Recombinant Schizosaccharomyces pombe Putative uncharacterized transmembrane protein PB15E9.06 (SPAPB15E9.06)

What is known about the structure and basic properties of SPAPB15E9.06?

SPAPB15E9.06 is a putative uncharacterized transmembrane protein in Schizosaccharomyces pombe with 89 amino acids. The complete amino acid sequence is: MKLNVCFRICNFLFQFSLEFFSISSLHSISSLHSISLSLSLFFLVAILYNIYIYLFRSKKKPKRILFAIPPLCPLCSPCFFFGTSSMLL . The protein contains hydrophobic regions characteristic of transmembrane domains, particularly in the N-terminal region. The protein appears to be conserved within the Schizosaccharomyces genus, suggesting functional importance, but its exact function remains uncharacterized.

For experimental work, recombinant versions can be produced with various tags, such as His-tags, to facilitate purification and detection . The protein should be stored in Tris-based buffer with 50% glycerol at -20°C for short-term storage or -80°C for extended storage to maintain stability .

Why use Schizosaccharomyces pombe as a model organism for studying transmembrane proteins?

S. pombe offers several advantages as a model organism for transmembrane protein research:

• It resembles human cells in terms of mitochondrial inheritance, transport mechanisms, and metabolism

• The organism has conserved regulatory processes and genetic features shared with metazoans

• S. pombe possesses a relatively small genome with lower redundancy compared to higher eukaryotes

• As a unicellular eukaryote, it combines experimental simplicity with relevant eukaryotic cellular organization

• It offers tractable genetics with well-established transformation protocols

• The petite-negative phenotype makes it particularly suitable for mitochondrial membrane protein studies

Furthermore, S. pombe has significantly contributed to biomedical research on fundamental cellular processes, with powerful experimental techniques and comprehensive database resources available .

What experimental design approach should be used when beginning work with SPAPB15E9.06?

When designing experiments to study SPAPB15E9.06, follow this systematic approach:

This approach ensures logical progression while maintaining flexibility to accommodate the challenges inherent in studying uncharacterized proteins.

What proteomics approaches are most effective for characterizing SPAPB15E9.06 function?

Several proteomics approaches can elucidate the function of uncharacterized transmembrane proteins like SPAPB15E9.06:

• Comparative Proteome Analysis: This powerful approach can identify targets for protein production and secretion in S. pombe . For SPAPB15E9.06:

  • Compare proteome profiles between wild-type and SPAPB15E9.06 knockout/overexpression strains

  • Analyze changes in abundance of functionally related proteins

  • Look for co-regulated proteins that may function in the same pathway

• Dynamic SILAC (Stable Isotope Labeling with Amino acids in Cell culture): This technique can determine protein half-lives and turnover rates :

  • Typical experimental setup involves culturing cells in media containing heavy isotope-labeled amino acids

  • Samples are collected at different time points to measure incorporation of labeled amino acids

  • Data analysis determines half-lives ranging from <1 to >20 days

  • Results can reveal whether SPAPB15E9.06 is short-lived (suggesting regulatory role) or long-lived (suggesting structural function)

• Immunoprecipitation-Mass Spectrometry: This approach can identify protein interaction partners :

  • Create strains with tagged SPAPB15E9.06 (e.g., GFP, FLAG, or His tag)

  • Perform immunoprecipitation under native conditions

  • Analyze co-precipitated proteins by LC-MS/MS

  • Validate interactions with reciprocal co-IP or proximity labeling

These methods provide complementary information that collectively can reveal functional insights into this uncharacterized protein.

How can genetic approaches help determine the function of SPAPB15E9.06?

Several genetic strategies can help decipher the function of SPAPB15E9.06:

• Synthetic Lethality Screening: This powerful approach can identify genetic interactions:

  • Create a strain with SPAPB15E9.06 deletion (if viable) or under regulated expression

  • Cross with arrays of deletion or temperature-sensitive mutants

  • Identify combinations that result in synthetic lethality or growth defects

  • Example methodology from search result :

    • Use plasmid shuffling with 5-FOA selection

    • Perform whole-genome sequencing on synthetic lethal strains

    • Validate candidates with complementation tests

• Transcription Factor Regulatory Network Analysis: Based on the comprehensive S. pombe TF atlas :

  • Identify transcription factors that bind near SPAPB15E9.06 locus

  • Examine expression changes in TF mutant strains

  • Map the regulatory network controlling SPAPB15E9.06 expression

• Flocculation Phenotype Analysis: As demonstrated in search result :

  • Test SPAPB15E9.06 deletion/overexpression for flocculation phenotypes

  • Perform microarray expression profiling and ChIP-chip analysis

  • Identify target genes and transcription factors in the regulatory network

These genetic approaches can provide valuable insights into the biological pathways and processes involving SPAPB15E9.06.

What challenges exist in characterizing uncharacterized transmembrane proteins like SPAPB15E9.06?

Researchers face several challenges when studying uncharacterized transmembrane proteins like SPAPB15E9.06:

• Biochemical Environment Requirements:

  • Transmembrane proteins reside in lipid bilayers, restricting their activity to specialized biochemical environments

  • They represent only 20-30% of human genes but account for over half of known drug targets

  • The lipid membrane constitutes only 6-12% of cytosolic volume, with plasma membrane representing only 2-5% of this total

• Lack of Reference Phenotypes:

  • Without known phenotypes, systematic screens are difficult to implement

  • Traditional approaches involving reconstituted biochemical assays and structural determination are technically challenging

• Expression and Purification Difficulties:

  • Maintaining proper folding during recombinant expression

  • Need for detergents or lipid environments for solubilization

  • Challenges in crystallization for structural studies

• Technical Approaches to Overcome These Challenges:

  • Bioinformatics integration with experimental approaches for focused characterization

  • Use of specialized expression systems for membrane proteins

  • Application of protein turnover studies to understand regulation

  • Employment of comparative proteomics across different conditions

How can dynamic protein turnover studies provide insights into SPAPB15E9.06 function?

Protein turnover studies can reveal critical functional insights for SPAPB15E9.06:

• Methodology for Dynamic SILAC:

  • Culture S. pombe cells in media containing heavy isotope-labeled amino acids

  • Collect samples at different time points (typically spanning hours to days)

  • Process samples for mass spectrometry analysis

  • Calculate protein half-lives based on heavy/light amino acid ratios

• Expected Insights from Turnover Studies:

  • Determine if SPAPB15E9.06 is short-lived (regulatory) or long-lived (structural)

  • Compare half-life in different cellular compartments

  • Examine turnover rates under different stress conditions

• Interpretation Framework based on Known Patterns:

  • Membrane proteins typically show shorter half-lives (SPAPB15E9.06 would likely follow this pattern)

  • Mitochondrial proteins generally have longer half-lives

  • Receptors and signaling molecules show shorter half-lives than structural proteins

  • Protein half-lives correlate with function and dynamics of protein complexes

• Comparative Turnover Data Analysis:
  From study , protein half-lives in rat primary hippocampal cultures showed:

  Protein Category	Typical Half-Life Range	Notes
  Membrane proteins	Relatively shorter	Including those of plasma membrane, ER, Golgi
  Mitochondrial proteins	Significantly longer	Possibly due to unique quality control mechanisms
  Receptors (e.g., glutamate)	Shorter than population average	Enabling faster regulation
  Signaling molecules	Short-lived	Allowing fine-tuned regulation
  Energy metabolism proteins	Long-lived (12-15 days)	Reflecting steady function

This comparative framework provides context for interpreting SPAPB15E9.06 turnover data.

What transcriptional regulation approaches would provide insights into SPAPB15E9.06 expression patterns?

Understanding the transcriptional regulation of SPAPB15E9.06 requires several complementary approaches:

• Chromatin Immunoprecipitation Sequencing (ChIP-seq):

  • Identify transcription factors binding near the SPAPB15E9.06 locus

  • Map the binding sites across different conditions

  • As demonstrated in search result :

    • Create strains with endogenously tagged transcription factors

    • Perform ChIP-seq to identify DNA binding sites

    • Analyze motifs and regulatory networks

• Transcription Factor Library Screening:
  A comprehensive approach used in recent S. pombe research :

  • Systematically test a library of 89 endogenously tagged S. pombe transcription factors

  • Map protein and chromatin interactions

  • Identify transcription factors that regulate SPAPB15E9.06

  • Discover DNA binding motifs and regulatory networks

• Expression Profiling Under Different Conditions:

  • Analyze SPAPB15E9.06 expression during different growth phases

  • Test expression under various stress conditions

  • Examine expression in different genetic backgrounds

• Regulatory Network Mapping:

  • Identify potential autoregulation mechanisms

  • Map inhibitory feed-forward loops involving SPAPB15E9.06

  • Discover cross-regulation with other genes

This multi-faceted approach can establish a comprehensive understanding of when and how SPAPB15E9.06 is expressed, providing clues to its function.

What are the best experimental approaches for studying SPAPB15E9.06 protein-protein interactions?

Several complementary methods can be employed to identify SPAPB15E9.06 interaction partners:

• Affinity Purification Coupled with Mass Spectrometry:

  • Express SPAPB15E9.06 with a tag (His, FLAG, or GFP)

  • Perform gentle lysis to maintain native interactions

  • Purify using affinity chromatography

  • Identify co-purified proteins via mass spectrometry

  • Validate interactions through reciprocal pull-downs

• Proximity Labeling Approaches:

  • Create fusion proteins with BioID or APEX2

  • These enzymes biotinylate proteins in close proximity

  • Purify biotinylated proteins using streptavidin

  • Identify labeled proteins by mass spectrometry

  • This method is particularly valuable for transient interactions

• Split-Reporter Systems:

  • Yeast two-hybrid (Y2H) screening with modified membrane protein protocols

  • Split-GFP or split-luciferase assays for in vivo validation

  • Bimolecular fluorescence complementation (BiFC) for subcellular localization

• Co-localization Studies:

  • Use fluorescently tagged SPAPB15E9.06

  • Perform co-localization studies with known organelle markers

  • Employ super-resolution microscopy for detailed localization

For transmembrane proteins like SPAPB15E9.06, special considerations include using appropriate detergents for solubilization and considering membrane-specific interaction environments.

How can undergraduate students effectively participate in research on uncharacterized proteins like SPAPB15E9.06?

Undergraduate students can meaningfully contribute to research on uncharacterized proteins through several approaches:

This structured approach enables meaningful undergraduate participation while building essential research skills.

What bioinformatics approaches are most valuable for predicting SPAPB15E9.06 function?

Bioinformatics offers powerful tools for predicting functions of uncharacterized proteins like SPAPB15E9.06:

• Sequence-Based Analysis:

  • Transmembrane domain prediction using TMHMM, Phobius, or TOPCONS

  • Signal peptide detection with SignalP

  • Domain identification using InterPro or Pfam

  • Sequence conservation analysis across species

  • Motif identification for potential post-translational modifications

• Structural Prediction and Analysis:

  • Secondary structure prediction with PSIPRED

  • 3D structure modeling using AlphaFold2 or RoseTTAFold

  • Molecular dynamics simulations in membrane environments

  • Binding site prediction for potential ligands

• Functional Inference from Networks:

  • Co-expression network analysis across conditions

  • Protein-protein interaction network integration

  • Phylogenetic profiling to identify functionally related proteins

  • Gene neighborhood analysis in related organisms

• Integrated Deorphanization Strategy:
  Based on search result , an effective approach includes:

  • Integration of experimental and bioinformatics approaches

  • Focused functional characterization within particular protein classes

  • Systematic screening with bioinformatics guidance

  • Landscape reference comparison across genomes

This multi-layered bioinformatics approach can generate testable hypotheses about SPAPB15E9.06 function before extensive experimental work.

How might SPAPB15E9.06 function in the context of cell membrane organization?

As a putative transmembrane protein, SPAPB15E9.06 likely plays a role in membrane organization that could be explored through:

• Membrane Domain Localization Studies:

  • Determine if SPAPB15E9.06 localizes to specific membrane microdomains

  • Analyze co-localization with lipid raft markers

  • Examine distribution during cell division or stress responses

• Membrane Protein Turnover Analysis:
  Based on search result , membrane proteins typically show distinct turnover patterns:

  Membrane Location	Typical Turnover Rate	Potential Significance
  Plasma membrane	Relatively short-lived	Adaptability to external signals
  ER membrane	Short to medium half-life	Quality control mechanisms
  Golgi apparatus	Short half-life	Dynamic sorting function
  Mitochondrial membrane	Longer half-life	Structural stability requirement

  Determining where SPAPB15E9.06 fits in this spectrum could provide functional insights.

• Membrane Stress Response Investigation:

  • Test whether SPAPB15E9.06 expression changes under membrane stress

  • Examine phenotypes of deletion/overexpression strains under conditions like:

    • Osmotic stress

    • Lipid composition alterations

    • Membrane-disrupting agents

• Potential Role in Specialized Membrane Functions:

  • Investigate involvement in membrane fusion/fission events

  • Examine potential roles in vesicular trafficking

  • Test for functions in cell wall synthesis or remodeling

These approaches can collectively reveal the membrane-related functions of this uncharacterized protein.

What comparative genomics strategies could provide evolutionary context for SPAPB15E9.06?

Understanding the evolutionary context of SPAPB15E9.06 requires systematic comparative genomics:

• Ortholog Identification and Analysis:

  • Identify orthologs across fungal species using reciprocal BLAST

  • Compare conservation patterns in other Schizosaccharomyces species

  • Examine presence/absence patterns across evolutionary distances

  • Analyze selection pressure through Ka/Ks ratios

• Synteny Analysis:

  • Examine conservation of genomic context around SPAPB15E9.06

  • Identify co-evolved gene clusters

  • Map chromosomal rearrangements affecting this locus

• Structural Conservation Analysis:

  • Compare predicted transmembrane topologies across species

  • Identify conserved residues that may be functionally important

  • Analyze conservation of potential protein-protein interaction interfaces

• Expression Pattern Evolution:

  • Compare regulation mechanisms across species

  • Identify conserved transcription factor binding sites

  • Analyze expression correlation patterns with known genes

This evolutionary perspective can provide crucial context for functional hypotheses and identify functionally important regions of the protein.

How could CRISPR-Cas9 genome editing be optimized for studying SPAPB15E9.06 function?

CRISPR-Cas9 offers powerful tools for studying SPAPB15E9.06, with specific considerations for S. pombe:

• Knockout Strategy Optimization:

  • Design specific gRNAs targeting SPAPB15E9.06

  • Include controls to verify editing efficiency

  • Create complete gene deletion versus domain-specific mutations

  • Validate knockouts through sequencing and protein detection

• Tagging Strategies for Functional Analysis:

  • C-terminal versus N-terminal tags based on predicted protein topology

  • Fluorescent protein fusions for localization studies

  • Affinity tags for interaction studies

  • Base editing for introducing specific mutations

• Conditional Systems for Essential Gene Analysis:

  • If SPAPB15E9.06 proves essential, implement:

    • Auxin-inducible degron system

    • Transcriptional repression strategies

    • Temperature-sensitive mutants

• Multiplexed Editing for Pathway Analysis:

  • Simultaneous editing of SPAPB15E9.06 and potential interacting partners

  • Creation of double/triple mutants to test genetic interactions

  • Systematic editing of predicted functional domains

• Genome-Wide Screens:

  • CRISPR interference (CRISPRi) for transcriptional repression

  • CRISPR activation (CRISPRa) for overexpression

  • Pooled screens to identify genetic interactions