Recombinant Saccharomyces cerevisiae Putative UPF0479 protein YBL113W-A (YBL113W-A)

What is YBL113W-A and what is known about its function?

YBL113W-A is classified as a putative UPF0479 family protein in Saccharomyces cerevisiae. It is currently annotated as a dubious open reading frame (ORF) that is unlikely to encode a functional protein, based on available experimental and comparative sequence data. It was identified through gene-trapping, microarray-based expression analysis, and genome-wide homology searching . While its precise function remains undetermined, it appears to have genetic and functional relationships with telomeric proteins in S. cerevisiae, particularly with helicase-like proteins encoded within the telomeric Y' element .

What protein interactions have been identified for YBL113W-A?

Protein interaction studies using the STRING database reveal that YBL113W-A potentially interacts with several proteins, including:

What expression systems are optimal for producing recombinant YBL113W-A protein?

Recombinant YBL113W-A is typically produced using one of several expression systems:

• E. coli expression system: The most common approach due to its simplicity, high yield, and cost-effectiveness . The protein can be expressed with an N-terminal or C-terminal His-tag to facilitate purification.

• Cell-free expression system: Offers advantages for potentially toxic or difficult-to-express proteins by avoiding cellular constraints . This system may be particularly useful for YBL113W-A given its classification as a dubious ORF.

• Yeast expression system: Using S. cerevisiae itself as the expression host can provide proper post-translational modifications and folding environment, although yields may be lower than in E. coli .

For optimal expression, researchers should consider testing multiple systems in parallel, as protein purity of ≥85% as determined by SDS-PAGE is typically achievable regardless of the chosen system .

What purification strategies are most effective for recombinant YBL113W-A?

Based on established protocols for similar S. cerevisiae recombinant proteins, an effective purification strategy includes:

• Immobilized metal affinity chromatography (IMAC): For His-tagged YBL113W-A, Ni-NTA superflow resin with a gradient or batch elution using increasing imidazole concentrations (20, 40, 60, 100, and 250 mM) is effective .

• Size exclusion chromatography: As a secondary purification step to improve purity, particularly if oligomeric states or aggregation is a concern .

• Buffer optimization: Proper buffer selection (typically 50 mM Tris-HCl, pH 8.0, with 300 mM NaCl) is critical for protein stability .

Protein concentration can be determined using a BCA protein assay kit, and purity assessed via SDS-PAGE . For validation, western blotting using anti-His antibodies or specific antibodies against YBL113W-A can confirm the identity of the purified protein .

How can researchers investigate potential functions of YBL113W-A despite its classification as a dubious ORF?

Despite being classified as a dubious ORF, there are several approaches to investigate potential functions:

• Comparative genomic analysis: Compare YBL113W-A with related UPF0479 family proteins across species to identify conserved domains or motifs .

• Synthetic genetic array (SGA) analysis: Systematic creation of double mutants with known telomeric proteins to identify genetic interactions and potential functional relationships .

• Proximity-based labeling: Methods such as BioID or APEX can identify proteins that physically interact with YBL113W-A in vivo, providing clues about its cellular context .

• Structural studies: Determining the three-dimensional structure through X-ray crystallography or cryo-EM could reveal functional domains not apparent from sequence analysis alone .

• Localization studies: Fluorescent tagging to determine subcellular localization, which may provide insights into function based on co-localization with known cellular structures .

Each approach should be implemented with appropriate controls, including comparisons with other dubious ORFs and known functional proteins to distinguish between technical artifacts and biologically meaningful results .

What experimental design approaches are most suitable for studying potential roles of YBL113W-A in telomere biology?

Given the putative association of YBL113W-A with telomeric Y' element proteins, a systematic experimental design approach should include:

• Two-factorial experimental design: Compare wild-type and YBL113W-A deletion strains under normal and telomere stress conditions . For example:

• Telomere-specific assays:

  • Southern blot analysis of telomere length

  • Chromatin immunoprecipitation (ChIP) to assess telomeric protein binding

  • Fluorescence in situ hybridization (FISH) to visualize telomere clustering

• DNA damage response assessment:

  • Sensitivity to DNA damaging agents (e.g., MMS, HU)

  • Homologous recombination frequency measurement

  • NHEJ efficiency assays

Statistical analysis should begin with determining main effects of YBL113W-A deletion on measured variables, followed by post-hoc analysis to examine interactions between experimental factors .

What are the challenges in expressing and purifying membrane-associated UPF0479 family proteins like YBL113W-A?

UPF0479 family proteins, including some members identified as membrane proteins, present specific challenges:

• Solubility issues: These proteins may form inclusion bodies in E. coli expression systems. Solutions include:

  • Use of solubility tags (SUMO, MBP, or TrxA)

  • Lower induction temperatures (16-20°C)

  • Co-expression with chaperones

  • Specialized membrane protein expression strains (e.g., C43(DE3))

• Detergent selection: If YBL113W-A has membrane association properties, proper detergent selection for extraction and purification is critical. Consider:

  • Mild non-ionic detergents (DDM, LMNG)

  • Detergent screening to optimize solubilization conditions

  • Nanodiscs or amphipols for maintaining native-like environment

• Stability optimization: Buffer supplements that can improve stability:

  • Glycerol (10-20%)

  • EGTA or EDTA for chelating metal ions

  • Reducing agents to prevent oxidation of cysteine residues

Each of these approaches should be systematically tested and optimized for YBL113W-A specifically, as optimal conditions may vary even among related proteins .

How can researchers validate antibodies against YBL113W-A for immunoprecipitation and immunofluorescence applications?

Validation of antibodies against YBL113W-A should follow a systematic approach:

• Western blot validation:

  • Test against recombinant protein (positive control)

  • Test against yeast whole cell extract (wild-type vs. deletion strain)

  • Verify specificity by competition with excess antigen

• Immunoprecipitation validation:

  • Perform pull-down assays followed by mass spectrometry

  • Confirm capture of endogenous protein from yeast lysates

  • Assess co-immunoprecipitation of known interacting partners

• Immunofluorescence validation:

  • Compare localization pattern with GFP-tagged protein expression

  • Perform peptide competition assays to confirm specificity

  • Include knockout/knockdown controls to verify signal specificity

• Cross-reactivity assessment:

  • Test against related UPF0479 family members

  • Assess potential cross-reactivity with other telomeric proteins

Researchers should expect approximately 85% purity for recombinant antibodies, with validation across multiple assay platforms to ensure reproducibility and specificity .

What are the advantages and limitations of using S. cerevisiae for studying proteins like YBL113W-A?

S. cerevisiae offers numerous advantages as a model organism for studying proteins like YBL113W-A:

Advantages:

• Genetic tractability: Easy to create gene deletions, insertions, and modifications .

• Well-characterized genome: The S. cerevisiae genome is fully sequenced with approximately 6,000 genes, facilitating comparative genomic analyses .

• Conservation of biological processes: Many fundamental biological processes are well-conserved between yeast and higher eukaryotes, including humans .

• Rapid growth and simple cultivation: Allows for quick experimental turnaround and high-throughput studies .

• Extensive genetic and proteomic tools: Availability of genome-wide deletion collections, overexpression libraries, and protein tagging resources .

Limitations:

• Evolutionary distance from humans: Some biological processes have diverged significantly .

• Absence of certain cellular processes: Some pathways present in multicellular organisms are absent in yeast .

• Different post-translational modifications: Some protein modifications may differ from those in higher eukaryotes .

• Telomere biology differences: While fundamental mechanisms are conserved, specific aspects of telomere maintenance differ between yeast and mammals .

For YBL113W-A specifically, researchers should consider that dubious ORFs in yeast may still have functional significance, possibly producing non-coding RNAs or small peptides that play regulatory roles .

What experimental conditions can enhance recombinant protein production in S. cerevisiae fed-batch cultures?

For optimizing recombinant protein production in S. cerevisiae fed-batch cultures:

• Glucose feed rate optimization: As demonstrated with β-galactosidase model protein, an average glucose feed rate of 1.31 g glucose h-1 results in maximum protein production rates of 831-950 units ml-1 h-1 and maximum cell production rates of 0.520-0.524 mg ml-1 h-1 .

• Temperature and pH control:

  • Optimal temperature: 28-30°C

  • Optimal pH: 5.5-6.0

  • Precise control of these parameters throughout cultivation is essential

• Media composition optimization:

  • Base medium components (per liter): 20 g glucose, 5 g (NH4)2SO4, 3 g KH2PO4, 0.5 g MgSO4·7H2O

  • Supplementation with trace elements and vitamins

  • Addition of amino acid supplements for auxotrophic strains

• Induction strategy:

  • For constitutive promoters: Focus on biomass accumulation before initiating fed-batch phase

  • For inducible promoters (GAL1, CUP1): Timing of inducer addition is critical and should be optimized

• Monitoring gene stability:

  • Assess segregational stability throughout cultivation

  • Monitor plasmid copy number if episomal vectors are used

These parameters should be systematically optimized for YBL113W-A expression specifically, as optimal conditions may vary for different recombinant proteins even within the same expression system .

How should researchers interpret protein-protein interaction data for YBL113W-A given its dubious ORF status?

When interpreting protein-protein interaction data for dubious ORFs like YBL113W-A:

• Apply stringent statistical thresholds: Use more conservative significance thresholds than for characterized proteins to minimize false positives .

• Cross-validate with multiple methods: Confirm interactions observed in computational predictions (e.g., STRING database) with experimental methods such as:

  • Yeast two-hybrid (Y2H)

  • Co-immunoprecipitation followed by mass spectrometry

  • Bimolecular fluorescence complementation (BiFC)

• Consider genomic context: Evaluate interactions in the context of telomeric location and potential overlap with other genes or regulatory elements .

• Compare with related UPF0479 family members: Analyze whether interaction patterns are conserved among family members or unique to YBL113W-A .

• Functional enrichment analysis: Determine if interacting partners are enriched for specific biological processes, which may suggest functional roles even for a dubious ORF .

The interpretation should acknowledge the preliminary nature of such findings and the possibility that observed interactions could result from:

• Actual biological function despite dubious ORF annotation

• Regulatory non-coding RNA encoded in the region

• Small peptides with regulatory functions

• Artifactual interactions due to experimental limitations

What statistical approaches are appropriate for analyzing experimental data from studies involving YBL113W-A?

When analyzing experimental data involving YBL113W-A, appropriate statistical approaches include:

• For comparative studies (wild-type vs. deletion):

  • Student's t-test for single variable comparisons

  • ANOVA for multi-variable comparisons

  • Post-hoc tests (Tukey's, Bonferroni) for multiple comparisons

  • Non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis) for non-normally distributed data

• For factorial experimental designs:

  • Two-way or three-way ANOVA to analyze main effects and interactions

  • Follow with appropriate post-hoc tests to identify specific significant comparisons

• For high-throughput data (e.g., RNA-seq comparing ΔyblL113W-A to wild-type):

  • Multiple testing correction (Benjamini-Hochberg procedure)

  • Fold change thresholds in addition to p-value cutoffs

  • Gene set enrichment analysis for pathway-level insights

• For protein-protein interaction studies:

  • Permutation tests to establish significance thresholds

  • Network analysis metrics (betweenness centrality, clustering coefficient)

  • Enrichment analysis of interaction networks

Data visualization should include appropriate error bars (standard deviation or standard error) and clear indication of statistical significance levels. Sample sizes should be reported, and power analyses performed to ensure adequate statistical power .

How might YBL113W-A be involved in telomere maintenance or DNA damage response pathways?

While direct evidence for YBL113W-A's role in telomere maintenance is limited, its genetic location and interaction patterns suggest potential involvement:

• Telomeric location context: YBL113W-A is located within the telomeric region and shows strong predicted interactions with helicase-like proteins encoded within the telomeric Y' element (YBL111C, YBL112C, YBL113C) .

• Potential parallels with Rev7: Recent studies have shown that S. cerevisiae Rev7 plays a role in DNA damage repair by promoting non-homologous end joining (NHEJ) while inhibiting homologous recombination (HR). Rev7 interacts with the Mre11-Rad50-Xrs2 (MRX) complex to regulate double-strand break repair pathway choice . YBL113W-A might function in similar regulatory pathways, possibly as part of telomeric protection mechanisms.

• Telomere position effect: YBL113W-A's location near telomeres suggests it could be subject to telomere position effect variegation, potentially functioning in response to telomere length changes or damage .

• Stress response context: Related telomeric proteins like YBL111C relocalize from mitochondrion to cytoplasm upon DNA replication stress, suggesting a potential role for this protein family in stress response .

Experimental approaches to investigate these possibilities would include:

• Telomere length analysis in deletion strains

• Sensitivity assays to DNA damaging agents

• Chromatin immunoprecipitation to assess telomeric association

• Double mutant analysis with known telomere maintenance genes

What approaches can be used to determine if YBL113W-A encodes a functional protein despite its dubious ORF annotation?

To determine if YBL113W-A encodes a functional protein despite its dubious ORF annotation:

• Ribosome profiling (Ribo-seq): This technique can detect translation of small ORFs and non-canonical translation products by sequencing ribosome-protected mRNA fragments. Evidence of ribosome occupancy across the YBL113W-A sequence would suggest active translation .

• Mass spectrometry-based proteomics:

  • Targeted peptide identification using synthetic reference peptides

  • Enrichment strategies for low-abundance proteins

  • Analysis of post-translational modifications

• Functional complementation assays:

  • Express YBL113W-A in deletion strains showing phenotypes

  • Test if expression rescues observed phenotypic defects

  • Include controls with mutated start codons or frameshift mutations

• Conservation analysis across yeast species:

  • Comparative analysis of orthologous regions in related yeast species

  • Calculation of dN/dS ratios to detect selective pressure

  • Identification of conserved domains or motifs

• RNA-based functions:

  • Investigate potential non-coding RNA functions of the transcript

  • RNA structure prediction and conservation analysis

  • RNA-protein interaction studies

Each approach should include appropriate controls and validation steps to distinguish between technical artifacts and true biological functions. The combination of multiple lines of evidence would provide the strongest support for functional significance .

What emerging technologies could advance our understanding of YBL113W-A and related UPF0479 family proteins?

Several cutting-edge technologies could significantly advance our understanding of YBL113W-A:

• CRISPR-based approaches:

  • CRISPRi for precise transcriptional repression

  • CRISPRa for targeted activation

  • Base editing for introduction of specific mutations without double-strand breaks

  • Prime editing for precise nucleotide changes

• Single-cell technologies:

  • Single-cell RNA-seq to detect cell-to-cell variability in expression

  • Single-cell proteomics to measure protein abundance in individual cells

  • Live-cell imaging with improved sensitivity for low-abundance proteins

• Structural biology advances:

  • Cryo-EM for determining structures of membrane-associated proteins

  • Integrative structural biology combining multiple experimental approaches

  • AlphaFold2 and related AI tools for structure prediction

• Proximity labeling techniques:

  • TurboID or miniTurbo for rapid proximity labeling

  • Split-TurboID for detecting protein-protein interactions

  • APEX2 for subcellular localization studies

• Long-read sequencing:

  • Nanopore direct RNA sequencing for transcript isoform detection

  • Full-length transcript analysis to identify potential regulatory elements

  • Methylation analysis to detect epigenetic regulation

These technologies, particularly when used in combination, could provide unprecedented insights into the potential functions and interactions of YBL113W-A, even if it represents a non-canonical gene or regulatory element rather than a traditional protein-coding gene .

How might studies of YBL113W-A contribute to our broader understanding of dubious ORFs in eukaryotic genomes?

Studies of YBL113W-A could significantly impact our understanding of dubious ORFs in several ways:

• Refinement of genome annotation criteria: Systematic functional analysis of YBL113W-A could help establish improved criteria for classifying ORFs as dubious versus functional, potentially revealing limitations in current annotation approaches .

• Discovery of non-canonical gene functions: Investigation might reveal:

  • Conditional expression under specific stress conditions

  • Production of functional small peptides

  • Regulatory roles of the transcript itself

  • Structural roles of the DNA or RNA sequence

• Evolutionary insights: Comparative analysis across yeast species could reveal:

  • Conservation patterns inconsistent with non-functionality

  • Recent pseudogenization events

  • Lineage-specific functionalization of previously non-coding sequences

• Development of systematic approaches: Methods developed to study YBL113W-A could be applied to:

  • Genome-wide analysis of other dubious ORFs

  • Identification of patterns in dubious ORF distributions

  • Integration of multiple data types for improved annotation

• Telomere biology context: Given the telomeric location of YBL113W-A, findings could contribute to understanding:

  • The role of subtelomeric regions in genome evolution

  • Potential adaptive functions of telomeric ORFs

  • Dynamic responses to environmental stresses