Recombinant Schizosaccharomyces pombe Protein rot1 (rot1)

What is the Schizosaccharomyces pombe rot1 protein and what is its significance in cellular function?

Rot1 (SPAC31G5.02) is an essential protein in Schizosaccharomyces pombe with 232 amino acids. According to the available data, rot1 has a full amino acid sequence that includes multiple functional domains with conserved motifs. The protein contains several structural elements including glycine-rich regions and potential phosphorylation sites that may be important for its function. Rot1 is believed to play crucial roles in cellular processes that are essential for fission yeast viability, likely involved in protein folding or quality control mechanisms similar to its distant homologs in other organisms. When working with recombinant rot1, researchers should note that the expression region typically used spans amino acids 22-232, omitting the putative signal sequence .

What are the optimal storage and handling conditions for recombinant S. pombe rot1 protein?

Recombinant S. pombe rot1 protein is typically stored in a Tris-based buffer containing 50% glycerol optimized specifically for this protein's stability. For long-term storage, the protein should be kept at -20°C or -80°C to maintain activity. Working aliquots can be stored at 4°C for up to one week to avoid repeated freeze-thaw cycles which may compromise protein integrity. It is not recommended to subject the protein to repeated freezing and thawing as this can lead to denaturation and loss of function . When designing experiments, consider the buffer composition when introducing the protein into different reaction systems to avoid compatibility issues with assay components.

How can I verify the purity and activity of recombinant rot1 protein for my experiments?

Verification of recombinant rot1 protein quality can be performed through several complementary approaches. Begin with SDS-PAGE analysis to confirm size and purity, expecting a band corresponding to approximately 26 kDa (depending on any tags present). Western blotting using antibodies specific to rot1 or to any included epitope tags provides further confirmation of identity. Functional assays should be designed based on the predicted activities of rot1, potentially including protein interaction studies with known binding partners from the Smc5/6 complex or related pathways. Mass spectrometry can provide definitive verification of protein identity and post-translational modifications. For activity assessment, consider using in vitro assays that evaluate rot1's putative functions in DNA repair or protein folding pathways based on S. pombe genetic studies of essential proteins .

What are the recommended approaches for integrating recombinant rot1 into S. pombe for functional studies?

For stable integration of recombinant rot1 into S. pombe, researchers should consider using the new series of stable integration vectors (SIVs) that avoid creating repetitive genomic regions which can lead to instability. These vectors target different prototrophy genes and produce non-repetitive, stable genomic loci that predominantly integrate as single copies. This approach is critical since commonly used fission yeast vectors often create unstable genomic loci . When designing your rot1 integration constructs, incorporate appropriate promoters, tags, and terminators from the modular toolbox available for heterologous sequence introduction. For functional studies of rot1, which is an essential gene, consider using conditional expression systems or partial loss-of-function mutations to avoid lethality. Integration should be verified using both PCR-based approaches and functional complementation assays to ensure proper expression and function of the recombinant protein .

How can I design mutation studies to analyze the functional domains of rot1 protein?

For mutational analysis of rot1 protein, implement a systematic approach using PCR-based mutagenesis techniques as demonstrated in similar S. pombe studies. Begin by identifying conserved motifs or predicted functional domains within the rot1 sequence for targeted mutation. The two-step PCR-based procedure can be employed, where two primers that border the DNA fragment within which a mutation is to be created are chosen. These primers should be designed to include unique restriction enzyme cutting sites, one upstream and one downstream of the mutation site. A third primer containing the desired base pair modifications in its central region is used to generate a megaprimer in the first PCR reaction. This megaprimer is then paired with another flanking primer in a second PCR reaction to create the mutant DNA fragment .

For comprehensive mutation analysis, consider the following systematic approach:

Verify mutant phenotypes using complementation assays in rot1 deletion backgrounds with plasmid-based expression systems under native or regulated promoters .

What methods are most effective for studying rot1 protein interactions with other cellular components?

To study rot1 protein interactions effectively, employ a multi-faceted approach combining both in vivo and in vitro techniques. For in vivo studies, consider implementing fluorescent tagging of rot1 using the toolbox of stable integration vectors in S. pombe that include various fluorescent markers. These can be used for co-localization studies with other cellular proteins to identify potential interaction partners . For biochemical confirmation of interactions, co-immunoprecipitation experiments using antibodies against rot1 or its tagged versions can identify protein complexes.

Yeast two-hybrid assays can be adapted for S. pombe to screen for potential interacting partners, though care must be taken with essential proteins like rot1. For more detailed analysis, consider bimolecular fluorescence complementation (BiFC) to visualize protein interactions in living cells. Mass spectrometry-based approaches after affinity purification (AP-MS) can identify components of protein complexes containing rot1.

For studying potential roles in DNA repair or replication processes, chromatin immunoprecipitation (ChIP) can determine if rot1 associates with specific genomic regions, particularly during stress conditions. Given the importance of protein phosphorylation in regulating S. pombe proteins during stress responses, as seen with Rad60's regulation by Cds1Chk2 , consider phospho-specific antibodies or phospho-proteomic approaches to study how rot1's interactions might be modulated by post-translational modifications.

How does rot1 protein function change during DNA damage response in S. pombe?

While direct evidence for rot1's role in DNA damage response is limited in the provided search results, insights can be drawn from studies of similar essential proteins in S. pombe. Like Rad60, which shows altered phosphorylation and cellular localization during hydroxyurea (HU)-induced replication stress , rot1 may undergo similar regulatory changes during DNA damage. To investigate these potential changes, researchers should examine rot1 phosphorylation status in response to various DNA damaging agents (UV, MMS, HU) using phospho-specific antibodies or mass spectrometry approaches.

For localization studies, fluorescently tagged rot1 constructs can be used to monitor protein redistribution following DNA damage, similar to how Rad60 becomes diffused throughout the cell in response to HU. Potential phosphorylation sites in rot1 can be identified using bioinformatics prediction tools and then mutated to evaluate their importance in the damage response. Interactions with known DNA repair factors like the Smc5/6 complex should be assessed both before and after damage induction .

The functional significance of any observed changes can be evaluated using phosphomimetic and phospho-deficient mutations, analyzing how they affect cell survival, chromosome stability, and repair pathway choice following various DNA damaging treatments.

What is the relationship between rot1 and the homologous recombination pathway in S. pombe?

Based on studies of other essential proteins in S. pombe, rot1 may have significant connections to the homologous recombination (HR) pathway. To investigate this relationship, researchers should first examine genetic interactions between rot1 and known HR factors like Rad51, Rad52, and components of the Smc5/6 complex. This can be done through synthetic genetic array analysis or targeted genetic crosses with conditional rot1 mutants.

Physical interactions with HR proteins can be assessed through co-immunoprecipitation experiments and proximity-based protein interaction assays. Functional assays measuring HR efficiency, such as direct-repeat recombination systems or site-specific DSB repair assays, can determine if rot1 mutants affect recombination rates. Chromatin immunoprecipitation (ChIP) can reveal whether rot1 is recruited to sites of DNA damage or stalled replication forks .

The impact of rot1 on HR regulation can be further explored by examining its potential roles in modulating Rad51 filament formation or dissolution, similar to how other factors in S. pombe limit crossover formation and inappropriate recombination. This is particularly relevant as inappropriate activation of HR can lead to genetic instability and chromosome rearrangements .

How can structural biology approaches be utilized to understand rot1 protein function?

Structural biology approaches offer powerful insights into rot1 protein function at the molecular level. Begin with bioinformatic structural predictions using tools like AlphaFold to generate hypothetical models of rot1 tertiary structure. For experimental structure determination, produce highly purified recombinant rot1 protein (full-length or functional domains) using E. coli or insect cell expression systems optimized for structural studies.

X-ray crystallography requires generation of diffraction-quality crystals of rot1, potentially in complex with binding partners or substrates to capture functionally relevant conformations. Alternatively, cryo-electron microscopy (cryo-EM) can determine structures of rot1 in various functional states without crystallization, particularly valuable for examining rot1 within larger protein complexes.

Nuclear magnetic resonance (NMR) spectroscopy can provide information about protein dynamics and identify regions involved in molecular interactions, especially useful for flexible regions of rot1 that might be involved in regulatory interactions. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map interaction surfaces and conformational changes upon binding to partners or substrates.

Integrate structural data with functional studies by designing targeted mutations based on structural features and examining their effects on protein function, stability, and interaction networks in vivo.

What are the common challenges in expressing and purifying functional recombinant rot1 protein?

Recombinant expression of S. pombe rot1 protein presents several technical challenges. The protein may form inclusion bodies in bacterial expression systems due to improper folding, requiring optimization of expression conditions (temperature, inducer concentration) or use of solubility tags (MBP, SUMO). Consider eukaryotic expression systems like insect cells if bacterial expression yields insoluble protein. The essential nature of rot1 may indicate it functions within protein complexes, potentially requiring co-expression with binding partners for stability.

During purification, rot1 may aggregate or be prone to degradation, necessitating the addition of protease inhibitors and careful buffer optimization. The presence of post-translational modifications important for function may require expression in eukaryotic systems capable of these modifications. If using tagged versions for purification, verify that the tag doesn't interfere with protein folding or function through comparative activity assays.

Verifying proper folding of purified rot1 can be assessed using circular dichroism spectroscopy or limited proteolysis. For proteins involved in specific biochemical activities, develop functional assays to confirm that the recombinant protein retains its native activities .

How can I optimize genetic manipulation techniques for studying rot1 function in S. pombe?

Optimizing genetic manipulation techniques for rot1 studies requires careful consideration of the protein's essential nature. For genome editing, CRISPR-Cas9 systems adapted for S. pombe can generate precise mutations, though care must be taken to maintain viability when targeting essential genes like rot1. Alternatively, the two-step PCR-based mutagenesis approach described for rad9 can be adapted for rot1 modifications .

For stable expression of modified rot1 variants, the stable integration vectors (SIVs) designed specifically for S. pombe offer significant advantages over traditional vectors that create repetitive, unstable genomic loci. These SIVs target different prototrophy genes and integrate predominantly as single copies, providing more consistent expression levels .

When designing conditional alleles to study essential functions, consider temperature-sensitive mutations, auxin-inducible degron systems, or promoter replacements with regulatable promoters like nmt1. For studying specific domains, a complementation approach using plasmid-expressed rot1 variants in a background with the endogenous rot1 under conditional control allows assessment of function without compromising viability.

Fluorescent tagging for localization studies should utilize the modular toolbox of tags optimized for S. pombe to minimize interference with protein function. Always confirm that tagged versions complement rot1 deletion to ensure functionality .

What considerations are important when investigating rot1 involvement in cellular stress responses?

When investigating rot1's potential involvement in cellular stress responses, consider both acute and chronic stress conditions relevant to S. pombe biology. Design experiments to examine rot1 protein levels, post-translational modifications, localization, and interaction partners under various stress conditions including oxidative stress, DNA damage, replication stress, and heat shock.

For phosphorylation analysis, similar to studies of Rad60 in response to hydroxyurea (HU), immunoprecipitate rot1 from stressed and unstressed cells followed by phospho-specific Western blotting or mass spectrometry . Monitor subcellular localization changes using fluorescently tagged rot1 constructs before and after stress induction, with particular attention to nuclear-cytoplasmic distribution shifts.

To identify stress-specific interaction partners, perform co-immunoprecipitation or proximity labeling experiments under various stress conditions. Genetic approaches including synthetic genetic interaction screens with known stress response factors can reveal functional relationships.

For functional studies, examine how rot1 mutations affect survival and recovery following different stressors. Consider the following experimental matrix:

Finally, investigate whether rot1 itself is transcriptionally regulated during stress responses using RT-qPCR or RNA-seq approaches under various stress conditions .

What emerging technologies could advance our understanding of rot1 function in S. pombe?

Several cutting-edge technologies show promise for deepening our understanding of rot1 function. Single-cell proteomics techniques could reveal cell-to-cell variability in rot1 levels and modifications that might be masked in population studies. Advanced microscopy methods including super-resolution techniques (PALM/STORM, STED) can provide unprecedented spatial resolution of rot1 localization and interactions with cellular structures.

Cryo-electron tomography of S. pombe cells could visualize rot1's native context within cellular complexes. Proximity labeling approaches like BioID or TurboID adapted for S. pombe would identify proteins in close proximity to rot1 under various conditions, potentially revealing transient interaction partners.

Integrative multi-omics approaches combining transcriptomics, proteomics, and metabolomics data from rot1 mutants could provide systems-level insights into rot1 function. Development of optogenetic tools for S. pombe would allow temporal control of rot1 activity to study immediate effects of its inactivation.

Emerging genome editing technologies beyond basic CRISPR-Cas9, such as base editors or prime editors, could enable more sophisticated genetic manipulations of rot1 without the need for double-strand breaks. High-throughput automated phenotyping platforms would facilitate comprehensive characterization of rot1 mutant libraries to identify specific functional regions .

How might comparative studies across different yeast species inform our understanding of rot1 function?

Comparative studies across yeast species can provide evolutionary context and functional insights into rot1. Begin by identifying rot1 orthologs in diverse yeast species including Saccharomyces cerevisiae, Candida albicans, and more distantly related species through phylogenetic analysis. Sequence conservation analysis can reveal evolutionarily conserved domains likely to be functionally critical.

Complementation studies testing whether rot1 from different species can rescue S. pombe rot1 mutants would identify functionally conserved regions. Similarly, determine if S. pombe rot1 can complement defects in orthologous genes in other yeast species. Comparative localization studies of rot1 orthologs across species could reveal conserved or divergent subcellular distributions indicative of functional conservation or specialization.

Create chimeric proteins combining domains from rot1 orthologs across species to map functionally interchangeable regions. Perform comparative interaction studies to identify conserved binding partners across species, which likely represent core functional relationships. Analyze species-specific genetic interactions to uncover potential functional divergence or adaptation to different cellular contexts.

Such comparative approaches could reveal whether rot1's functions in DNA repair, stress response, or other cellular processes are evolutionarily conserved or represent species-specific adaptations, providing context for understanding its role in S. pombe biology.