Recombinant Schizosaccharomyces pombe Uncharacterized protein C1687.08 (SPAC1687.08)

What are the basic structural characteristics of SPAC1687.08?

SPAC1687.08 is a transmembrane protein from Schizosaccharomyces pombe (fission yeast) consisting of 96 amino acids. The protein has UniProt accession number O14067 and is characterized by the amino acid sequence: MFIFNVLTIRCTFHVLFAICYFCDHLLQYISNSRDSKAGLKIFLVFELAVTIFNTVMLQL ANRVKNGLTLAILIVSVVMFVYHQQLIVNCKKMLAL. Based on its sequence analysis, it contains transmembrane domains that suggest its localization to cellular membranes, though its specific subcellular location requires experimental verification. The protein is currently classified as "uncharacterized," indicating limited knowledge about its specific biological functions .

What expression systems are suitable for producing recombinant SPAC1687.08?

The recombinant SPAC1687.08 protein can be successfully produced using in vitro E. coli expression systems. For research applications, the protein is typically produced with an N-terminal 10xHis-tag to facilitate purification through affinity chromatography. The full-length protein (amino acids 1-96) has been successfully expressed in this system, indicating that E. coli can properly produce this relatively small protein. When designing expression constructs, researchers should consider including appropriate tags for detection and purification while ensuring these modifications don't interfere with the protein's native structure or function .

What are the optimal storage conditions for recombinant SPAC1687.08?

For short-term storage (up to one week), working aliquots of recombinant SPAC1687.08 can be maintained at 4°C. For intermediate-term storage, the protein should be stored at -20°C. For extended storage periods, the protein should be conserved at -20°C or -80°C. It's important to note that repeated freezing and thawing is not recommended as it can lead to protein degradation and loss of activity. The shelf life of the liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form maintains stability for approximately 12 months at these temperatures. Storage buffer composition (typically Tris-based buffer with 50% glycerol) is optimized for this specific protein to maintain stability .

What strategies can be employed to investigate the function of an uncharacterized protein like SPAC1687.08?

To investigate SPAC1687.08's function, researchers should employ a multi-faceted approach. Begin with bioinformatic analysis to identify conserved domains and potential orthologs in other species. Follow with PCR-based gene deletion to observe phenotypic effects, though this may be challenging as some genomic regions in S. pombe are refractory to standard deletion methods. Complementary approaches include gene tagging for localization studies using fluorescent proteins, promoter analysis to understand expression patterns, and yeast two-hybrid screening to identify interacting partners. Proteomics approaches such as co-immunoprecipitation followed by mass spectrometry can help identify protein complexes containing SPAC1687.08. RNA-seq analysis comparing wild-type and deletion strains can reveal affected pathways. Additionally, phenotypic screens exposing deletion strains to various stressors (temperature, oxidative stress, DNA damage agents) may provide functional insights .

What methods are recommended for detecting protein-protein interactions involving SPAC1687.08?

For identifying protein-protein interactions involving SPAC1687.08, researchers should implement complementary approaches to overcome limitations of individual methods. Affinity purification coupled with mass spectrometry (AP-MS) using the His-tagged recombinant protein can capture stable interactors. For in vivo interactions, tagging the endogenous gene with epitopes like HA or FLAG allows co-immunoprecipitation experiments. Yeast two-hybrid screening provides an alternative that can detect direct binary interactions, though it may generate false positives. Proximity-dependent biotin identification (BioID) or APEX labeling are valuable for detecting transient or weak interactions in the native cellular environment. Validation of identified interactions should include reciprocal co-IPs and functional assays to determine biological relevance. Carefully designed controls are essential, including tag-only controls and testing interactions in both orientations for two-hybrid experiments .

How does SPAC1687.08 potentially relate to mitotic recombination processes in S. pombe?

The relationship between SPAC1687.08 and mitotic recombination requires investigation using specialized assays developed for S. pombe. Researchers should consider employing chromosome-based recombination assays like those using the truncated Ch.16 system, which can detect chromosome loss, gene conversion, and break-induced replication events following DNA damage. Non-tandem repeat assays can reveal if SPAC1687.08 influences recombination at repetitive elements. Replication fork stalling assays would be particularly valuable for examining if this transmembrane protein affects fork progression or restart. Deletion or conditional expression of SPAC1687.08 followed by analysis using these established assays could reveal phenotypes related to homologous recombination efficiency, DNA damage sensitivity, or chromosome stability. Monitoring for changes in spontaneous and induced recombination rates in various genetic backgrounds (wild-type versus SPAC1687.08 mutants) would provide insights into its potential role in genome maintenance pathways .

What is the evolutionary significance of uncharacterized proteins like SPAC1687.08 in S. pombe?

The evolutionary significance of uncharacterized proteins like SPAC1687.08 can be analyzed through comprehensive phylogenetic studies. Research has shown that whether a gene is essential correlates with its timing of appearance in evolutionary history and its conservation across evolutionary branches. Comparative genomic analyses should be performed to identify potential orthologs in other species, particularly examining conservation between S. pombe, S. cerevisiae, and higher eukaryotes including humans. The pilot gene deletion study revealed that none of the investigated ancient genes in fission yeast that were lost in the S. cerevisiae lineage were essential, suggesting evolutionary flexibility for some conserved genes. For SPAC1687.08 specifically, researchers should examine its presence or absence across diverse fungal species and other eukaryotes to determine if it represents a species-specific adaptation or a conserved cellular component with potentially undiscovered functions in higher organisms .

How might transmembrane localization influence the functional characterization of SPAC1687.08?

The transmembrane nature of SPAC1687.08 presents specific challenges and opportunities for functional characterization. Researchers must consider that standard cytosolic protein analysis techniques may yield limited results. Membrane fractionation followed by Western blotting can confirm subcellular localization. For functional studies, researchers should investigate potential roles in membrane-associated processes such as signal transduction, transport, or organelle integrity. The protein's membrane topology should be determined using protease protection assays or fluorescent protein tagging at different termini. Since transmembrane proteins often function in complexes, BN-PAGE (Blue Native-PAGE) can identify native membrane protein complexes containing SPAC1687.08. Additionally, researchers should examine phenotypes specifically related to membrane function when analyzing deletion strains, including membrane fluidity, stress responses, and lipid composition. The relatively small size (96 amino acids) suggests possible roles in membrane structure maintenance rather than enzymatic activity .

What are the methodological challenges in working with recombinant transmembrane proteins like SPAC1687.08?

Working with recombinant transmembrane proteins presents several methodological challenges that researchers must address. Expression optimization in E. coli systems often requires testing multiple constructs with varying tags, fusion partners, and expression conditions to prevent protein aggregation. Purification protocols should incorporate detergents suitable for transmembrane proteins (e.g., DDM, CHAPS, or digitonin) to maintain solubility and native conformation. Refolding strategies may be necessary if the protein forms inclusion bodies. Functional assays must be designed considering the membrane environment, potentially utilizing liposome reconstitution or nanodiscs to study the protein in a lipid bilayer context. Structural studies present additional challenges, often requiring specialized approaches like cryo-electron microscopy rather than traditional crystallography. When designing experiments, researchers should include comprehensive controls that account for detergent effects and potential artifacts introduced by purification methods .

How can researchers design experiments to determine if SPAC1687.08 is essential for S. pombe viability?

Determining the essentiality of SPAC1687.08 requires careful experimental design due to the challenges in gene deletion for certain genomic regions. A comprehensive approach begins with attempting PCR-based gene deletion in diploid strains followed by tetrad analysis to observe segregation patterns. If direct deletion yields no viable haploid knockout colonies, researchers should implement conditional expression systems such as the nmt1 promoter (repressible by thiamine) to control gene expression. The auxin-inducible degron (AID) system provides an alternative for rapidly depleting the protein post-translationally. Complementation assays using plasmid-borne wild-type genes can confirm whether lethality is specifically due to SPAC1687.08 disruption. Terminal phenotype analysis of cells undergoing protein depletion can provide insights into the cellular processes affected. Researchers should be aware that approximately 17.5% of S. pombe genes are essential, and some genomic regions (like the 18kb segment identified in chromosome II) may be particularly refractory to standard deletion techniques, requiring multiple attempts or alternative approaches .

What high-throughput experimental approaches could accelerate functional characterization of uncharacterized proteins like SPAC1687.08?

To accelerate functional characterization of SPAC1687.08, researchers should employ integrated high-throughput experimental pipelines. Systematic genetic interaction screens using synthetic genetic array (SGA) methodology can reveal functional relationships by identifying genes that, when mutated alongside SPAC1687.08, produce enhanced or suppressed phenotypes. Parallel creation of comprehensive mutant libraries through CRISPR-Cas9 or transposon mutagenesis allows for rapid phenotypic screening under diverse conditions. High-content microscopy screening of GFP-tagged SPAC1687.08 against chemical or genetic perturbations can reveal functional context through localization changes. Metabolomic profiling comparing wild-type and mutant strains can identify affected biochemical pathways. Proteome-wide thermal stability assays can detect proteins whose stability is affected by SPAC1687.08 deletion. Additionally, computational approaches using machine learning algorithms can integrate diverse datasets (expression profiles, protein-protein interactions, localization data) to predict protein function. This multi-faceted approach allows parallel investigation of multiple hypotheses, dramatically accelerating the characterization process .

How does the study of uncharacterized proteins in S. pombe compare to similar research in S. cerevisiae and other model organisms?

The study of uncharacterized proteins differs significantly between model organisms due to their unique biological characteristics and available research tools. S. pombe offers distinct advantages over S. cerevisiae for certain research questions, particularly those related to chromosome structure and function. S. pombe diverged from S. cerevisiae approximately one billion years ago, yet many S. pombe genes show greater similarity to human disease genes than their S. cerevisiae counterparts. For uncharacterized proteins like SPAC1687.08, S. pombe's higher conservation in chromosome structure and function genes makes it particularly valuable for studying potential roles in these processes. Methodologically, while both yeasts allow for facile genetic manipulation, S. pombe's chromosome structure with regional centromeres more closely resembles higher eukaryotes than S. cerevisiae's point centromeres. When designing comparative studies, researchers should consider these evolutionary relationships and structural differences, potentially examining functional conservation of SPAC1687.08 homologs across species when they exist .

What insights can be gained from comparing SPAC1687.08 with other uncharacterized proteins in the S. pombe genome?

Comparing SPAC1687.08 with other uncharacterized proteins in the S. pombe genome can provide valuable functional insights through guilt-by-association principles. Researchers should perform clustering analyses based on expression profiles, specifically examining co-regulation patterns under different conditions (stress, cell cycle phases, developmental stages). Similarity in regulatory elements in promoter regions may suggest shared transcriptional control mechanisms. Protein sequence analysis focusing on shared domains or motifs among uncharacterized proteins may reveal functional groupings. Systematic comparison of knockout phenotypes can identify functionally related uncharacterized proteins based on similar cellular defects. Network analysis incorporating known protein-protein interactions can place SPAC1687.08 in the context of specific cellular pathways, even when its direct function remains unknown. This comparative approach can help prioritize hypotheses about SPAC1687.08 function and potentially identify functional clusters of previously uncharacterized proteins acting in common biological processes .

How should contradictory experimental results regarding SPAC1687.08 function be reconciled?

When confronted with contradictory experimental results regarding SPAC1687.08 function, researchers should implement a systematic reconciliation approach. Begin by critically evaluating experimental conditions, as transmembrane proteins are particularly sensitive to environmental factors like temperature, pH, and ionic strength. Verify strain backgrounds, as genetic interactions can drastically alter phenotypic outcomes. Consider protein expression levels, as both deletion and overexpression can yield misleading results through compensation or dominant-negative effects. Examine the specific assays used, as different methodologies may probe distinct aspects of protein function. Temporal considerations are crucial—apparent contradictions may reflect different stages of cellular processes. When integrating findings from multiple studies, prioritize results with the most robust controls and replication. To resolve discrepancies, design experiments that directly test competing hypotheses under identical conditions, and consider that SPAC1687.08 may possess multiple functions depending on cellular context. Document all conditions meticulously to ensure reproducibility and facilitate accurate interpretation of seemingly contradictory outcomes .

What emerging technologies could advance understanding of proteins like SPAC1687.08?

Emerging technologies offer promising avenues for characterizing proteins like SPAC1687.08. Cryo-electron microscopy advances now permit structural determination of small membrane proteins at near-atomic resolution, potentially revealing functional insights through structure. Genome-wide CRISPR interference/activation screens in S. pombe can systematically probe genetic interactions under various conditions. Single-cell approaches including RNA-seq and proteomics can reveal cell-to-cell variability in expression and function, potentially identifying specialized roles in subpopulations. Proximity labeling techniques like TurboID or APEX2 can map the protein's immediate environment in living cells. Advances in computational prediction tools combining evolutionary information with deep learning algorithms increasingly predict protein functions with higher accuracy. Super-resolution microscopy techniques can track protein dynamics with unprecedented spatial resolution. Mass spectrometry innovations enable detection of post-translational modifications that may regulate function. Integration of these technologies through computational frameworks will likely provide complementary insights, accelerating functional characterization of previously uncharacterized proteins .

What are the implications of characterizing SPAC1687.08 for understanding fundamental cellular processes?

Characterizing SPAC1687.08 could yield insights into fundamental cellular processes, particularly those involving membrane biology and potentially mitotic recombination. As a transmembrane protein, SPAC1687.08 might function in membrane organization, signaling, or transport processes essential for cellular homeostasis. If genetic studies reveal connections to DNA damage response pathways, this could expand our understanding of how membrane proteins influence genome stability, potentially uncovering novel mechanisms for nucleus-cytoplasm communication during stress responses. The protein's evolutionary conservation pattern could provide insights into specialized functions that emerged in the fission yeast lineage. From a systems biology perspective, understanding SPAC1687.08 fills a knowledge gap in comprehensive cellular models. Even if the protein serves a highly specialized function, its characterization contributes to completing the functional annotation of the S. pombe genome, advancing our understanding of essential and non-essential gene networks and their evolutionary significance .

How can researchers design comprehensive functional genomics studies to systematically characterize proteins like SPAC1687.08?

A comprehensive functional genomics approach for characterizing SPAC1687.08 should integrate multiple experimental dimensions. Begin with strain construction, creating fluorescent protein fusions for localization studies and conditionally regulated alleles for temporal functional analysis. Perform systematic phenotyping under diverse conditions (temperature, pH, osmotic stress, DNA damaging agents) to identify sensitivity profiles. Implement genome-wide genetic interaction mapping using both positive (suppressor) and negative (synthetic) interaction screens to place the protein in functional networks. In parallel, conduct transcriptome and proteome profiling comparing wild-type and mutant strains under multiple conditions to identify affected pathways. Employ chromatin profiling techniques if preliminary data suggests nuclear functions. Utilize comparative genomics to identify potential functional orthologs in other species for evolutionary insights. Design the study with standardized protocols and detailed metadata collection to facilitate integration with existing datasets. This multi-layered approach ensures that even if individual assays yield limited insights, the integration of diverse data types can reveal functional patterns and generate testable hypotheses about SPAC1687.08's cellular roles .