Recombinant Schizosaccharomyces pombe Nuclear control of ATPase protein 2 (nca2)

What is Schizosaccharomyces pombe Nuclear control of ATPase protein 2 (nca2)?

Nuclear control of ATPase protein 2 (nca2) is a 573-amino acid protein (UniProt ID: O74963) found in the fission yeast Schizosaccharomyces pombe, a well-established model organism for eukaryotic cell studies . The protein is encoded by the nca2 gene (also designated as SPBC4B4.02c) and plays roles in cellular processes related to ATPase regulation. S. pombe has become increasingly important as a model organism following its genome sequencing, particularly for studies related to cell cycle, chromosome biology, and other fundamental cellular processes . Within this context, recombinant nca2 protein serves as a valuable tool for investigating specific protein functions and interactions in controlled experimental settings.

How does S. pombe serve as a model organism for protein functional studies?

S. pombe has emerged as a powerful model organism for molecular cell biology due to several advantageous characteristics:

• Ease of genetic manipulation and cell biology techniques

• Well-characterized cell cycle (particularly through Cdc2 studies)

• Fully sequenced genome

• Established research foundations in chromosome biology, mitosis, cytokinesis, and cell morphology

• Conservation of many fundamental cellular mechanisms with higher eukaryotes

These attributes make S. pombe an attractive system for studying protein function through both genetic and biochemical approaches . The organism's popularity increased substantially in the past 20 years following major discoveries related to cell cycle regulation, offering researchers a complementary model to Saccharomyces cerevisiae for investigating eukaryotic cellular processes. For nca2 studies specifically, S. pombe provides a native context for examining the protein's biological roles.

What expression systems are commonly used for recombinant S. pombe nca2 protein?

Recombinant nca2 protein is typically expressed in bacterial systems, with E. coli being the most common host . The commercially available recombinant full-length S. pombe nca2 protein is produced in E. coli with an N-terminal His-tag to facilitate purification . This approach allows for high-yield production of the protein for biochemical and structural studies.

For expression systems, researchers should consider:

The choice of expression system should align with the specific experimental requirements and the protein's characteristics.

What purification methods are most effective for His-tagged nca2 protein?

For His-tagged recombinant nca2 protein, the following purification workflow is recommended:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co2+ resins

• Intermediate Purification: Ion exchange chromatography based on the protein's theoretical pI

• Polishing Step: Size exclusion chromatography to remove aggregates and ensure homogeneity

The commercial recombinant nca2 preparations achieve >90% purity using SDS-PAGE analysis . To maintain protein stability and activity during purification, researchers should use buffers containing:

• Tris or PBS-based buffer systems (typically pH 8.0)

• Stabilizing agents like trehalose (6%) to prevent aggregation

• Protease inhibitors to prevent degradation

After purification, proper storage in aliquots with glycerol (typically 5-50%) is recommended to prevent freeze-thaw damage .

How does nca2 function relate to S. pombe cell cycle regulation?

While the specific role of nca2 in cell cycle regulation has not been fully characterized, it should be considered in the context of S. pombe's well-studied cell cycle regulatory mechanisms. S. pombe has become a model organism for cell cycle studies, with microarray analyses identifying approximately 750 genes with strong cell cycle regulation .

Cell cycle regulation in S. pombe involves:

• Coordinated expression of genes during specific phases

• Regulation by transcription factors including Cdc10 (MBF subunit), Sep1, and Ace2

• Complex promoter structures with multiple regulatory motifs

To investigate potential nca2 involvement in cell cycle processes, researchers could:

• Analyze nca2 expression patterns across synchronized cell populations

• Examine phenotypes of nca2 mutants in relation to cell cycle progression

• Study interactions between nca2 and known cell cycle regulators

The methodological approach used by Rustici et al. and others for cell cycle gene identification could serve as a model for characterizing nca2's potential role in this process .

What are the recommended approaches for studying nca2 protein interactions?

For comprehensive analysis of nca2 protein interactions, researchers should employ multiple complementary approaches:

• Yeast Two-Hybrid Screening:

  • Can identify direct protein-protein interactions

  • Similar to the approach used to identify interactions between Dna2 and other replication proteins in S. pombe

  • Should include appropriate controls to minimize false positives

• Co-Immunoprecipitation:

  • Verifies interactions in more native conditions

  • Can be performed with tagged recombinant nca2 or antibodies against the native protein

  • Western blotting confirms specific interacting partners

• Protein Complex Isolation:

  • Tandem affinity purification (TAP) tagging of nca2

  • Mass spectrometry identification of co-purifying proteins

  • Quantitative analysis to distinguish specific from non-specific interactions

When analyzing potential interactions, researchers should consider that S. pombe proteins often participate in complexes for coordinated functions, as seen with the Dna2 protein which interacts with polymerase delta subunits (Cdc1 and Cdc27), DNA ligase I (Cdc17), and Fen-1 (Rad2) .

What are the optimal storage and handling conditions for recombinant nca2 protein?

Proper storage and handling are critical for maintaining recombinant nca2 protein activity. Based on established protocols for similar recombinant proteins, follow these guidelines :

Storage Recommendations:

• Store lyophilized protein at -20°C/-80°C upon receipt

• After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles

• For short-term storage, keep working aliquots at 4°C for up to one week

• For long-term storage, add glycerol (final concentration 5-50%) and store at -20°C/-80°C

• The default final concentration of glycerol is typically 50%

Reconstitution Protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol for stability if preparing for long-term storage

• Aliquot to minimize freeze-thaw cycles

Handling Precautions:

• Avoid repeated freeze-thaw cycles which can denature the protein

• Maintain cold chain during experiments

• For experimental use, dilute stocks in appropriate buffers immediately before use

How can researchers verify the functional activity of purified nca2 protein?

Verifying functional activity of recombinant nca2 is essential before using it in downstream applications. Since the specific enzymatic activity assays for nca2 may not be well-established, consider these general approaches:

• Structural Integrity Assessment:

  • Circular dichroism (CD) spectroscopy to confirm proper folding

  • Size exclusion chromatography to verify monomeric state or expected oligomerization

  • Thermal shift assays to assess protein stability

• Binding Assays:

  • Surface plasmon resonance (SPR) to measure interactions with known or predicted binding partners

  • Isothermal titration calorimetry (ITC) for quantitative binding parameters

  • Fluorescence polarization assays for smaller ligand interactions

• ATPase-Related Activity:

  • Since nca2 is involved in ATPase regulation, consider adapting ATPase activity assays

  • Measure ATP hydrolysis rates in the presence/absence of nca2

  • Test effects of nca2 on known ATPases in S. pombe

When developing functional assays, researchers might draw inspiration from methodologies used to study other S. pombe proteins like Ubc13 and Mms2, which were functionally characterized using in vitro ubiquitin conjugation assays .

What genetic approaches can be used to study nca2 function in vivo?

To investigate nca2 function within living S. pombe cells, researchers can employ several genetic approaches:

• Gene Disruption/Deletion:

  • Create nca2Δ strains to observe loss-of-function phenotypes

  • Analyze effects on cell morphology, growth rate, and specific cellular processes

  • This approach was successfully used to study dna2+ function in S. pombe

• Temperature-Sensitive Mutants:

  • Generate conditional temperature-sensitive (ts) alleles of nca2+

  • Similar to studies of dna2(ts) mutants that revealed late S-phase arrest phenotypes

  • Allows for temporal control of protein inactivation

• Overexpression Studies:

  • Use nmt1 promoter-based vectors (e.g., pREP series) for controlled overexpression

  • Compare with methodologies used for rho2+ overexpression studies

  • Analyze gain-of-function phenotypes that may reveal protein function

• Fluorescent Tagging:

  • Create GFP or other fluorescent protein fusions to track nca2 localization

  • Monitor dynamic changes in localization during cell cycle or stress responses

  • Combine with time-lapse microscopy for real-time analysis

When designing genetic experiments, researchers should consider that disruption of essential genes in S. pombe often leads to specific phenotypes that provide clues to function, as seen with the chromosome fragmentation observed in dna2+ disruption strains .

How can researchers address solubility challenges with recombinant nca2 protein?

Recombinant proteins from S. pombe can present solubility challenges during expression and purification. To improve nca2 solubility:

• Optimization of Expression Conditions:

  • Test multiple induction temperatures (15°C, 18°C, 25°C, 30°C)

  • Vary IPTG concentration (0.1-1.0 mM) for fine-tuning expression levels

  • Adjust induction time (4h to overnight) to balance yield and solubility

• Solubility Enhancement Approaches:

  • Expression with solubility-enhancing fusion partners (MBP, SUMO, Trx)

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Addition of solubility-enhancing additives to lysis buffer (detergents, amino acids, osmolytes)

• Refolding Strategies if Necessary:

  • Denaturation with urea or guanidine HCl followed by controlled refolding

  • Pulse refolding or dilution methods to minimize aggregation

  • Inclusion of stabilizing agents like arginine or trehalose during refolding

• Buffer Optimization:

  • Screen various pH conditions (typically 6.5-8.5)

  • Test different salt concentrations (100-500 mM NaCl)

  • Evaluate various buffer systems (Tris, HEPES, Phosphate)

The successful production of soluble His-tagged nca2 protein in E. coli suggests that with proper optimization, researchers can achieve sufficient yields of soluble protein .

What are the most informative assays for studying potential roles of nca2 in ATPase regulation?

Given its classification as Nuclear control of ATPase protein 2, nca2 likely plays a role in regulating ATPase activity. To investigate this function, consider these approaches:

• In vitro ATPase Activity Modulation:

  • Measure ATP hydrolysis rates of candidate ATPases with/without nca2

  • Use colorimetric assays (malachite green) or radiometric methods with γ-32P-ATP

  • Test concentration-dependent effects of nca2 on ATPase activity

• Identification of ATPase Targets:

  • Pull-down assays using immobilized recombinant nca2

  • Mass spectrometry identification of interacting ATPases

  • Confirmation of specific interactions using purified proteins

• Regulation Mechanism Investigation:

  • Study post-translational modifications of nca2 and their impact on function

  • Analyze structural changes in target ATPases in presence of nca2

  • Determine if nca2 acts directly on ATPases or indirectly through other factors

• Cellular Energy Metabolism Analysis:

  • Measure ATP/ADP ratios in wild-type vs. nca2 mutant cells

  • Investigate mitochondrial function in nca2 mutants

  • Analyze growth under different carbon sources or metabolic stress conditions

When designing these assays, researchers can draw inspiration from methodologies used to study other regulatory proteins in S. pombe, such as the approach used to characterize Rho2p GTPase's regulation of cell wall α-glucan biosynthesis .

How does S. pombe nca2 compare to homologous proteins in other yeast species?

Comparative analysis of nca2 across different yeast species can provide valuable insights into conserved functions and evolutionary adaptations:

• Sequence Comparison Analysis:

  • Align S. pombe nca2 with potential homologs in S. cerevisiae and other fungi

  • Identify conserved domains that may indicate functional importance

  • Map conservation patterns to predict functional regions

• Functional Complementation Studies:

  • Test if S. pombe nca2 can rescue phenotypes of mutants in homologous genes from other species

  • Express potential homologs from other yeasts in S. pombe nca2Δ strains

  • Compare complementation efficiency to infer functional conservation

• Evolutionary Rate Analysis:

  • Calculate evolutionary rates across different protein regions

  • Identify domains under positive or negative selection

  • Correlate evolutionary patterns with known or predicted functions

When conducting comparative analyses, researchers should consider that many cellular processes are conserved between S. pombe and S. cerevisiae but with important differences, as noted in studies of PCNA modification where PCNA is ubiquitinated during S phase in unperturbed S. pombe cells but sumoylated in S. cerevisiae .

How can nca2 studies be integrated with broader omics approaches?

Integrating nca2-focused research with broader omics approaches can provide a systems-level understanding of its function:

• Transcriptomics Integration:

  • RNA-seq analysis comparing wild-type and nca2 mutant strains

  • Identify genes differentially expressed in response to nca2 perturbation

  • Similar to approaches used for cell cycle gene identification in S. pombe

• Proteomics Applications:

  • Quantitative proteomics to identify proteins affected by nca2 deletion/overexpression

  • Phosphoproteomics to identify signaling pathways connected to nca2 function

  • Interactome mapping using proximity labeling methods (BioID, APEX)

• Metabolomics Approaches:

  • Characterize metabolic changes in nca2 mutants

  • Focus on energy metabolism intermediates given potential ATPase regulatory role

  • Correlate metabolic signatures with phenotypic observations

• Integrative Data Analysis:

  • Network analysis to position nca2 within cellular interaction networks

  • Pathway enrichment analysis of multi-omics datasets

  • Machine learning approaches to predict nca2 functions from integrated data

When designing multi-omics experiments, consider the approach used by researchers studying cell cycle-regulated genes, who integrated time course data with specific perturbation experiments to build a comprehensive understanding of regulatory mechanisms .