Recombinant Saccharomyces cerevisiae Putative UPF0479 protein YOR396C-A (YOR396C-A)

What is YOR396C-A protein and how is it classified?

YOR396C-A is a protein of unknown function in Saccharomyces cerevisiae (budding yeast), comprising approximately 160 amino acids. It was initially identified through gene-trapping techniques, microarray-based expression analysis, and genome-wide homology searching . The protein belongs to the UPF0479 family, which contains proteins with conserved sequences but largely uncharacterized functions. While computational analysis places it in this family, functional studies remain limited, making it a subject of ongoing research interest in yeast biology.

The UPF (Uncharacterized Protein Family) designation indicates that while the protein has been identified at the sequence level, its biological role remains to be fully elucidated. Researchers approaching this protein should consider it as part of broader studies investigating novel yeast protein functions and potential contributions to cellular processes.

What experimental methods are most effective for studying the expression of YOR396C-A?

When investigating YOR396C-A expression, researchers should employ a multi-faceted approach combining:

• RNA-seq and quantitative PCR: These techniques allow precise quantification of YOR396C-A transcript levels under various conditions, providing insight into transcriptional regulation.

• Western blotting with epitope tagging: Since specific antibodies against YOR396C-A may not be commercially available, expressing the protein with epitope tags (e.g., FLAG, HA, or GFP) enables detection and quantification.

• Microarray analysis: This approach can reveal expression patterns across different conditions and in relation to other genes, potentially identifying co-regulated genes .

• Gene-trapping approaches: These methods have proven successful in identifying YOR396C-A expression patterns and can be replicated to verify previous findings .

For comprehensive expression analysis, researchers should consider examining expression under various stress conditions, throughout different growth phases, and in various genetic backgrounds to identify potential regulatory pathways.

How does YOR396C-A relate to other members of the UPF0479 protein family?

YOR396C-A shows sequence homology with other members of the UPF0479 family, including YLR466C-A and YLR467C-A in S. cerevisiae, with similarity scores of 0.695 according to STRING database analysis . The functional relationship between these proteins remains under investigation, but their sequence similarity suggests potential redundancy or related functions.

When designing experiments to investigate YOR396C-A function, researchers should consider:

• Creating deletion mutants for multiple UPF0479 family members to assess potential functional redundancy

• Performing complementation assays with different family members

• Conducting comparative structural analysis to identify conserved domains

• Analyzing expression patterns of all family members under identical conditions

The co-occurrence of these proteins across different yeast strains and related species can provide evolutionary context for understanding functional conservation.

What synthetic recombination approaches are optimal for studying YOR396C-A functional variants?

When generating recombinant variants of YOR396C-A for functional studies, researchers should consider two primary approaches based on the synthetic recombinant population construction methods:

S-type approach: This method involves careful crossing designs in which founding lines are crossed in pairs. For YOR396C-A studies, this approach offers better representation of founder genotypes. The protocol involves:

• Pairing haploid strains of opposite mating types

• Isolating successful diploid colonies

• Inducing sporulation in 1% potassium acetate media for 72 hours at 30°C

• Dissecting tetrads to collect meiotic products

• Verifying proper segregation of markers through replica plating

K-type approach: This more straightforward method involves:

• Growing newly mated diploid cells in YPD media

• Inducing sporulation

• Disrupting asci using methods such as Y-PER reagent treatment, zymolyase digestion, and mechanical agitation

• Allowing the isolated spores to mate randomly

The S-type approach, while more labor-intensive, produces populations with more equal founder haplotype representation and consequently higher levels of genetic variation, which is particularly valuable for studying proteins of unknown function like YOR396C-A .

How can researchers effectively analyze the potential interactions between YOR396C-A and its predicted functional partners?

Based on STRING database analysis, YOR396C-A has several predicted functional partners, including YRF1-8 (score 0.929), YOR394C-A (score 0.900), YLR466C-A (score 0.695), and YLR467C-A (score 0.695) . Researchers investigating these interactions should implement a systematic approach:

• Co-immunoprecipitation (Co-IP) studies: Epitope-tag YOR396C-A and its predicted partners to verify physical interactions. Use mild detergents for membrane proteins and optimize buffer conditions for nuclear proteins like YRF1-8.

• Yeast two-hybrid assays: Employ both N and C-terminal fusions to account for potential structural interference with interaction domains.

• Fluorescence microscopy with co-localization analysis: Create fluorescently tagged versions of YOR396C-A and partners to assess spatial proximity in vivo.

• Genetic interaction mapping: Generate single and double deletion/overexpression strains to identify synthetic lethality, sickness, or rescue phenotypes.

• Transcriptional co-regulation analysis: Examine expression patterns across conditions to identify coordinated expression.

The interaction with YRF1-8, a telomeric Y' element-encoded DNA helicase, suggests potential involvement in telomere maintenance or DNA replication stress response . This high confidence score (0.929) warrants particular attention when designing interaction studies.

What genomic and proteomic techniques can best resolve the functional ambiguity of YOR396C-A?

To address the functional ambiguity of this protein, researchers should implement a multi-omics approach:

Genomic approaches:

• CRISPR-Cas9 mediated precise mutations: Create targeted mutations in conserved residues predicted through structural analysis

• Synthetic genetic array (SGA) analysis: Identify genetic interactions by crossing YOR396C-A deletion mutants with genome-wide deletion collections

• Genome-wide transcriptional profiling: Compare wild-type and YOR396C-A mutant strains under various conditions

Proteomic approaches:

• Proximity-dependent biotin identification (BioID): Identify proteins in close proximity to YOR396C-A in vivo

• Stable isotope labeling with amino acids in cell culture (SILAC): Quantitatively compare proteomes of wild-type and YOR396C-A mutant strains

• Thermal proteome profiling: Identify potential ligands or substrates by monitoring thermal stability shifts

Structural approaches:

• Cryo-electron microscopy: Determine high-resolution structure

• Hydrogen-deuterium exchange mass spectrometry: Identify dynamic regions potentially involved in interactions

The lack of detailed functional knowledge about YOR396C-A necessitates this comprehensive approach to identify its cellular role and biochemical function.

How should researchers design experiments to study the role of YOR396C-A in different yeast growth conditions?

When investigating the conditional function of YOR396C-A, experimental design should follow these methodological principles:

• Control strain selection: Include the parental wild-type strain, YOR396C-A deletion strain, and a complementation strain expressing YOR396C-A from a plasmid to control for secondary mutations.

• Growth condition matrix: Test a comprehensive panel of conditions including:

• Time-course analysis: Monitor growth, gene expression, and phenotypic changes at multiple time points rather than single endpoints.

• Multiple independent transformants: Use at least three independent transformants for each genetic construct to account for clonal variation.

• Quantitative phenotypic analysis: Employ high-throughput phenotyping methods such as growth curve analysis in plate readers with technical and biological replicates .

This systematic approach will help uncover condition-specific functions that might be missed in standard laboratory conditions.

What are the optimal protocols for expressing and purifying recombinant YOR396C-A for in vitro studies?

For researchers requiring purified YOR396C-A protein for biochemical and structural studies, the following optimized protocol is recommended:

Expression system selection:
The choice between bacterial (E. coli) and yeast expression systems should be guided by the research questions:

Purification protocol:

• Tag selection: A dual-tag approach is recommended - His₆ tag for initial affinity purification and a cleavable tag (TEV protease site) for tag removal.

• Lysis conditions: For yeast expression, use glass bead disruption in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM PMSF, and protease inhibitor cocktail.

• Chromatography sequence:

  • Immobilized metal affinity chromatography (IMAC)

  • Tag cleavage with TEV protease

  • Size exclusion chromatography

• Storage conditions: Store the purified protein in 50 mM Tris-based buffer with 50% glycerol at -20°C for short-term or -80°C for extended storage to maintain stability. Avoid repeated freeze-thaw cycles .

• Quality control: Verify protein identity through mass spectrometry and purity through SDS-PAGE. Assess structural integrity through circular dichroism before functional studies.

For researchers working with YOR396C-A, special attention should be paid to potential membrane association or hydrophobic regions that might affect solubility during purification.

How can researchers effectively analyze and present data from YOR396C-A functional studies?

When analyzing and presenting data from YOR396C-A studies, researchers should follow these methodological guidelines:

Data analysis approach:

• Statistical rigor: Apply appropriate statistical tests based on data distribution. For growth experiments, consider area under the curve (AUC) analysis rather than single time-point comparisons.

• Normalization methods: When comparing expression levels or protein abundance across conditions, normalize to stable reference genes verified to be unaffected by your experimental conditions.

• Batch effects consideration: Design experiments to distribute conditions across batches and include batch as a factor in statistical models.

Data presentation guidelines:

• Table design: When presenting large datasets, organize information into clear categories with descriptive column headers. Tables should be self-explanatory without reference to the text .

• Appropriate data visualization: Choose the right format based on data type following this guide:

• Reproducibility considerations: Provide detailed methods, including strain construction, primers, and growth conditions to enable other researchers to reproduce your findings .

• Integration of diverse data types: When presenting multi-omics data, use integrative visualizations that show relationships between different data types (e.g., expression levels, genetic interactions, and phenotypic effects) .

Following these guidelines will enhance the clarity and impact of research findings related to this poorly characterized protein.

What are the current knowledge gaps and future research directions for YOR396C-A studies?

Current research on YOR396C-A reveals significant knowledge gaps that present opportunities for future investigation. The protein remains functionally ambiguous despite genomic and proteomic identification . Key areas for future research include:

• Evolutionary conservation and divergence: Comparative genomics across Saccharomyces species and other fungi could reveal selective pressures and functional constraints on YOR396C-A.

• Condition-specific essentiality: While not essential under standard laboratory conditions, YOR396C-A may play crucial roles under specific environmental stresses or genetic backgrounds.

• Integration with cellular pathways: The connection between YOR396C-A and telomeric DNA helicase YRF1-8 suggests potential involvement in genome maintenance pathways that remains to be explored .

• Structural biology approaches: Determining the three-dimensional structure of YOR396C-A would provide insights into potential binding partners and biochemical functions.

• Systems biology integration: Placing YOR396C-A within the broader context of yeast genetic and protein interaction networks through comprehensive genetic screens and proteomic analyses.

Researchers are encouraged to consider these directions when designing studies, as addressing these knowledge gaps will contribute significantly to our understanding of this uncharacterized protein family and potentially reveal novel cellular mechanisms in yeast.