Recombinant Schizosaccharomyces pombe Uncharacterized membrane protein C21B10.06c (SPBC21B10.06c)

What is the predicted function of SPBC21B10.06c (INP2) in Schizosaccharomyces pombe?

SPBC21B10.06c, also known as INP2, is characterized as a myosin binding vezatin family protein involved in peroxisome inheritance in Schizosaccharomyces pombe . The protein contains domains that are consistent with membrane association and protein-protein interactions that facilitate peroxisome transport during cell division. Experimental evidence suggests its role is conserved across fungal species, though specific functional mechanisms in S. pombe require further experimental validation through knockout studies and localization experiments.

How is INP2 classified within the cellular component ontology of S. pombe?

According to gene ontology classifications, INP2 (SPBC21B10.06c) is associated with multiple cellular components . While specific GO Component data is not comprehensively detailed in the available literature, research indicates its association with membrane structures and peroxisomes. Researchers should consider using fluorescent tagging approaches to definitively establish the subcellular localization pattern throughout the cell cycle, particularly during mitosis when peroxisome inheritance occurs.

What experimental evidence supports the annotation of INP2 as a peroxisome inheritance factor?

The annotation of INP2 as a peroxisome inheritance factor appears to be largely predictive based on sequence homology and domain architecture analysis rather than direct experimental evidence in S. pombe specifically. The protein contains characteristic domains of the vezatin family, which are known to interact with myosin motor proteins in other organisms . To validate this function experimentally, researchers should consider:

• Creating INP2 deletion strains and examining peroxisome distribution patterns

• Performing co-immunoprecipitation experiments to confirm myosin binding

• Conducting live-cell imaging with fluorescently tagged peroxisomes in wild-type versus Δinp2 strains

• Complementation studies with orthologs from related fungal species

What are the optimal conditions for expressing recombinant SPBC21B10.06c in S. pombe expression systems?

For optimal expression of recombinant SPBC21B10.06c in S. pombe, researchers should consider the following methodological approach:

• Vector selection: Use an expression vector containing the nmt1 promoter (no message in thiamine) which provides tight regulation and high-level expression when thiamine is removed from the medium.

• Tagging strategy: When adding epitope or fluorescent tags, consider both N and C-terminal fusions, as membrane proteins often have topological constraints that may interfere with proper folding or localization.

• Expression conditions:

  Parameter	Optimal Condition	Notes
  Culture temperature	30°C	Lower temperature (25°C) may improve folding
  Induction time	16-24 hours	After thiamine removal
  Media	EMM2	Minimal medium without thiamine
  Cell density	Mid-log phase	OD600 of 0.5-0.8

• Cell lysis approach: Use gentle detergent-based methods rather than mechanical disruption to preserve membrane protein structure and interactions.

• Validation: Confirm expression through Western blotting and proper localization via fluorescence microscopy.

This approach accounts for the membrane protein nature of INP2 and the established experimental design principles for S. pombe expression systems .

How can researchers effectively design experiments to study INP2 interactions with the cytoskeleton?

When designing experiments to study INP2 interactions with the cytoskeleton, particularly with myosin motor proteins, researchers should implement a multi-tiered experimental approach:

• Define clear variables: The independent variable should be the specific cytoskeletal component being examined (e.g., different myosin isoforms, actin structures), while the dependent variable should be a measurable aspect of INP2 function or localization .

• Develop specific, testable hypotheses about INP2-cytoskeleton interactions based on the known functions of vezatin family proteins.

• Implement an experimental design that includes:

  • Co-immunoprecipitation assays to identify direct protein-protein interactions

  • Yeast two-hybrid screens to map interaction domains

  • Live-cell imaging with dual-labeled components (INP2 and cytoskeletal elements)

  • Conditional mutants of cytoskeletal components to observe effects on INP2 localization

• Control for confounding variables such as cell cycle stage, as cytoskeletal organization varies dramatically throughout the cell cycle in S. pombe .

• Analyze data using quantitative approaches such as co-localization coefficients, FRET efficiency measurements, or biochemical binding affinities.

This systematic approach follows established experimental design principles while addressing the specific challenges of studying membrane protein-cytoskeleton interactions .

What methods are most effective for analyzing INP2 membrane topology?

To effectively analyze the membrane topology of INP2, researchers should employ a complementary set of biochemical and imaging techniques:

• Computational prediction: Begin with transmembrane domain prediction software (TMHMM, MEMSAT, etc.) to generate a theoretical topology model.

• Protease protection assays: Perform selective membrane permeabilization followed by protease treatment and immunoblotting to determine which portions of the protein are accessible.

• Glycosylation mapping: Insert glycosylation sites at various positions throughout the protein sequence; only sites exposed to the ER lumen will be glycosylated.

• Fluorescence-based approaches:

  • Split-GFP complementation to determine orientation of specific domains

  • pH-sensitive fluorescent tags that distinguish between luminal and cytosolic environments

• Cysteine accessibility methods: Introduce cysteine residues throughout the protein and test their accessibility to membrane-impermeable sulfhydryl reagents.

This multi-method approach provides convergent evidence for topology determination, which is critical for membrane proteins like INP2 where structure directly relates to function.

How is INP2 expression regulated during the S. pombe cell cycle?

The regulation of INP2 expression during the S. pombe cell cycle requires consideration within the broader context of cell cycle-regulated genes. Based on patterns observed in other S. pombe genes:

• Cell cycle regulation patterns: The expression of many S. pombe genes oscillates throughout the cell cycle, with two major waves occurring in early/mid G2 phase and near the G2/M transition . Analysis of INP2's promoter sequence should be performed to identify potential cell cycle-responsive elements.

• Transcription factor binding: Examination of the INP2 promoter for forkhead transcription factor binding sites (consensus sequence GTAAACAAA) may suggest regulation similar to the Cdc15 cluster of genes . Additionally, look for MBF-like transcription factor binding sites, which regulate many S. pombe cell cycle genes.

• Experimental approach to determine regulation:

  • Synchronize S. pombe cells using centrifugal elutriation or a cdc25-22 temperature-sensitive block-release

  • Collect samples at regular intervals throughout the cell cycle

  • Measure INP2 mRNA levels by RT-qPCR and protein levels by Western blotting

  • Correlate expression with cell cycle phases using established markers

• Regulatory mechanisms may involve multiple transcription factors, including Sep1, Fkh2, or complexes containing Cdc10, Res1, or Res2 .

A comprehensive understanding of INP2 cell cycle regulation would help explain its role in peroxisome inheritance, which is inherently linked to the cell division process.

What interaction partners of INP2 have been identified, and what methodologies are recommended for discovering new interactions?

• Affinity purification-mass spectrometry (AP-MS):

  • Express epitope-tagged INP2 in S. pombe

  • Perform immunoprecipitation under different detergent conditions optimized for membrane proteins

  • Identify co-precipitating proteins by mass spectrometry

  • Validate interactions via reciprocal IP experiments

• Proximity-based labeling methods:

  • Create fusions of INP2 with BioID or APEX2 enzymes

  • Allow in vivo biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

• Split-ubiquitin yeast two-hybrid system:

  • Specifically designed for membrane proteins

  • Screen against S. pombe genomic or cDNA libraries

  • Validate positive hits in vivo

• Genetic interaction screening:

  • Create an INP2 deletion strain

  • Cross with deletion or temperature-sensitive mutants of candidate interactors

  • Analyze synthetic phenotypes that suggest functional relationships

• Co-localization studies:

  • Fluorescently tag INP2 and candidate interactors

  • Perform high-resolution confocal or super-resolution microscopy

  • Quantify co-localization using appropriate statistical methods

When analyzing GTPase interactions in particular, researchers should design experiments to test whether INP2 preferentially interacts with GTP-bound forms, similar to protein kinase C homologs in S. pombe that interact with rho1p and rho2p only when bound to GTP .

How do interactions between INP2 and GTPases contribute to peroxisome inheritance?

While specific data on INP2-GTPase interactions is not fully characterized in the literature, insights can be drawn from related systems in S. pombe:

• Potential mechanism: Similar to how protein kinase C homologues (pck1p and pck2p) interact with GTP-bound rho1p and rho2p in S. pombe , INP2 may interact with small GTPases as part of a signaling pathway that regulates peroxisome movement during cell division.

• Experimental approach to investigate GTPase interactions:

  • Create recombinant GTPases locked in GTP-bound or GDP-bound conformations

  • Perform binding assays with purified INP2 protein or domains

  • Map interaction domains through truncation analysis

  • Test if binding has functional consequences for peroxisome positioning

• Functional relevance: GTPase interactions may regulate:

  • Timing of peroxisome inheritance during the cell cycle

  • Association of peroxisomes with the cytoskeleton

  • Proper segregation of peroxisomes between mother and daughter cells

• Proposed model: GTP-bound GTPases may recruit INP2 to the peroxisome membrane, where it can then engage myosin motors for transport along actin filaments.

This model draws upon established principles of GTPase-effector interactions observed in S. pombe and applies them to the predicted function of INP2 in peroxisome inheritance.

How can researchers distinguish between direct and indirect effects when studying INP2 function in peroxisome inheritance?

Distinguishing between direct and indirect effects in INP2 function requires rigorous experimental design and controls:

• Acute vs. chronic depletion strategies:

  • Generate conditional INP2 systems using auxin-inducible degrons or temperature-sensitive alleles

  • Compare the immediate effects of INP2 loss (direct) with long-term adaptation (indirect)

  • Use time-resolved imaging to establish the sequence of cellular events following INP2 depletion

• Structure-function analysis:

  • Create a series of domain deletion and point mutation variants

  • Test each variant for:
    a) Peroxisome localization
    b) Myosin binding capacity
    c) Ability to rescue peroxisome inheritance defects

  • Identify separation-of-function mutations that affect specific aspects of INP2 activity

• Bypass suppression experiments:

  • Test whether artificial tethering of myosin motors to peroxisomes can bypass the need for INP2

  • If successful, this would support a direct adapter function rather than a regulatory role

• Biochemical reconstitution:

  • Purify components (peroxisomes, INP2, myosin, ATP) and test if transport can be reconstituted in vitro

  • Only direct components will be required for the reconstituted system to function

• Epistasis analysis:

  • Determine the genetic relationship between INP2 and other peroxisome inheritance factors

  • Position INP2 within a functional pathway based on double mutant phenotypes

These approaches follow sound experimental design principles and will help establish the direct mechanistic role of INP2 in peroxisome inheritance versus indirect effects through other cellular processes.

What considerations should researchers take into account when designing CRISPR/Cas9 genome editing experiments for INP2 in S. pombe?

When using CRISPR/Cas9 for genome editing of INP2 in S. pombe, researchers should consider the following methodological aspects:

• Guide RNA design and specificity:

  • Select guide RNAs with minimal off-target sites in the S. pombe genome

  • Verify the uniqueness of the target sequence using S. pombe genome databases

  • Design guides with appropriate GC content (40-60%) for optimal Cas9 activity

  • Target conserved functional domains for knockouts

• Homology-directed repair (HDR) considerations:

  • Use homology arms of at least 500bp for efficient integration

  • When introducing tags or mutations, position them at least 10bp away from the Cas9 cut site

  • Include silent mutations in the PAM site or guide sequence to prevent re-cutting after HDR

• Experimental design for validation:

  Validation Step	Technique	Purpose
  Genotyping	PCR and sequencing	Confirm correct editing
  Expression	Western blot	Verify protein production (for tags) or absence (for knockouts)
  Localization	Fluorescence microscopy	Confirm expected subcellular distribution
  Functionality	Peroxisome inheritance assay	Assess biological effect of the modification

• Control considerations:

  • Include wild-type controls processed identically except for CRISPR components

  • Generate revertant strains to confirm phenotypes are due to the targeted modification

  • Create multiple independent clones to rule out off-target or clonal effects

• Technical optimization for S. pombe:

  • Use codon-optimized Cas9 for expression in S. pombe

  • Consider using a ribonucleoprotein (RNP) delivery approach instead of plasmid-based expression

  • Optimize transformation protocols for high efficiency (lithium acetate method with PEG)

This approach incorporates proper experimental design principles while addressing the specific challenges of CRISPR editing in S. pombe.

How can researchers integrate multi-omics data to understand the broader functional context of INP2?

To comprehensively understand INP2 within its broader cellular context, researchers should implement an integrated multi-omics approach:

• Data integration strategy:

  • Start with a clear hypothesis about INP2 function based on existing knowledge

  • Design experiments that generate complementary data types

  • Use computational methods to integrate diverse datasets

  • Validate key predictions with focused experimental approaches

• Multi-omics experimental design:

  Omics Approach	Technique	Insight Provided
  Genomics	Comparative genome analysis across fungi	Evolutionary conservation and divergence
  Transcriptomics	RNA-seq of INP2 deletion vs. wild-type	Affected gene expression networks
  Proteomics	IP-MS, global proteome analysis	Protein interactions and abundance changes
  Metabolomics	LC-MS profiling	Metabolic consequences of peroxisome misregulation
  Phenomics	High-content screening	Cellular phenotypes under various conditions

• Network analysis approaches:

  • Construct protein-protein interaction networks centered on INP2

  • Identify enriched pathways and biological processes

  • Compare with known peroxisome inheritance factors

  • Look for unexpected connections to other cellular processes

• Temporal dynamics consideration:

  • Analyze data across the cell cycle to capture dynamic regulation

  • Consider INP2's role in the context of the two waves of cell cycle transcription in S. pombe

  • Map expression patterns to specific cell cycle regulatory networks

• Validation of network predictions:

  • Test key nodes in the network through targeted experiments

  • Use genetic approaches (synthetic lethality, suppressor screens)

  • Create reporter systems to monitor pathway activities

This integrated approach leverages diverse data types to position INP2 within the complex cellular networks of S. pombe, providing a systems-level understanding of its function.

What are common challenges in purifying functional INP2 protein, and how can they be addressed?

Purifying functional membrane proteins like INP2 presents several technical challenges that researchers should anticipate and address:

• Solubilization challenges:

  • Traditional detergents may disrupt protein structure or strip away essential lipids

  • Solution: Screen a panel of detergents including mild non-ionic options (DDM, LMNG) and newer amphipathic polymers (SMALPs, nanodiscs)

  • Test detergent concentrations systematically (typically 1-3× CMC)

  • Consider detergent exchange during purification to improve stability

• Expression system optimization:

  • S. pombe expression may yield authentic post-translational modifications but low quantities

  • Solution: Test both homologous (S. pombe) and heterologous (E. coli, insect cells) expression systems

  • For E. coli, use specialized strains (C41/C43, Lemo21) designed for membrane proteins

  • Consider cell-free expression systems with supplied lipids or detergents

• Protein stability issues:

  Problem	Solution	Rationale
  Aggregation	Add glycerol (10-20%)	Prevents non-specific aggregation
  Proteolysis	Include protease inhibitors	Prevents degradation during purification
  Oxidation	Add reducing agents	Maintains cysteine residues in reduced state
  Denaturation	Purify at 4°C	Slows unfolding and aggregation

• Functionality assessment:

  • Develop activity assays based on predicted functions:
    a) Binding assays with myosin components
    b) Lipid interaction assays
    c) GTPase interaction measurements

  • Use biophysical methods (CD, thermal shift) to confirm folded state

• Protein orientation and reconstitution:

  • For functional studies, reconstitute into liposomes with controlled orientation

  • Verify orientation using protease protection or antibody accessibility assays

  • Consider nanodiscs for a more native-like membrane environment

These methodological considerations address the specific challenges of membrane protein purification while applying sound experimental design principles .

How can researchers resolve contradictory data when studying INP2 function across different experimental systems?

When faced with contradictory data regarding INP2 function across different experimental systems, researchers should implement a systematic approach to resolve discrepancies:

• Evaluate experimental variables systematically:

  • Compare exact experimental conditions between contradictory studies

  • Identify differences in strain backgrounds, tags, expression levels, or assay conditions

  • Test whether the contradictions persist when these variables are harmonized

• Consider context-dependent functions:

  • Design experiments to test if INP2 function varies with:
    a) Cell cycle stage
    b) Nutritional status
    c) Stress conditions
    d) Genetic background

• Technique-specific limitations assessment:

  • Evaluate inherent limitations of each experimental approach

  • Determine if contradictions arise from technical artifacts

  • Develop independent methodologies that don't share the same limitations

• Resolving approaches:

  Contradiction Type	Resolution Approach	Example
  Localization discrepancies	Super-resolution microscopy	Distinguish between closely associated structures
  Binding partner conflicts	In vitro vs. in vivo validation	Test if interactions are direct or indirect
  Phenotypic differences	Acute vs. chronic depletion	Separate immediate from adaptive effects
  Functional assignment conflicts	Domain-specific mutations	Map functions to specific protein regions

• Data integration strategy:

  • Develop models that accommodate seemingly contradictory data

  • Test predictions of these integrated models with new experiments

  • Use genetic approaches (suppressor screens, synthetic interactions) to resolve functional relationships

This approach applies principles of robust experimental design to systematically address contradictions and develop a more nuanced understanding of INP2 function.

What are the most promising future research directions for understanding SPBC21B10.06c function?

Based on current knowledge of INP2 (SPBC21B10.06c) and related proteins in S. pombe, several promising research directions emerge:

• Structural biology approaches to elucidate INP2's membrane integration and interaction interfaces with binding partners, particularly myosin motors and potential GTPases.

• Cell cycle regulation studies to determine how INP2 expression and activity are coordinated with peroxisome inheritance during cell division, potentially connecting to the well-characterized transcriptional networks in S. pombe .

• Comparative studies across fungal species to understand the evolutionary conservation and divergence of peroxisome inheritance mechanisms, similar to how cell wall metabolism genes have been studied .

• Systems biology approaches to position INP2 within the broader cellular networks, particularly in relation to membrane trafficking, organelle inheritance, and cell cycle progression.

• Development of in vitro reconstitution systems to directly test the sufficiency of INP2 and identified partners in driving peroxisome movement along cytoskeletal elements.

These research directions build upon the established knowledge while addressing key gaps in our understanding of this membrane protein's function in S. pombe.