Recombinant Schizosaccharomyces pombe UPF0618 protein C8C9.19 (SPAC8C9.19)

What are the storage requirements for Recombinant S. pombe UPF0618 protein C8C9.19?

The recombinant Schizosaccharomyces pombe UPF0618 protein C8C9.19 should be stored at -20°C for standard storage periods . For extended storage periods, it is recommended to maintain the protein at either -20°C or -80°C to preserve stability and functional integrity . Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing cycles should be avoided as they may compromise protein integrity and function . The shelf life varies depending on storage conditions: liquid formulations typically maintain stability for 6 months at -20°C/-80°C, while lyophilized preparations can remain stable for 12 months under the same temperature conditions .

What is the amino acid sequence of UPF0618 protein C8C9.19?

The amino acid sequence of the UPF0618 protein C8C9.19 from Schizosaccharomyces pombe is: MLPNLRRIFASFRTEEEERSYSRKAFFHLIGYITCSVLFSWLVRKKVISSPVVSSPIHAL S . This 61-amino acid sequence represents the full-length protein as expressed in recombinant systems . The protein is classified as a transmembrane protein according to its structural characteristics, suggesting it contains membrane-spanning domains that may be critical for its cellular function . Understanding this sequence is essential for designing experimental approaches involving site-directed mutagenesis, structural studies, or antibody production.

What expression systems are commonly used for producing Recombinant S. pombe UPF0618 protein C8C9.19?

The predominant expression system used for producing Recombinant S. pombe UPF0618 protein C8C9.19 is an in vitro E. coli expression system . This bacterial expression platform allows for high-yield production of the recombinant protein with appropriate post-translational modifications. Commercial preparations typically include an N-terminal 10xHis-tag to facilitate purification and potential downstream applications . While E. coli remains the standard expression system, alternative approaches may include yeast-based expression systems, particularly when studying S. pombe proteins in their native context . For comparative studies, researchers may consider both heterologous (E. coli) and homologous (S. pombe) expression systems to evaluate potential differences in protein folding, modification, and activity.

How can proteome analysis be optimized to study UPF0618 protein C8C9.19 expression patterns?

Optimizing proteome analysis for studying UPF0618 protein C8C9.19 expression patterns requires a multidimensional approach. Researchers should employ two-dimensional liquid chromatography coupled offline to MALDI MS for comprehensive protein separation and identification . To reduce measurement time while maintaining data quality, implement a pooling scheme for multidimensional separation as demonstrated in comparative S. pombe proteome studies . Quantitative information should be generated through isobaric labeling using the iTRAQ approach, with application of a global internal standard to ensure consistent comparison across experimental conditions .

For cell lysate preparation, follow these validated steps:

• Harvest cells by centrifugation (3,000 × g for 15 min at 4°C)

• Transfer supernatant to 2 ml Eppendorf tubes

• Repeat extraction steps to maximize protein yield

• Determine protein concentration using Bradford assay

• Alkylate proteins with IAA (375 mM) at room temperature for 30 minutes in darkness

• Concentrate and buffer-exchange using 0.5 ml Amicon ultra centrifugal filter units (MWCO: 3 kDa)

• Centrifuge at 14,000 × g (25 min, 4°C) and re-buffer with appropriate solutions

This comprehensive approach enables detection of subtle changes in protein expression levels in response to various experimental conditions or genetic modifications.

What approaches can be used to study the potential role of UPF0618 protein C8C9.19 in replication fork dynamics?

Investigating the potential role of UPF0618 protein C8C9.19 in replication fork dynamics requires specialized experimental approaches. Researchers should consider employing a site-specific replication fork barrier (RFB) system similar to the RTS1 (Replication Termination Sequence 1) paradigm established for S. pombe studies . This approach involves creating constructs that allow for controlled replication fork arrest and restart, enabling observation of protein recruitment and function during these critical processes.

A methodological framework should include:

• Generation of S. pombe strains with modified expression of UPF0618 protein C8C9.19 (deletion, overexpression, or mutation)

• Implementation of a site-specific RFB system to study replication dynamics

• Application of 2D gel analysis of replicating plasmids to visualize replication intermediates

• Analysis of Y-intermediates to assess replication fork progression and stability

• Cell synchronization using ATP-analogue sensitive alleles (such as cdc2asM17) to study cell-cycle specific effects

• Incorporation of reversion frequency assays to quantify genetic instability associated with replication fork collapse

This experimental framework allows for detailed mechanistic studies on how UPF0618 protein C8C9.19 might function in the context of DNA replication, potentially revealing roles in fork stabilization, restart, or quality control.

How can researchers investigate the potential transmembrane functions of UPF0618 protein C8C9.19?

Investigating the transmembrane functions of UPF0618 protein C8C9.19 requires specialized approaches that address both structural and functional aspects of membrane proteins. Given its classification as a transmembrane protein , researchers should employ a multifaceted strategy:

• Topology Mapping: Utilize computational prediction tools combined with experimental approaches such as glycosylation mapping or protease protection assays to determine transmembrane segments and their orientation.

• Membrane Localization Studies: Employ fluorescent protein tagging (ensuring tags don't disrupt membrane insertion) followed by confocal microscopy to determine subcellular localization within S. pombe cells.

• Membrane Fluidity Analysis: As membrane composition significantly impacts protein function and secretion efficiency in S. pombe , researchers should analyze how changes in membrane fluidity affect UPF0618 protein C8C9.19 activity through fluorescence anisotropy measurements.

• Interactome Analysis: Implement proximity-based labeling techniques (such as BioID or APEX) specifically adapted for membrane proteins to identify interacting partners within the membrane environment.

• Functional Reconstitution: For definitive functional characterization, purify the protein and reconstitute it into liposomes or nanodiscs to study its activity in a controlled membrane environment.

This integrated approach will provide insights into both the localization and functional aspects of this transmembrane protein within the context of S. pombe cellular biology.

What methods can be applied to study potential post-translational modifications of UPF0618 protein C8C9.19?

Studying post-translational modifications (PTMs) of UPF0618 protein C8C9.19 requires sophisticated analytical approaches that can detect and characterize diverse modification types. A comprehensive methodology should include:

• Mass Spectrometry-Based Identification: Employ high-resolution MS/MS analysis after enrichment for specific PTMs (phosphorylation, glycosylation, ubiquitination). The two-dimensional LC coupled to MALDI MS approach described for S. pombe proteome studies can be adapted specifically for PTM analysis .

• Site-Directed Mutagenesis: Systematically modify predicted PTM sites in the protein sequence (MLPNLRRIFASFRTEEEERSYSRKAFFHLIGYITCSVLFSWLVRKKVISSPVVSSPIHAL S) to assess functional consequences .

• Modification-Specific Antibodies: Develop antibodies that specifically recognize modified forms of the protein for western blotting and immunoprecipitation studies.

• Inhibitor Studies: Apply specific inhibitors of PTM-mediating enzymes to assess dynamic regulation of UPF0618 protein C8C9.19 modification status.

• In Vitro Modification Assays: Perform enzyme-substrate reactions with purified kinases, glycosyltransferases, or other modification enzymes to confirm direct modification capability.

• Cycloheximide Chase Experiments: Assess protein stability and turnover in relation to modification status using methods similar to those described for S. pombe protein analysis, with cycloheximide treatment at 100 μg/ml .

This multifaceted approach will provide comprehensive insights into the PTM landscape of UPF0618 protein C8C9.19 and its functional significance in cellular processes.

How does UPF0618 protein C8C9.19 expression correlate with global protein secretion patterns in S. pombe?

The relationship between UPF0618 protein C8C9.19 expression and global protein secretion patterns in S. pombe should be investigated through a systems biology approach that integrates multiple levels of analysis. Recent comparative proteome studies in S. pombe have demonstrated that high-level protein secretion causes global changes in protein expression profiles throughout the cell . To establish correlations between UPF0618 protein C8C9.19 and secretion patterns, researchers should:

• Perform quantitative proteomics using isobaric labeling (iTRAQ approach) to compare wild-type strains with those overexpressing or depleted of UPF0618 protein C8C9.19 .

• Analyze changes across multiple biological pathways, particularly focusing on secretory pathway components, membrane composition factors, and amino acid biosynthesis networks that have been identified as critical for efficient protein secretion in S. pombe .

• Integrate findings with targeted supplementation experiments, as proteomic results have shown that amino acid biosynthesis and membrane fluidity can be strategically modified to enhance protein secretion .

• Consider the impact of media composition and growth conditions, as these environmental factors interact significantly with cellular secretion machinery.

This integrated approach will help determine whether UPF0618 protein C8C9.19 functions as a limiting factor or regulatory component within the broader secretory network of S. pombe.

What RNA splicing mechanisms might regulate UPF0618 protein C8C9.19 expression?

RNA splicing regulation of UPF0618 protein C8C9.19 expression may involve several mechanisms that can be investigated using approaches validated in S. pombe research. Recent studies have demonstrated that factors like Rtf2 play critical roles in efficient splicing of specific introns in S. pombe . To investigate splicing regulation of UPF0618 protein C8C9.19, researchers should:

• Intron Retention Analysis: Use PCR amplification of cDNA to detect potential intron retention events, similar to methods used to analyze rtf1 intron retention in S. pombe (using paired primers to amplify across intron-exon boundaries) .

• Splicing Factor Interactions: Investigate interactions between UPF0618 protein C8C9.19 mRNA and known splicing regulators using RNA immunoprecipitation techniques.

• Cell Cycle-Dependent Regulation: Implement synchronization protocols using ATP-analogue sensitive alleles (such as cdc2asM17) with 3-BrB-PP1 treatment to analyze cell cycle-dependent splicing patterns .

• Stress Response Analysis: Examine how various cellular stresses affect splicing efficiency and accuracy for UPF0618 protein C8C9.19.

• Comparative Genomics: Analyze intron conservation across related Schizosaccharomyces species to identify potentially regulatory intronic sequences.

This multi-faceted approach will provide insights into the post-transcriptional regulation mechanisms that may control UPF0618 protein C8C9.19 expression levels and isoform diversity in response to varying cellular conditions.

What protein extraction protocols are optimal for UPF0618 protein C8C9.19 recovery from S. pombe?

Optimizing protein extraction for UPF0618 protein C8C9.19 from S. pombe requires specialized protocols that account for its transmembrane nature . The following methodology has been validated for efficient recovery:

• Cell Lysis: Harvest S. pombe cells in logarithmic growth phase (approximately 5×10^7 cells) and resuspend in 200 μl of 20% trichloroacetic acid (TCA) for initial protein precipitation and stabilization .

• Mechanical Disruption: Lyse cells using glass beads and a Ribolyser (or equivalent) with three 30-second cycles at 6.5 m/s to ensure complete membrane disruption without excessive protein degradation .

• Protein Solubilization: Following bead removal, resuspend the pellet in 200 μl of 1X Protein Loading Buffer (250 mM Tris pH 6.8, 8% SDS, 20% glycerol, 20% β-mercaptoethanol, and 0.4% bromophenol blue) and heat at 95°C for 10 minutes to fully denature and solubilize membrane proteins .

• Detergent Optimization: For transmembrane proteins like UPF0618 protein C8C9.19, consider testing alternative detergents (DDM, CHAPS, or digitonin) if SDS extraction yields insufficient recovery.

• Purification Strategy: For recombinant protein, leverage the N-terminal 10xHis-tag for affinity purification using nickel or cobalt resins under denaturing or native conditions depending on downstream applications .

This extraction protocol maximizes recovery while maintaining protein integrity, enabling subsequent analytical procedures including western blotting, mass spectrometry, or functional assays.

What are the key considerations for designing experiments to study UPF0618 protein C8C9.19 interactions with other cellular components?

Designing experiments to study UPF0618 protein C8C9.19 interactions requires careful consideration of its transmembrane nature and potential integration into cellular processes. Researchers should address the following key considerations:

• Environment-Appropriate Methods: Select interaction detection methods compatible with membrane environments, such as membrane yeast two-hybrid systems, split-ubiquitin assays, or proximity-based labeling approaches rather than conventional yeast two-hybrid screens.

• Expression Level Control: Utilize established S. pombe expression systems like the nmt1 promoter, which allows for regulated expression through plasmid-based or chromosomal integration approaches . This ensures physiologically relevant interaction studies without artifacts from extreme overexpression.

• Control Constructs: Design appropriate controls including:

  • Tag-only constructs to identify tag-mediated false positives

  • Known non-interacting membrane proteins as negative controls

  • Validated interacting pairs as positive controls

• Cell Synchronization: Implement cell cycle synchronization using ATP-analogue sensitive alleles (cdc2asM17) with 3-BrB-PP1 treatment to identify cell cycle-dependent interactions .

• Data Validation Pipeline:

  • Initial screening through high-throughput methods

  • Secondary validation through co-immunoprecipitation or pull-down assays

  • Tertiary validation through functional assays to establish biological relevance

  • Final verification through reciprocal tagging experiments

This comprehensive experimental design framework ensures robust identification of physiologically relevant interaction partners while minimizing both false positives and negatives.

How can UPF0618 protein C8C9.19 research be integrated with broader S. pombe proteome studies?

Integrating UPF0618 protein C8C9.19 research with broader S. pombe proteome studies requires a systematic approach that positions this specific protein within the context of global cellular networks. Researchers should:

• Apply Standardized Proteomics Workflows: Utilize the established two-dimensional LC coupled offline to MALDI MS methodology with iTRAQ labeling for quantitative comparisons across conditions . This ensures compatibility with existing S. pombe proteome datasets.

• Implement Global Internal Standard Approaches: Apply the global internal standard methodology described in comparative proteome analyses to enable accurate quantification across multiple experimental conditions and integration with existing datasets .

• Connect with Key Cellular Pathways: Specifically investigate connections to:

  • Protein secretion pathways, which show global changes during high-level expression

  • Amino acid biosynthesis pathways, identified as limiting factors for protein production

  • Membrane composition networks, which impact protein function and secretion efficiency

• Link to DNA Replication Processes: Explore potential connections to replication fork dynamics using established S. pombe experimental systems like the RTS1 replication fork barrier .

• Data Integration Strategy: Develop computational approaches to integrate UPF0618 protein C8C9.19-specific data with publicly available proteome datasets using standardized identifiers like UniProt accession numbers (Q4ZGE1) .

This integrated approach positions UPF0618 protein C8C9.19 research within the broader context of S. pombe cellular biology, enabling discovery of novel functional connections and regulatory networks.

What computational tools are most effective for predicting UPF0618 protein C8C9.19 function based on structural analysis?

Predicting UPF0618 protein C8C9.19 function through computational structural analysis requires a multi-tool approach that addresses the challenges of transmembrane protein analysis. Researchers should employ:

• Transmembrane Topology Prediction:

  • TMHMM, HMMTOP, and Phobius for predicting transmembrane segments

  • SignalP for signal peptide identification

  • TopPred for topology orientation prediction

• Structural Modeling Pipeline:

  • AlphaFold2 or RoseTTAFold for ab initio structure prediction, particularly effective for smaller proteins like UPF0618 protein C8C9.19 (61 amino acids)

  • Membrane-specific refinement using MEMOIR or other membrane protein-specific modeling tools

  • Model validation through ProCheck and WHATIF

• Functional Site Prediction:

  • ConSurf for evolutionary conservation mapping

  • FTMap for binding site identification

  • COACH for ligand binding site prediction

  • PredictProtein for functional residue identification

• Comparative Approaches:

  • HHpred for remote homology detection

  • DALI for structural similarity searches

  • InterProScan for domain and motif identification

• Integration with Experimental Data:

  • Utilize sequence information (MLPNLRRIFASFRTEEEERSYSRKAFFHLIGYITCSVLFSWLVRKKVISSPVVSSPIHAL S) as input for all analyses

  • Refine predictions using available experimental constraints

This comprehensive computational approach maximizes the predictive power for this challenging transmembrane protein, generating testable hypotheses about its structure-function relationships that can guide subsequent experimental investigation.