Recombinant Saccharomyces cerevisiae Uncharacterized protein YIL134C-A (YIL134C-A)

What is YIL134C-A and why is it classified as a "hypothetical protein"?

YIL134C-A is an open reading frame (ORF) in the Saccharomyces cerevisiae genome that is predicted to encode a protein based on computational analysis, but has not been experimentally characterized regarding its function, structure, or biological role. The designation as "hypothetical" indicates that while bioinformatic evidence supports its existence as a protein-coding gene, direct experimental evidence confirming its expression and function remains limited.

According to the genomic data, YIL134C-A appears among significantly upregulated genes in differential expression studies, showing an almost 12-fold increase in expression under certain stress conditions compared to wild-type yeast . The protein is documented in yeast databases such as the Saccharomyces Genome Database (SGD) and specialized resources like TopologYeast , suggesting ongoing research interest in determining its cellular function.

What experimental methods are used to confirm the existence of uncharacterized proteins like YIL134C-A?

Confirming the existence of hypothetical proteins requires a multi-faceted approach:

• Transcriptional verification: RT-PCR and RNA-seq can confirm whether the gene is transcribed into mRNA under various conditions. Expression data from stress response experiments has already detected YIL134C-A transcripts with significant upregulation (11.969-fold increase) .

• Proteomic validation: Mass spectrometry-based proteomics can identify peptides matching the predicted amino acid sequence. For low-abundance proteins like YIL134C-A, enrichment techniques may be required before MS analysis.

• Epitope tagging: Adding tags (FLAG, HA, GFP) to the genomic sequence through homologous recombination allows for visualization and detection of the expressed protein.

• Antibody development: Generating antibodies against synthetic peptides derived from the predicted sequence enables detection via Western blotting, immunoprecipitation, or immunolocalization.

• Heterologous expression: Similar to recombinant approaches used for Ras proteins in therapeutic contexts , expressing the coding sequence in alternative systems can confirm protein production capability.

How do researchers differentiate between basic and advanced characterization of YIL134C-A?

Basic characterization of YIL134C-A involves:

• Sequence analysis: Bioinformatic prediction of domains, motifs, and secondary structures.

• Expression profiling: Determining when and where the gene is expressed, as demonstrated by the expression data showing 11.969-fold upregulation under certain conditions .

• Subcellular localization: Identifying where the protein resides within the cell, which might utilize databases like TopologYeast that specialize in protein localization .

• Conservation analysis: Comparing sequences across species to identify conserved regions that may indicate functional importance.

Advanced characterization extends to:

• Phenotypic analysis of deletion/overexpression: Systematically analyzing phenotypes in knockout or overexpression strains across various environmental conditions.

• Interaction mapping: Identifying binding partners through techniques like yeast two-hybrid, affinity purification-mass spectrometry, or proximity labeling.

• Structural determination: Using X-ray crystallography, NMR, or cryo-EM to determine three-dimensional structure.

• Post-translational modification analysis: Investigating modifications like prenylation, which has been extensively studied in other yeast proteins such as Ydj1 .

• Functional reconstitution: Expressing the protein in heterologous systems to confirm biochemical activity.

What stress conditions influence YIL134C-A expression and what methodologies should be used to study these responses?

YIL134C-A shows significant upregulation (11.969-fold increase) in DM (mutant) compared to WT cells , suggesting stress-responsive regulation. To comprehensively study its stress response profile:

Experimental methodologies:

• Systematic stress exposure: Expose yeast cultures to a panel of stressors (oxidative, osmotic, temperature, nutrient limitation, pH changes) and measure YIL134C-A expression using RT-qPCR or RNA-seq.

• Time-course analysis: Monitor expression changes at different time points after stress induction to distinguish between early and late response genes.

• Promoter analysis: Perform chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the YIL134C-A promoter under stress conditions.

• Reporter constructs: Fuse the YIL134C-A promoter to reporter genes (GFP, luciferase) to visualize expression patterns in real-time.

• Comparison with known stress-responsive genes: Compare expression patterns with well-characterized stress genes like HSP12 (3.227-fold), CTT1 (3.076-fold), and CIN5 (3.522-fold) that appear in the same dataset .

The gene's significant upregulation suggests it may participate in similar stress response pathways as other highly induced genes in the dataset, such as RCK1 (16.716-fold), which encodes a putative serine/threonine protein kinase involved in oxidative stress response .

How might YIL134C-A potentially interact with protein modification pathways such as the CaaX prenylation system?

The CaaX prenylation pathway is an important post-translational modification system in yeast that affects protein localization and function. To investigate potential interactions between YIL134C-A and this pathway:

Methodological approaches:

• Sequence analysis for modification motifs: Examine the YIL134C-A sequence for CaaX motifs (where "C" is cysteine, "a" are aliphatic amino acids, and "X" can be various amino acids) . These motifs are recognition sequences for farnesyltransferase or geranylgeranyltransferase.

• Metabolic labeling: Incubate cells with radioactive prenyl precursors (³H-mevalonate, ³H-farnesol) to detect incorporation into YIL134C-A.

• Mobility shift assays: Compare electrophoretic mobility of native versus potentially prenylated forms of the protein.

• Inhibitor studies: Treat cells with prenylation inhibitors and observe effects on YIL134C-A localization and function.

• Genetic interaction screens: Test for synthetic phenotypes between YIL134C-A deletion and mutations in prenylation pathway components (RAM1, RAM2, STE24, RCE1).

Studies of the CaaX pathway have revealed flexibility in substrate recognition and processing outcomes, as demonstrated in Ydj1 thermotolerance screening that identified approximately 140 CaaX sequences that undergo prenylation without subsequent processing steps . Similar approaches could determine if YIL134C-A undergoes complete or partial CaaX processing.

What approaches can be used to study potential roles of YIL134C-A in disease models?

While YIL134C-A's function remains uncharacterized, exploring its potential relevance to disease models requires innovative approaches:

Methodological strategies:

• Homology identification in higher organisms: Search for sequence or structural homologs in mammals that might have disease associations.

• Heterologous expression systems: Express YIL134C-A in recombinant systems similar to those used for Ras proteins in cancer immunotherapy approaches , which utilized whole recombinant S. cerevisiae yeast to express target proteins that stimulate immune responses.

• Disease-relevant functional assays: If YIL134C-A shows stress-responsive properties, test its effects in cellular models of diseases involving stress responses (neurodegenerative disorders, inflammatory conditions).

• Antibody development and screening: Similar to Anti-Saccharomyces cerevisiae Antibodies (ASCA) used in inflammatory bowel disease diagnostics , develop antibodies against YIL134C-A and screen patient samples for reactivity.

• Immune response characterization: If considering YIL134C-A for recombinant expression systems, characterize potential immune responses using proliferation assays with tritiated thymidine incorporation similar to those used in Ras protein studies .

The approach used in clinical trials with recombinant S. cerevisiae expressing Ras proteins for cancer immunotherapy provides a methodological framework that could be adapted for studying uncharacterized proteins like YIL134C-A in disease contexts .

What proteomics approaches would be most effective for studying the regulation and interactome of YIL134C-A?

Understanding the regulation and interaction network of YIL134C-A requires sophisticated proteomics strategies:

Advanced methodological approaches:

• Proximity-dependent biotin identification (BioID): Fuse YIL134C-A to a biotin ligase (BirA) to biotinylate proximal proteins, which can then be purified and identified by mass spectrometry.

• Tandem affinity purification-mass spectrometry (TAP-MS): Generate strains expressing TAP-tagged YIL134C-A to isolate stable protein complexes.

• Thermal proteome profiling (TPP): Monitor thermal stability changes of YIL134C-A and other proteins under different conditions to infer functional relationships.

• Cross-linking mass spectrometry (XL-MS): Use chemical cross-linkers to capture transient interactions before mass spectrometry analysis.

• Parallel reaction monitoring (PRM): Develop targeted mass spectrometry assays to quantify YIL134C-A across conditions, particularly important given its 11.969-fold upregulation in stress conditions .

• Post-translational modification mapping: Use enrichment strategies (phosphopeptide enrichment, ubiquitin remnant antibodies) coupled with mass spectrometry to identify modifications that may regulate YIL134C-A function.

These approaches would be particularly valuable given YIL134C-A's apparent stress responsiveness, potentially revealing connections to stress response networks that include other highly regulated proteins in the same dataset, such as RCK1, FRE8, and GTO3 .

Expression Data for YIL134C-A in Stress Response Studies

The following table shows the expression fold change of YIL134C-A compared to other stress-responsive genes:

This data indicates that YIL134C-A is among the most highly upregulated genes under specific stress conditions, suggesting an important role in stress response mechanisms .

Methodological Comparison for Protein Characterization Approaches