Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YIR014W (YIR014W)

What genomic context and sequence features characterize YIR014W in S. cerevisiae?

YIR014W is an open reading frame located on chromosome IX of the S. cerevisiae genome. Initial characterization begins with bioinformatic analysis using tools such as BLAST, Pfam, and InterPro to identify conserved domains and potential functional motifs. Analysis should include evaluation of sequence conservation across fungal species, identification of potential transmembrane domains, signal peptides, and post-translational modification sites.

To experimentally verify expression, RT-qPCR is recommended using primers specific to the YIR014W sequence. For optimal results, extract RNA from cells grown under multiple conditions (logarithmic phase, stationary phase, stress conditions) to determine if expression is constitutive or condition-dependent. This approach mirrors methods used for studying other uncharacterized yeast proteins and provides baseline data for subsequent functional studies .

What are the most effective approaches to determine subcellular localization of YIR014W?

To determine subcellular localization, several complementary approaches should be employed:

• Fluorescent Protein Tagging: Generate C-terminal or N-terminal GFP fusion constructs using CRISPR/Cas9-based genome editing technology . When designing fusion proteins, carefully consider:

  • Tag position (C vs. N-terminal) to minimize functional disruption

  • Flexible linker sequences to reduce steric hindrance

  • Native promoter usage to maintain physiological expression levels

• Immunolocalization: Develop antibodies against YIR014W or use epitope tagging (HA, Myc, FLAG) followed by immunofluorescence microscopy using fixed yeast cells.

• Subcellular Fractionation: Perform differential centrifugation to isolate cellular components followed by Western blot analysis to detect YIR014W in specific fractions.

• Co-localization Studies: Use known organelle markers to confirm localization pattern observed with fluorescent tagging.

Quantitative analysis of localization should include measurement of signal intensity across cellular compartments under different growth conditions to detect potential translocation events.

How can I verify if YIR014W is involved in cell wall organization?

Given that many uncharacterized yeast proteins play roles in cell wall organization, this represents a compelling avenue for investigation:

• Cell Wall Integrity Assays: Expose YIR014W deletion or overexpression strains to cell wall-perturbing agents such as Congo Red (CR), calcofluor white, or SDS . Quantify growth differences compared to wild-type strains through spot assays or growth curve analysis.

• Cell Wall Composition Analysis: Extract and analyze cell wall components (β-glucans, mannoproteins, chitin) using enzymatic digestion followed by HPLC or mass spectrometry.

• Electron Microscopy: Examine cell wall ultrastructure in YIR014W mutants compared to wild-type using transmission electron microscopy.

• Cell Surface Protein Analysis: Use comparative secretome analysis to identify changes in cell wall proteins when YIR014W is disrupted or overexpressed . This can reveal functional relationships between YIR014W and known cell wall proteins.

The observation that disruption of certain cell wall proteins like CWP2 affects resistance to Congo Red suggests similar approaches may be valuable for characterizing YIR014W .

What expression systems and vectors are optimal for recombinant production of YIR014W?

For optimal expression of recombinant YIR014W, consider the following methodological approaches:

• Expression System Selection:

  • Homologous expression: Using S. cerevisiae offers correct post-translational modifications and proper folding environment.

  • Heterologous expression: E. coli or P. pastoris systems may provide higher yields but require optimization for yeast proteins.

• Vector Design Considerations:

  • Promoter selection: For constitutive expression, use PGK1 or TDH3 promoters ; for inducible expression, GAL1 promoter provides tight regulation.

  • Affinity tags: N-terminal or C-terminal 6xHis, FLAG, or GST tags facilitate purification. Include a TEV protease cleavage site for tag removal.

  • Signal sequences: If secretion is desired, incorporate the α-mating factor or SUC2 signal peptide.

• Strain Selection:

  • Protease-deficient strains (pep4Δ) increase stability of recombinant proteins.

  • Strains with impaired cell wall integrity (e.g., cwp2Δ) may enhance protein secretion if YIR014W is difficult to extract .

• Optimization Strategies:

  • Codon optimization for S. cerevisiae if expressing in homologous system.

  • Temperature (20-30°C) and expression time (24-96 hours) should be systematically tested.

  • Media composition affects expression levels; compare YPD, synthetic defined media, and specialized expression media.

Expression levels should be monitored by SDS-PAGE and Western blot at various timepoints during culture growth.

How can I optimize extraction and purification of YIR014W while maintaining protein integrity?

Efficient extraction and purification of YIR014W requires careful consideration of:

• Cell Lysis Methods:

  • Mechanical disruption: Glass bead beating or homogenization is effective for yeast cells. Use brief pulses with cooling intervals to prevent protein denaturation.

  • Enzymatic lysis: Zymolyase treatment followed by gentle osmotic shock preserves protein structure.

  • Chemical lysis: Detergent-based methods using Triton X-100 or CHAPS may be necessary if YIR014W is membrane-associated.

• Buffer Optimization:

  Buffer Component	Range to Test	Purpose
  pH	6.0-8.0	Stability optimization
  NaCl	100-500 mM	Reduce non-specific interactions
  Glycerol	5-15%	Enhance protein stability
  Reducing agents	1-5 mM DTT or β-ME	Prevent disulfide bond formation
  Protease inhibitors	PMSF, EDTA, cocktail	Prevent degradation

• Purification Strategy:

  • Initial capture: Affinity chromatography using the engineered tag (Ni-NTA for His-tagged proteins).

  • Intermediate purification: Ion exchange chromatography based on predicted pI of YIR014W.

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

• Quality Control:

  • Assess purity by SDS-PAGE with Coomassie or silver staining.

  • Verify identity by mass spectrometry or Western blotting.

  • Check activity using functional assays developed based on bioinformatic predictions.

For membrane-associated proteins, consider detergent screening (n-dodecyl-β-D-maltoside, digitonin, CHAPS) to identify optimal solubilization conditions.

How can CRISPR/Cas9 genome editing be effectively applied to characterize YIR014W function?

CRISPR/Cas9 technology offers powerful approaches for functional characterization of YIR014W:

• Gene Disruption Strategy:

  • Design gRNAs targeting the YIR014W open reading frame using tools like CHOPCHOP or E-CRISP.

  • Introduce a stop codon (TAA) early in the ORF via homology-directed repair to create null mutants .

  • Alternatively, delete the entire ORF by designing gRNAs targeting regions flanking YIR014W.

• Targeted Modifications:

  • Site-directed mutagenesis of predicted functional domains or motifs to assess their importance.

  • Integration of reporter genes or epitope tags at the endogenous locus to maintain native regulation.

  • Introduction of inducible or repressible promoters to control YIR014W expression levels.

• Implementation Protocol:

  • Transform S. cerevisiae with Cas9 expression plasmid and gRNA vector simultaneously .

  • Include donor DNA fragments containing desired modifications with 40-bp homology arms .

  • Select transformants with appropriate antibiotics (e.g., G418, HygB) .

  • Confirm mutations by sequencing and remove Cas9/gRNA plasmids via serial transfer in non-selective media .

• Phenotypic Analysis of Mutants:

  • Growth assays under various conditions (temperature, pH, osmotic stress, carbon sources).

  • Microscopic examination of morphology and cell division patterns.

  • Metabolic profiling to detect alterations in cellular pathways.

  • Stress response assays, particularly focusing on cell wall integrity if bioinformatic analysis suggests this function.

When analyzing phenotypes, employ both quantitative (growth rates, biomass yields) and qualitative (morphological changes) assessments to capture the full spectrum of functional consequences.

What interactome analysis approaches can identify YIR014W binding partners?

Understanding protein-protein interactions provides critical insights into YIR014W function:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Express epitope-tagged YIR014W at endogenous levels to maintain physiological interactions.

  • Perform gentle cell lysis to preserve protein complexes.

  • Use tandem affinity purification (TAP) or single-step pull-downs depending on desired stringency.

  • Analyze co-purified proteins by LC-MS/MS and filter against appropriate controls to identify specific interactors.

• Yeast Two-Hybrid (Y2H) Screening:

  • Clone YIR014W as both bait (DNA-binding domain fusion) and prey (activation domain fusion) to screen for interactions.

  • Use domain truncations to map interaction surfaces if full-length protein shows self-activation.

  • Verify positive interactions by co-immunoprecipitation or bimolecular fluorescence complementation.

• Proximity-Dependent Biotin Identification (BioID):

  • Fuse YIR014W to a promiscuous biotin ligase (BirA*) that biotinylates proximal proteins.

  • Extract biotinylated proteins with streptavidin and identify by mass spectrometry.

  • This approach captures both stable and transient interactions in the native cellular environment.

• Crosslinking Mass Spectrometry (XL-MS):

  • Utilize chemical crosslinkers to stabilize protein-protein interactions in vivo.

  • Analyze crosslinked peptides by mass spectrometry to identify interaction partners and contact sites.

For data analysis, construct interaction networks and perform Gene Ontology enrichment analysis to identify biological processes and cellular components associated with YIR014W interactors.

How can transcriptomic analysis help elucidate the functional role of YIR014W?

Transcriptomic analysis provides system-level insights into YIR014W function:

• Experimental Design Considerations:

  • Compare YIR014W deletion, overexpression, and wild-type strains under multiple conditions.

  • Include time-course experiments to capture dynamic transcriptional responses.

  • Design biological and technical replicates appropriate for statistical power.

• RNA-Seq Protocol Optimization:

  • Extract total RNA with methods optimized for yeast (hot phenol or commercial kits).

  • Assess RNA quality using Bioanalyzer (RIN > 8 recommended).

  • Select poly(A) enrichment for mRNA analysis or rRNA depletion for including non-coding RNAs.

  • Use strand-specific library preparation to differentiate sense and antisense transcription.

• Differential Expression Analysis:

  • Employ DESeq2 or edgeR for statistical analysis with appropriate false discovery rate control.

  • Validate key differentially expressed genes using RT-qPCR.

  • Perform cluster analysis to identify co-regulated gene sets.

• Interpretation Framework:

  • Gene Ontology enrichment to identify affected biological processes.

  • Pathway analysis to map transcriptional changes onto metabolic and signaling networks.

  • Comparison with existing datasets from related conditions or perturbations.

  • Integration with protein-protein interaction data to construct regulatory networks.

Comparative transcriptomic analysis revealed that overexpression of MIG1spsc01, which affects cell wall functions, led to downregulation of cell wall protein genes CWP2 and YGP1 . Similar approaches could identify if YIR014W expression affects cell wall-related genes, providing functional insights.

What strategies can determine if YIR014W participates in cell wall biogenesis or protein secretion pathways?

Given the importance of cell wall integrity and protein secretion in S. cerevisiae, these represent high-priority areas for investigation:

• Cell Wall Structure Analysis:

  • Quantify cell wall components (β-1,3-glucan, β-1,6-glucan, chitin, mannoproteins) in YIR014W mutants.

  • Perform transmission electron microscopy to measure cell wall thickness and ultrastructure.

  • Assess sensitivity to cell wall-perturbing agents like Congo Red, similar to approaches used for characterizing CWP2 function .

• Protein Secretion Assessment:

  • Express a reporter protein (e.g., cellobiohydrolase) in YIR014W mutant backgrounds and quantify extracellular activity .

  • Fractionate cellular compartments to determine if secretory proteins accumulate within the cell.

  • Analyze glycosylation patterns of secreted proteins using endoglycosidase H treatment .

• Genetic Interaction Studies:

  • Combine YIR014W mutations with deletions in known cell wall genes (CWP2, YGP1, UTH1) to identify aggravating or alleviating genetic interactions .

  • Perform genome-wide synthetic genetic array (SGA) analysis to systematically identify genetic interactions.

  • Overexpress YIR014W in cell wall mutant backgrounds to test for phenotypic rescue.

• Vesicle Trafficking Analysis:

  • Use fluorescent markers for secretory compartments (ER, Golgi, secretory vesicles) to detect trafficking defects.

  • Employ temperature-sensitive sec mutants to determine if YIR014W functions at specific stages of the secretory pathway.

  • Consider whether YIR014W interacts with proteins like SED5, which has been shown to affect protein secretion when overexpressed .

The observation that simultaneous manipulation of multiple genes (e.g., YGP1 and SED5) can synergistically enhance protein secretion suggests similar combinatorial approaches may be valuable for characterizing YIR014W function .

How can I address low expression or detection issues when working with YIR014W?

Low expression or detection of YIR014W requires systematic troubleshooting:

• Expression Optimization Strategies:

  • Codon optimization: Adjust codon usage to match highly expressed S. cerevisiae genes.

  • Promoter selection: Test constitutive (PGK1, TDH3) versus inducible (GAL1, CUP1) promoters .

  • Culture conditions: Systematically vary temperature (20-30°C), media composition, and harvest time.

  • Strain engineering: Consider deletion of proteases (pep4Δ) or proteins that may compete for cellular resources.

• Protein Stability Enhancement:

  Approach	Implementation	Considerations
  Fusion partners	MBP, SUMO, thioredoxin	May enhance solubility and expression
  Stabilizing mutations	Based on computational prediction	Requires structure-based design
  Culture additives	Glycerol, sorbitol, chemical chaperones	Optimize concentration for each additive
  Temperature reduction	16-20°C expression	Slows protein synthesis and improves folding

• Detection Method Optimization:

  • Antibody development: Use multiple antigenic peptides from different regions of YIR014W.

  • Epitope tagging: Test multiple tags (HA, FLAG, Myc) at both N and C termini.

  • Signal amplification: Consider using enhanced chemiluminescence or fluorescent secondary antibodies.

  • Sample preparation: Test different extraction methods (mechanical, enzymatic, detergent-based) to ensure complete release from cellular structures.

• Alternative Expression Systems:

  • Pichia pastoris for potentially higher expression of yeast proteins.

  • Cell-free expression systems for difficult-to-express proteins.

  • E. coli with specialized folding-promoting strains (SHuffle, Origami).

When implementing these strategies, maintain a systematic approach with appropriate controls and documentation of conditions to identify optimal parameters.

What approaches can resolve contradictory results between different functional assays for YIR014W?

Contradictory results are common when characterizing uncharacterized proteins and require careful resolution:

• Systematic Validation Protocol:

  • Replicate experiments with increased biological and technical replicates.

  • Standardize growth conditions, strain backgrounds, and experimental procedures.

  • Implement blinding where possible to eliminate unconscious bias.

  • Use multiple methodological approaches to answer the same question.

• Strain Background Considerations:

  • Compare results across multiple strain backgrounds (laboratory vs. industrial strains).

  • The observation that UTH1 disruption produced different results than previously reported illustrates how strain genetic background can influence phenotypes .

  • Create clean knockout strains in new backgrounds to verify phenotypes.

• Conditional Functionality Assessment:

  • Test function under diverse environmental conditions (temperature, pH, carbon sources).

  • Consider that YIR014W may have different roles under different cellular states.

  • Implement time-course experiments to capture dynamic rather than endpoint effects.

• Integrative Data Analysis:

  • Develop a weighted evaluation framework that considers the reliability and relevance of each assay.

  • Use statistical meta-analysis techniques to integrate results from multiple experiments.

  • Employ computational modeling to identify conditions that reconcile apparently contradictory results.

  • Consider whether contradictions actually reveal complex cellular strategies (e.g., bifunctional proteins, condition-specific functions).

When facing contradictions between functional assays, develop a clear hierarchy of evidence based on assay directness, reproducibility, and physiological relevance.

How can multi-omics approaches be integrated to comprehensively characterize YIR014W function?

Integrated multi-omics provides a systems biology perspective on YIR014W function:

• Data Generation Strategy:

  • Coordinate sample collection across platforms to ensure data comparability.

  • Design experiments to capture both wild-type and YIR014W mutant responses under identical conditions.

  • Include time-course measurements to capture dynamic responses.

• Individual Omics Approaches:

  • Transcriptomics: RNA-seq to identify differentially expressed genes.

  • Proteomics: Quantitative MS to measure protein abundance changes.

  • Metabolomics: Targeted and untargeted approaches to identify altered metabolic pathways.

  • Interactomics: Protein-protein interaction mapping using AP-MS or Y2H.

  • Phenomics: High-throughput phenotypic assays under diverse conditions.

• Integration Methods:

  • Correlation networks: Identify relationships between molecules across different data types.

  • Pathway enrichment: Map multi-omics data onto known biological pathways.

  • Machine learning: Develop predictive models incorporating multiple data types.

  • Causal network inference: Determine directional relationships between molecules.

• Visualization and Interpretation:

  • Develop multi-layered network visualizations that incorporate different data types.

  • Implement dimensionality reduction techniques to identify major patterns across datasets.

  • Use comparative analysis with known proteins to identify functional similarities.

The integration of transcriptomic analysis with secretome studies, as demonstrated in the characterization of MIG1spsc01 function , illustrates how combining multiple approaches can reveal unexpected connections between genes and cellular processes.