Recombinant Saccharomyces cerevisiae Uncharacterized protein YIR021W-A (YIR021W-A)

What is YIR021W-A and what are its fundamental characteristics?

YIR021W-A is an uncharacterized protein from Saccharomyces cerevisiae (baker's yeast) consisting of 70 amino acids. Its complete amino acid sequence is: MSFSVSCKTPKTTKLLVSSISESAVALIIITIRILFSIGKSDFKKIISKEINGAETIYYR NIPESKPQGS. The protein is cataloged in UniProt with ID Q3E739 and is part of the significant portion (approximately 25%) of the S. cerevisiae genome that remains functionally unannotated . This protein represents an opportunity for novel functional characterization studies in yeast biology.

How is recombinant YIR021W-A typically produced for research applications?

Recombinant YIR021W-A can be successfully expressed in E. coli expression systems with an N-terminal histidine tag. The full-length protein (amino acids 1-70) is commonly produced as a His-tagged fusion protein to facilitate purification through affinity chromatography. The expressed protein is typically supplied as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE analysis .

What are the recommended storage and handling conditions for recombinant YIR021W-A?

For optimal stability and activity maintenance, recombinant YIR021W-A should be stored at -20°C to -80°C upon receipt. The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, preferably with 5-50% glycerol (50% is optimal) as a cryoprotectant for long-term storage. Repeated freeze-thaw cycles should be avoided to prevent protein degradation. Working aliquots may be stored at 4°C for up to one week . The reconstituted protein is typically stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

How should experiments be designed to characterize the function of YIR021W-A?

Characterizing uncharacterized proteins like YIR021W-A requires a comprehensive experimental design approach. Begin by clearly defining variables: independent variables might include experimental conditions (temperature, pH, substrate concentrations), while dependent variables would be measurable outcomes (binding affinity, enzymatic activity, phenotypic changes in deletion strains) .

A systematic approach should include:

• Sequence analysis and structural predictions

• Localization studies using tagged versions of the protein

• Phenotypic analyses of deletion mutants under various stress conditions

• Protein-protein interaction studies

• Comparative analysis with similar proteins in other organisms

Control experiments are essential, including both positive controls (well-characterized proteins) and negative controls (unrelated proteins or buffer-only conditions). Randomization and minimizing confounding variables are crucial for obtaining reliable results .

What are the recommended approaches for creating YIR021W-A deletion mutants for functional studies?

To study the function of YIR021W-A through deletion analysis, researchers should consider the following methodology:

• Gene replacement strategy: Use homologous recombination to replace the YIR021W-A gene with a selectable marker (such as kanMX for G418 resistance).

• Confirmation verification: Confirm successful deletion through:

  • PCR verification of the deletion site

  • Southern blot analysis

  • RT-PCR to confirm absence of transcript

  • Western blot analysis if antibodies are available

• Strain background consideration: Create deletions in multiple strain backgrounds (e.g., BY4741, Σ1278b) as phenotypes may be strain-dependent.

• Phenotypic analysis: Systematically test the deletion strain under various conditions (different carbon sources, stress conditions, nutrient limitations) following established protocols for genome-wide deletion mutant analysis .

• Complementation testing: Reintroduce the wild-type gene on a plasmid to confirm that observed phenotypes are due to the deletion.

What bioinformatic methods can predict potential functions of YIR021W-A?

Advanced bioinformatic analysis can provide valuable insights into the potential functions of uncharacterized proteins like YIR021W-A. A comprehensive approach should include:

• Sequence similarity networks: Create sensitive sequence similarity predictions by comparing YIR021W-A against multiple databases of known proteins across diverse organisms .

• Structural prediction and domain analysis: Utilize tools like AlphaFold2 to predict protein structure and identify potential functional domains.

• Phylogenetic profiling: Analyze the evolutionary conservation pattern of YIR021W-A across species to identify functional relationships.

• Integration with "pathway holes": Identify biochemical pathways in yeast with missing enzymatic components and evaluate if YIR021W-A could be a candidate to fill these gaps, similar to the approach used for Yhr202w in the NAD degradation pathway .

• Co-expression network analysis: Identify genes with similar expression patterns to infer potential functional relationships.

This multi-layered approach can generate testable hypotheses about YIR021W-A function that can guide subsequent experimental validation.

How can phosphoproteomics and other -omics approaches be applied to understand YIR021W-A function?

Comprehensive -omics approaches can reveal the functional context of YIR021W-A within cellular pathways:

• Phosphoproteomics:

  • Quantitative phosphoproteomics using SILAC (Stable Isotope Labeling with Amino acids in Cell culture) can identify if YIR021W-A is phosphorylated under specific conditions

  • Analysis can determine if YIR021W-A is a target of specific kinases involved in stress response pathways, similar to studies on pseudohyphal growth regulation

• Metabolomics:

  • Untargeted metabolomics comparing wild-type, YIR021W-A deletion, and overexpression strains can reveal metabolic pathways affected

  • Focus on specific metabolite classes based on preliminary functional predictions

  • Comparative analysis similar to that performed for Yhr202w can identify substrate-product relationships

• Transcriptomics:

  • RNA-seq analysis of YIR021W-A deletion strains under various conditions to identify altered gene expression patterns

  • Transcriptional profiling can reveal if YIR021W-A is involved in specific stress responses, similar to findings for Ksp1 kinase

• Interactomics:

  • Affinity purification coupled with mass spectrometry to identify protein interaction partners

  • Yeast two-hybrid screening to detect direct protein-protein interactions

These multi-omics approaches should be integrated computationally to develop a comprehensive functional model for YIR021W-A.

What are the recommended procedures for determining the subcellular localization of YIR021W-A?

Determining the subcellular localization of YIR021W-A is crucial for understanding its function. A comprehensive localization study should include:

• Fluorescent protein fusion approach:

  • C-terminal and N-terminal GFP or other fluorescent protein tags

  • Verification that fusion proteins maintain functionality

  • Live-cell imaging under different growth conditions and stress treatments

  • Co-localization with known compartment markers

• Biochemical fractionation:

  • Differential centrifugation to separate cellular compartments

  • Western blot analysis of fractions using anti-His antibodies for recombinant protein detection

  • Comparison with known compartment marker proteins

• Immunolocalization:

  • Production of specific antibodies against YIR021W-A

  • Immunofluorescence microscopy with appropriate fixation methods

  • Gold-labeled antibodies for electron microscopy

• In silico prediction validation:

  • Use of localization prediction tools (TargetP, PSORT, etc.)

  • Experimental verification of predicted localization signals

Each approach has strengths and limitations, so using multiple complementary methods is recommended for conclusive localization determination.

How can protein-protein interaction studies be optimized for characterizing YIR021W-A?

To comprehensively characterize the protein interaction network of YIR021W-A:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Use His-tagged recombinant YIR021W-A as bait

  • Perform pull-downs under various physiological conditions

  • Include appropriate controls (untagged strains, irrelevant His-tagged proteins)

  • Use quantitative MS approaches (SILAC, TMT) to distinguish true interactors from background

• Yeast two-hybrid screening:

  • Use both N-terminal and C-terminal fusions to activation/binding domains

  • Screen against comprehensive yeast genomic libraries

  • Validate interactions with targeted assays and co-immunoprecipitation

• Proximity-dependent labeling:

  • Create fusions with BioID or APEX2 enzymes

  • Analyze the proximal proteome under different conditions

  • Compare results with known stress granule-associated proteins, as uncharacterized proteins may localize to stress granules similar to findings with Ksp1 kinase pathways

• Co-evolution and computational prediction validation:

  • Use tools that predict protein-protein interactions based on evolutionary data

  • Validate top computational predictions experimentally

Correlation of protein interaction data with phenotypic analyses of deletion mutants can provide functional insights into the biological role of YIR021W-A.

How should researchers integrate diverse experimental data to propose functions for YIR021W-A?

Integrating diverse experimental data for uncharacterized proteins like YIR021W-A requires a systematic approach:

• Create a comprehensive data matrix:

  • Compile all experimental results (localization, interaction, phenotypic, -omics data)

  • Normalize and standardize diverse data types

  • Apply statistical methods appropriate for each data type

• Hierarchical data integration:

  • Start with highest-confidence data points

  • Build a model incorporating various lines of evidence

  • Use Bayesian approaches to assign confidence scores to functional predictions

• Network-based analysis:

  • Place YIR021W-A in the context of known interaction networks

  • Identify network motifs and functional modules it might participate in

  • Apply guilt-by-association principles to well-characterized neighbors

• Comparative analysis with characterized proteins:

  • Compare experimental signatures with proteins of known function

  • Use tools similar to those that successfully characterized Yhr202w as an extracellular AMP hydrolase

• Iterative hypothesis refinement:

  • Generate testable functional hypotheses

  • Design experiments to validate or refute these hypotheses

  • Refine models based on new experimental data

This integrative approach can transform disparate data points into coherent functional models, similar to how Yhr202w was successfully characterized in the NAD degradation pathway .

What are the best practices for resolving contradictory experimental results about YIR021W-A?

When confronted with contradictory experimental results regarding YIR021W-A:

• Systematic error identification:

  • Evaluate experimental design for potential confounding variables

  • Assess statistical power and significance of each result

  • Consider strain background differences that might influence outcomes

• Condition-dependent functionality analysis:

  • Test if contradictions arise from different experimental conditions

  • Systematically vary parameters (temperature, media, stress conditions)

  • Consider that YIR021W-A may have different functions under different conditions

• Technical validation across platforms:

  • Reproduce key findings using orthogonal techniques

  • Vary expression levels of the protein (endogenous, overexpression, deletion)

  • Use both tagged and untagged versions to rule out tag interference

• Collaborative verification:

  • Engage other laboratories to independently verify key findings

  • Use standardized protocols to minimize lab-specific variations

  • Pool raw data for meta-analysis when possible

• Context-dependent interpretation framework:

  • Develop a model that accommodates seemingly contradictory results

  • Consider multifunctionality as an explanation for divergent findings

  • Evaluate if YIR021W-A function depends on specific protein complexes or modifications

This systematic approach can transform apparent contradictions into deeper insights about context-dependent protein functions, similar to how complex signaling pathways in pseudohyphal growth were elucidated .

What emerging technologies might accelerate functional characterization of YIR021W-A?

Several cutting-edge technologies hold promise for characterizing uncharacterized proteins like YIR021W-A:

• CRISPR-based functional genomics:

  • CRISPRi for tunable repression to study dosage effects

  • CRISPRa for context-specific overexpression

  • Base editing for studying effects of specific amino acid changes

  • Perturb-seq for high-throughput phenotyping of genetic perturbations

• Single-cell analyses:

  • Single-cell transcriptomics to identify cell-to-cell variability in response to YIR021W-A modulation

  • Single-cell proteomics to detect rare cell populations with distinct YIR021W-A functions

  • Spatial transcriptomics to map expression patterns in colonies or pseudohyphal structures

• Advanced structural biology:

  • Cryo-EM for structural determination of YIR021W-A and its complexes

  • Hydrogen-deuterium exchange mass spectrometry for dynamic structural analysis

  • Integrative structural biology combining multiple data sources

• In situ techniques:

  • APEX2-mediated proximity labeling for in situ interactome mapping

  • Live-cell biosensors to track YIR021W-A activity in real-time

  • Super-resolution microscopy for detailed localization studies

• Systems biology approaches:

  • Multi-omics data integration frameworks

  • Machine learning for functional prediction from complex datasets

  • Genome-scale metabolic models incorporating YIR021W-A

These emerging technologies, when applied systematically, can accelerate the functional characterization of YIR021W-A beyond what is possible with conventional approaches.

How can researchers design experiments to determine if YIR021W-A is involved in stress response pathways?

To investigate YIR021W-A's potential role in stress response pathways:

• Comprehensive stress exposure panel:

  • Expose wild-type and ΔyirO21w-a strains to diverse stressors (oxidative, osmotic, temperature, nutrient limitation)

  • Quantify growth rates, viability, and morphological changes

  • Compare with known stress response mutants

• Stress-induced transcriptional regulation:

  • Monitor YIR021W-A expression under various stress conditions

  • Identify transcription factors that regulate its expression

  • Map its position in stress response transcriptional networks

• Protein modification and relocalization:

  • Track post-translational modifications of YIR021W-A during stress

  • Monitor potential relocalization to stress granules or other compartments

  • Compare with known stress response proteins like those regulated by Ksp1 kinase

• Genetic interaction mapping:

  • Perform synthetic genetic array analysis with YIR021W-A deletion

  • Focus on interactions with known stress response pathways

  • Identify condition-specific genetic interactions

• Pathway-specific assays:

  • Test specific hypotheses about pathway involvement with targeted biochemical assays

  • Measure key metabolites in deletion and overexpression strains

  • Apply methodology similar to that used for characterizing Yhr202w in the NAD degradation pathway

This systematic approach can determine if YIR021W-A functions in specific stress response pathways, potentially revealing connections to filamentous growth, TORC1, MAPK, PKA, or AMPK signaling pathways as seen with other yeast proteins .