selection: Integrative vectors like pCAD1 or episomal vectors like pREP1 can be used, depending on expression stability requirements .

• Promoter choice: For high-level expression, strong promoters like nmt1 are recommended, while moderate expression may benefit from attenuated versions.

• Fusion tags: His-tags facilitate purification but consider N-terminal versus C-terminal placement based on structural predictions.

• Host strain selection: NCYC 2036 has proven effective for recombinant protein expression .

A typical cloning strategy involves:

• PCR amplification of the vps28 gene with flanking restriction sites

• Restriction digestion and ligation into appropriate vectors

• Transformation into cryocompetent S. pombe cells

• Selection and verification of transformants

For optimal expression, culture conditions should be carefully controlled, with special attention to media composition and induction timing.

What methodologies are most effective for investigating VPS28 function in membrane trafficking?

Advanced investigation of VPS28 function in membrane trafficking requires a multi-faceted approach:

• CRISPR/Cas9 mutagenesis: Generate specific mutations in the vps28 gene. This approach has been effectively used to create a truncated VPS28 protein (terminating at amino acid position 118) that lacks the conserved domain, resulting in significant phenotypic changes .

• Fluorescent protein tagging: Creating VPS28-GFP fusion constructs under native promoters allows for real-time visualization of protein localization and trafficking. This method has been successfully implemented in transgenic models like Tg(Vps28:eGFP) zebrafish .

• Electron microscopy analysis: Transmission electron microscopy provides high-resolution visualization of subcellular structures affected by VPS28 dysfunction, particularly multivesicular bodies. Quantitative analysis of MVB density and morphology serves as a critical readout of VPS28 functionality .

• Co-immunoprecipitation assays: To identify VPS28 interaction partners within the ESCRT-I complex and beyond.

• RNA interference: For temporal control of VPS28 depletion, allowing for assessment of acute versus chronic loss of function.

How should researchers address contradictory data when studying VPS28 function?

When faced with data that contradicts established hypotheses about VPS28 function, researchers should implement a systematic approach:

• Thorough data examination: Carefully analyze all results to identify specific discrepancies between expected and observed outcomes. This includes statistical analysis of outliers that may influence results .

• Validation with alternative methods: Confirm unexpected findings using different technical approaches. For example, if protein localization results from fluorescence microscopy contradict expectations, validate with subcellular fractionation or immunoelectron microscopy.

• Cross-species comparison: VPS28 is evolutionarily conserved, so comparing function across model organisms can provide context for unexpected results. Differences may represent species-specific adaptations rather than experimental errors.

• Re-evaluation of experimental conditions: Consider whether specific growth conditions, strain backgrounds, or experimental parameters might explain contradictory results.

• Hypothesis refinement: Develop new models that incorporate both the expected and unexpected data, potentially revealing more complex regulatory mechanisms.

When publishing contradictory findings, researchers should clearly document all methodological details and present both expected and observed results with appropriate statistical analysis .

What are the optimal purification strategies for recombinant S. pombe VPS28 protein?

Purification of recombinant S. pombe VPS28 requires careful optimization to maintain protein structure and function:

• Cell lysis conditions: For S. pombe, mechanical disruption using glass beads in appropriate buffer systems (typically containing protease inhibitors and reducing agents) yields optimal results.

• Affinity chromatography: His-tagged VPS28 can be purified using immobilized metal affinity chromatography (IMAC). Optimizing imidazole concentration in washing and elution buffers is critical to balance purity and yield .

• Secondary purification: Following IMAC, size exclusion chromatography further enhances purity and allows assessment of oligomeric state.

• Protein stability assessment: Monitor protein stability through thermal shift assays or limited proteolysis to identify optimal buffer conditions for downstream applications.

• Quality control: Final preparations should achieve >90% purity as assessed by SDS-PAGE and may be further validated by western blotting and mass spectrometry .

For structural studies, additional considerations include:

• Buffer optimization to prevent aggregation

• Removal of affinity tags if they interfere with function

• Assessment of protein homogeneity by dynamic light scattering

How can researchers effectively analyze the role of VPS28 in extracellular vesicle biogenesis and secretion?

Analysis of VPS28's role in extracellular vesicle (EV) biogenesis requires specialized methodologies:

• EV isolation: Implement differential ultracentrifugation, size exclusion chromatography, or polymer precipitation to isolate EVs from cell culture supernatants or biological fluids.

• EV characterization:

  • Nanoparticle tracking analysis for size distribution and concentration

  • Electron microscopy for morphological assessment

  • Western blotting for EV marker proteins (CD63, TSG101, Alix)

• Cargo analysis: Utilize proteomics and RNA sequencing to identify proteins and RNAs selectively packaged into EVs in the presence or absence of functional VPS28.

• Functional assays: Assess the biological activity of EVs derived from control versus VPS28-deficient cells on recipient cells.

Research has demonstrated that VPS28 loss-of-function dramatically decreases EV secretion by influencing MVB formation both in vitro and in vivo. Specifically, EVs secreted by neurons contain VEGF-A and participate in central nervous system vascularization, with VPS28 playing a critical role in this process .

This multifaceted approach allows for comprehensive assessment of VPS28's contribution to EV biology.

What controls should be included when investigating VPS28 function through gene knockout or knockdown?

When designing experiments to investigate VPS28 function through knockout or knockdown approaches, several critical controls must be included:

• Validation of knockout/knockdown efficiency:

  • Quantitative PCR to measure mRNA levels

  • Western blot analysis to confirm protein depletion

  • These validations should be performed at multiple timepoints when using inducible systems

• Rescue experiments:

  • Reintroduction of wild-type VPS28 to confirm phenotype specificity

  • Domain-specific mutants (e.g., truncated at amino acid 118) to identify functional regions

  • Species-specific rescue to test evolutionary conservation

• Off-target effect controls:

  • Multiple independent siRNA/shRNA sequences targeting different regions of VPS28

  • Non-targeting control siRNA/shRNA with similar chemical properties

  • For CRISPR/Cas9, multiple guide RNAs and off-target analysis

• Functional readouts:

  • MVB formation assessment using MVB markers like HGS

  • Quantification of EV secretion using nanoparticle tracking analysis

  • Cargo sorting efficiency for known VPS28-dependent proteins

Including these controls ensures that observed phenotypes are specifically attributable to VPS28 loss rather than experimental artifacts or off-target effects.

How can proteomics approaches be utilized to study VPS28-dependent cellular pathways in S. pombe?

Comparative proteomics offers powerful insights into VPS28-dependent cellular pathways in S. pombe:

• Sample preparation optimization:

  • Efficient protein extraction from S. pombe requires optimization of cell wall disruption methods

  • Subcellular fractionation to enrich for endosomal/vesicular compartments

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling for quantitative comparison

• Mass spectrometry approaches:

  • Shotgun proteomics for broad protein identification

  • Targeted proteomics for focused analysis of ESCRT pathway components

  • Phosphoproteomics to identify regulatory mechanisms

• Data analysis strategies:

  • Pathway enrichment analysis to identify affected cellular processes

  • Protein-protein interaction network construction

  • Temporal profiling to distinguish primary from secondary effects

Research has demonstrated that high-level protein secretion causes global changes in protein expression levels in S. pombe, and comparative proteome analysis has proven effective in identifying targets for improving protein production and secretion .

• Validation approaches:

  • Western blotting of key identified proteins

  • Functional assays based on proteomics insights

  • Genetic interaction studies of identified pathway components

Data from such studies should be deposited in public repositories like ProteomeXchange to facilitate further analysis by the research community .

How does S. pombe VPS28 function compare to orthologs in other model organisms?

VPS28 function shows both conservation and divergence across species:

• Structural conservation:

  • The core VPS28 domain (approximately amino acids 40-220 in S. pombe) is highly conserved across eukaryotes

  • N-terminal and C-terminal regions show greater sequence divergence

• Functional conservation:

  • ESCRT-I complex formation is conserved from yeast to humans

  • MVB biogenesis role appears universal

  • Protein sorting mechanisms show similar principles across species

• Species-specific adaptations:

  • In zebrafish, VPS28 shows neuron-specific expression patterns and functions in neurovascular communication through VEGF-A trafficking

  • In mammalian systems, VPS28 has additional interaction partners not present in yeast

• Experimental considerations:

  • Different model systems may require adjusted experimental approaches

  • Cross-species complementation can test functional conservation experimentally

Understanding these similarities and differences helps researchers select appropriate model systems and interpret results in an evolutionary context.

What are the implications of VPS28 research for understanding membrane trafficking disorders?

Research on VPS28 provides significant insights into membrane trafficking disorders:

• Neurological disorders:

  • VPS28's role in neuronal EV secretion suggests potential involvement in neurodegenerative diseases where protein aggregation is a feature

  • The demonstrated role in VEGF-A trafficking connects VPS28 dysfunction to potential vascular abnormalities in the central nervous system

• Cancer biology:

  • Altered EV composition and secretion are hallmarks of many cancers

  • VPS28's function in cargo sorting may affect tumor microenvironment communication

• Developmental disorders:

  • The zebrafish studies demonstrate that VPS28 mutation disrupts central nervous system vascularization, suggesting potential roles in developmental vascular disorders

  • Similar mechanisms may operate in mammalian development

• Therapeutic implications:

  • Understanding VPS28-dependent pathways may reveal new therapeutic targets

  • Potential for engineering EVs with specific cargo for therapeutic delivery

• Diagnostic applications:

  • VPS28 dysfunction biomarkers could be developed for associated disorders

  • EV profiling from patient samples may reveal disease-specific signatures

What emerging technologies could advance our understanding of VPS28 function?

Several cutting-edge technologies show promise for advancing VPS28 research:

• Cryo-electron microscopy:

  • High-resolution structural analysis of VPS28 within the ESCRT-I complex

  • Visualization of conformational changes during membrane deformation

• Live-cell super-resolution microscopy:

  • Tracking VPS28 dynamics during MVB formation with nanometer precision

  • Multi-color imaging to visualize interactions with other ESCRT components

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins to identify transient interaction partners

  • Spatial proteomics to map the VPS28 interaction network in different cellular compartments

• Organoid and tissue-specific models:

  • Investigation of VPS28 function in complex multicellular contexts

  • Tissue-specific knockout models to understand specialized functions

• Single-cell analysis techniques:

  • Single-cell proteomics and transcriptomics to capture heterogeneity in VPS28 function

  • Correlation of VPS28 expression with cellular phenotypes at single-cell resolution

These technologies will help address fundamental questions about VPS28 dynamics, interactions, and tissue-specific functions that current methods cannot fully resolve.

How can researchers effectively troubleshoot problems in VPS28 expression and purification?

Common challenges in VPS28 expression and purification can be addressed through systematic troubleshooting:

• Low expression levels:

  • Optimize codon usage for S. pombe

  • Test different promoters (nmt1, adh1) and induction conditions

  • Consider fusion partners known to enhance solubility (e.g., SUMO tag)

• Protein insolubility:

  • Modify lysis buffer conditions (salt concentration, pH, detergents)

  • Test expression at lower temperatures (16-25°C)

  • Consider co-expression with known interaction partners

• Degradation issues:

  • Include appropriate protease inhibitors in all buffers

  • Optimize purification speed to minimize exposure time

  • Consider N-terminal versus C-terminal tag placement

• Purification challenges:

  • Optimize imidazole concentrations in wash and elution buffers for His-tagged VPS28

  • Test multiple chromatography approaches (ion exchange, hydrophobic interaction)

  • Consider on-column refolding for difficult cases

• Protein activity assessment:

  • Develop functional assays to confirm biological activity

  • Structural analysis (circular dichroism, thermal shift) to confirm proper folding

Maintaining detailed laboratory records of conditions tested and outcomes observed will facilitate efficient problem-solving and protocol optimization.