Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YNL235C (YNL235C)

What is YNL235C and what is currently known about its structural features?

YNL235C is a putative uncharacterized protein from Saccharomyces cerevisiae (strain ATCC 204508 / S288c), commonly known as baker's yeast. The protein consists of 143 amino acids with the sequence: MRTLGILEERNSRHCHSHFFLLSREVKCLKQFYTKLCYSTNNQPSISKSIPEHMHSLVYMVGHLLVWMLVGTIVLSLDIIIFPALVTEPHLLHLLSFPSDISDTLSLSQVTSSYSNLLKDLEVLFFMGSLSVSIINPSSNGCNK . The protein is also known by the ORF name N1139 . While its specific function remains unknown, analysis of the amino acid sequence suggests it may contain hydrophobic regions potentially indicative of membrane association or transmembrane domains.

The sequence contains a mix of hydrophobic and hydrophilic residues with potential structural motifs that could inform function. Computational structure prediction tools would be necessary to generate hypotheses about its three-dimensional conformation, while experimental approaches like X-ray crystallography or NMR would provide definitive structural data.

What commercial research tools are available for studying YNL235C?

Several commercial research tools are available for investigating YNL235C:

• Antibodies: Polyclonal antibodies raised in rabbits against recombinant YNL235C protein are commercially available (e.g., Product Code CSB-PA346116XA01SVG) . These antibodies have been tested for applications including ELISA and Western blot.

• Recombinant Proteins: Purified recombinant YNL235C protein is available in quantities such as 50 μg, typically supplied in a Tris-based buffer with 50% glycerol .

• Expression Vectors: While not explicitly mentioned in the search results, standard yeast expression vectors can be adapted for YNL235C studies.

When using these resources, researchers should follow manufacturer-recommended storage conditions (-20°C or -80°C) and avoid repeated freeze-thaw cycles that could compromise protein or antibody integrity .

What are the optimal transformation methods for genetic studies of YNL235C?

Recent advances in yeast transformation technology offer significant advantages for YNL235C studies. The dual heat-shock and electroporation approach (HEEL) is particularly promising as it creates high-quality DNA libraries by increasing the fraction of mono-transformed yeast cells from 20% to over 70% . This method allows for near-perfect phenotype-to-genotype associations, which is crucial for accurate functional characterization.

The HEEL methodology allows more than 10^7 yeast cells per reaction to be transformed with a circular plasmid molecule, representing an almost 100-fold improvement over conventional transformation techniques . This high efficiency, combined with the reduced occurrence of multiple plasmid uptake, makes HEEL particularly valuable for library-based screening approaches where accurate phenotype-to-genotype mapping is essential.

How can researchers distinguish between technical artifacts and true phenotypes when studying uncharacterized proteins like YNL235C?

When investigating an uncharacterized protein like YNL235C, distinguishing genuine phenotypes from technical artifacts requires a multi-faceted validation approach:

• Use multiple independent mutant strains or clones to verify that observed phenotypes are consistent.

• Implement complementation tests where the wild-type YNL235C gene is reintroduced to confirm phenotype rescue.

• Employ different tagging strategies (N-terminal vs. C-terminal) to ensure that tagged versions retain functionality.

• Create point mutations rather than complete deletions to distinguish loss-of-function from structural disruption effects.

• Utilize orthogonal methods to confirm findings, such as combining genetic approaches with biochemical validation.

The potential for multi-plasmid uptake during transformation represents a significant technical challenge that can be addressed using the HEEL methodology, which minimizes this problem during high-throughput yeast DNA transformations .

What high-throughput approaches can identify potential functions of YNL235C?

Several complementary high-throughput approaches can accelerate functional discovery for uncharacterized proteins like YNL235C:

• Synthetic genetic array (SGA) analysis - This approach systematically creates double mutants combining YNL235C deletion with other yeast gene deletions to identify genetic interactions that suggest functional relationships.

• Transcriptomic profiling - RNA-seq analysis of YNL235C deletion or overexpression strains can reveal affected pathways. For example, analysis of the YBR238C knockout (another uncharacterized yeast gene) revealed 326 upregulated and 61 downregulated genes, suggesting specific pathway involvement .

• Proteome-wide interaction mapping - Techniques such as affinity purification followed by mass spectrometry can identify physical interaction partners.

• Chemical genomics screening - Testing YNL235C mutants against diverse chemical compounds can reveal specific sensitivity or resistance profiles that suggest biological functions.

• Dual-barcode design with high-diversity regions - This approach allows for robust identification and quantification of unique genotypes within heterogeneous populations using standard Sanger sequencing .

How might YNL235C function be related to mitochondrial activity based on studies of similar uncharacterized proteins?

While YNL235C's specific relationship to mitochondrial function is not directly established in the search results, insights can be drawn from research on other uncharacterized yeast proteins like YBR238C. Methodological approaches to investigate such a connection would include:

• Mitochondrial function assays:

  • Measure oxygen consumption rates in YNL235C mutants

  • Assess mitochondrial membrane potential

  • Quantify ATP production and respiratory capacity

• Gene expression analysis:

  • Examine expression of nuclear-encoded mitochondrial genes in YNL235C mutants

  • Analyze transcription factors like HAP4 that regulate mitochondrial function (HAP4 has been shown to mediate effects of other uncharacterized proteins)

• Functional validation:

  • Create double deletions with known mitochondrial regulators

  • Test epistatic relationships with mitochondrial pathways

  • Examine YNL235C expression in response to mitochondrial stressors

Research on YBR238C has demonstrated that uncharacterized yeast genes can significantly impact mitochondrial function and cellular aging through specific regulatory pathways . YBR238C deletion increases cellular lifespan by enhancing mitochondrial function, while its overexpression accelerates aging via mitochondrial dysfunction . Similar mechanisms could potentially involve YNL235C.

How should researchers design experiments to compare YNL235C with other uncharacterized yeast proteins?

A systematic comparative analysis of YNL235C with other uncharacterized yeast proteins requires a multi-dimensional experimental design:

• Phenotypic profiling matrix:

  • Create a standardized panel of growth conditions (temperature, carbon source, stressors)

  • Test deletion and overexpression strains of multiple uncharacterized proteins

  • Develop quantitative metrics for comparing phenotypic signatures

• Transcriptomic comparative analysis:

  • Perform RNA-seq on multiple uncharacterized gene mutants under identical conditions

  • Identify shared and divergent expression patterns

  • Cluster genes based on expression profile similarities

• Localization and interaction mapping:

  • Systematically tag proteins with identical reporters

  • Perform standardized localization studies

  • Compare protein-protein interaction networks

• Evolutionary analysis:

  • Assess conservation patterns across yeast species

  • Identify co-evolution patterns among uncharacterized genes

  • Evaluate selection pressures through dN/dS analysis

This approach would allow positioning of YNL235C within the broader context of uncharacterized genes and potentially reveal functional clusters.

What bioinformatic approaches can predict functional relationships between YNL235C and characterized yeast genes?

Several bioinformatic strategies can generate testable hypotheses about YNL235C function:

• Co-expression network analysis:

  • Analyze publicly available transcriptomic datasets

  • Identify genes consistently co-expressed with YNL235C

  • Apply guilt-by-association principles to infer function

• Protein domain and motif prediction:

  • Scan for known functional domains and motifs

  • Identify potential post-translational modification sites

  • Predict secondary structure elements and transmembrane regions

• Structural modeling and comparison:

  • Generate homology models if distant homologs exist

  • Analyze structural features for functional clues

  • Perform structural alignment with characterized proteins

• Phylogenetic profiling:

  • Identify co-occurring genes across multiple species

  • Infer functional relationships from evolutionary co-presence/absence patterns

  • Detect potential pathway associations

• Text mining and literature-based discovery:

  • Apply natural language processing to extract indirect relationships

  • Identify bridging concepts between YNL235C and characterized pathways

  • Generate network models from literature-derived associations

How might YNL235C be involved in aging and longevity pathways?

Although YNL235C's direct role in aging is not established in the search results, insights from research on other uncharacterized yeast genes like YBR238C suggest potential investigative approaches:

• Lifespan analysis:

  • Measure chronological lifespan (CLS) in YNL235C deletion and overexpression strains

  • Determine replicative lifespan (RLS) using micromanipulation

  • Assess the impact of caloric restriction on YNL235C mutant lifespans

• TORC1 pathway interactions:

  • Examine YNL235C expression in response to rapamycin treatment

  • Investigate genetic interactions with TORC1 pathway components

  • Assess TORC1 activity markers in YNL235C mutants

Research on YBR238C has demonstrated that uncharacterized genes can significantly impact lifespan through TORC1 signaling and mitochondrial pathways. YBR238C was identified as the only uncharacterized gene that increases both chronological and replicative lifespan upon deletion and is downregulated by rapamycin . This gene affects aging through a feedback loop between TORC1 and mitochondria (the TORC1-MItochondria-TORC1 or TOMITO signaling process) .

• Stress response pathway analysis:

  • Examine transcription factors like MSN4 that regulate stress responses

  • Measure reactive oxygen species (ROS) levels in YNL235C mutants

  • Test resistance to oxidative stress (e.g., H₂O₂ exposure)

YBR238C deletion mutants show reduced ROS levels and increased resistance to H₂O₂-induced oxidative stress toxicity , suggesting potential mechanisms through which uncharacterized proteins might influence aging.

What considerations should researchers take when designing CRISPR-Cas9 experiments for YNL235C modification?

CRISPR-Cas9 approaches for YNL235C modification require careful design considerations:

• Guide RNA design strategy:

  • Perform thorough off-target analysis specific to the S. cerevisiae genome

  • Select target sites that ensure complete functional disruption

  • Consider multiple guide RNAs targeting different regions to compare phenotypes

• Repair template optimization:

  • Design homology arms of appropriate length (40-60 bp for yeast)

  • Include silent mutations in the PAM site or seed region to prevent re-cutting

  • Plan for seamless epitope tagging if protein localization/interaction studies are intended

• Transformation considerations:

  • Use high-efficiency protocols like HEEL to maximize transformation while minimizing multi-plasmid uptake

  • Employ appropriate selection strategies to isolate modified clones

  • Consider inducible CRISPR systems for temporal control of modifications

• Validation approach:

  • Sequence the modified locus to confirm precise editing

  • Verify the absence/modification of YNL235C expression

  • Compare phenotypes with traditional deletion methods to rule out CRISPR-specific artifacts

How should researchers investigate potential RNA binding properties of YNL235C?

Given that YNL235C belongs to a family of proteins with potential RNA binding properties, several specialized techniques could be employed:

• RNA immunoprecipitation (RIP) approach:

  • Express tagged versions of YNL235C

  • Perform immunoprecipitation under RNA-preserving conditions

  • Identify bound RNAs through RNA-seq or targeted RT-PCR

  • Include appropriate controls for non-specific binding

• In vitro RNA binding assays:

  • Express and purify recombinant YNL235C protein

  • Perform electrophoretic mobility shift assays (EMSA) with candidate RNAs

  • Use fluorescence anisotropy to measure binding affinities

  • Employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to identify preferred binding sequences

• Cross-linking approaches:

  • Apply UV cross-linking and immunoprecipitation (CLIP) methodologies

  • Implement PAR-CLIP for enhanced crosslinking efficiency

  • Map binding sites at nucleotide resolution

• Structural studies of RNA-protein complexes:

  • Use NMR or X-ray crystallography to characterize interaction domains

  • Employ mutagenesis to identify critical residues for RNA binding

  • Validate structural predictions through functional assays

What controls and validation steps are essential when examining YNL235C's potential involvement in stress response pathways?

When investigating YNL235C's role in stress response, rigorous controls and validation steps are necessary:

• Experimental controls:

  • Include multiple well-characterized stress response mutants as benchmarks

  • Apply a standardized panel of stressors (oxidative, temperature, osmotic)

  • Implement time-course experiments to distinguish primary from secondary effects

  • Use isogenic strains to eliminate background effects

• Phenotypic validation:

  • Quantify growth parameters under normal and stress conditions

  • Measure cellular markers of stress (e.g., HSP expression, ROS levels)

  • Assess cell viability using multiple independent methods

  • Determine stress recovery kinetics after removal of stressor

• Genetic validation:

  • Perform complementation with wild-type YNL235C

  • Create point mutants affecting specific protein domains

  • Implement pathway-specific suppressor screens

  • Test epistatic relationships with known stress response regulators

• Molecular validation:

  • Quantify transcriptional changes of stress response genes

  • Assess protein levels of key stress response factors like Msn4

  • Examine post-translational modifications associated with stress

  • Determine whether YNL235C itself is modified during stress response

What are the key physical and biochemical properties of YNL235C protein?

Based on available information, the following table summarizes the key properties of YNL235C:

What research resources and reagents are available for YNL235C studies?

The following resources can facilitate YNL235C research:

What are the major knowledge gaps regarding YNL235C and how might they be addressed?

Several significant knowledge gaps exist regarding YNL235C that warrant systematic investigation:

• Biological function:

  • Primary molecular role (enzymatic activity, structural, regulatory)

  • Cellular pathways and processes involving YNL235C

  • Conditions under which YNL235C becomes essential

• Structural information:

  • Three-dimensional structure

  • Functional domains and motifs

  • Structure-function relationships

• Regulation mechanisms:

  • Transcriptional and post-transcriptional regulation

  • Post-translational modifications

  • Protein turnover and stability factors

• Interaction landscape:

  • Direct protein and/or RNA interaction partners

  • Genetic interaction network

  • Subcellular localization and dynamics

• Evolutionary context:

  • Conservation pattern across species

  • Functional homologs in other organisms

  • Evolutionary history and selection pressures

These gaps could be addressed through integrative approaches combining classical genetics, molecular biology, biochemistry, structural biology, and computational analysis. Particular attention should be given to stress conditions and aging models, given the emerging connections between uncharacterized yeast genes and longevity pathways .

How might research on YNL235C contribute to broader understanding of eukaryotic cellular processes?

Research on uncharacterized proteins like YNL235C has the potential to make significant contributions to our understanding of fundamental eukaryotic cellular processes:

• Completion of the functional map of the yeast proteome:

  • Approximately 1000 yeast genes remain functionally uncharacterized

  • Each characterization advances our understanding of the minimal eukaryotic genome

• Discovery of novel regulatory mechanisms:

  • Uncharacterized proteins often reveal unexpected cellular regulatory circuits

  • New connections between established pathways may be uncovered

• Evolutionary insights:

  • Understanding of protein function conservation across species

  • Identification of yeast-specific adaptations and their significance

• Translational potential:

  • Many yeast genes have human homologs with disease relevance

  • Mechanistic insights can inform human disease studies

The case of YBR238C illustrates how characterizing previously unknown genes can reveal novel regulatory mechanisms with broad implications. YBR238C was found to be an effector of TORC1 that modulates mitochondrial function and affects cellular aging . Similar breakthrough discoveries could emerge from YNL235C research, particularly in understanding the integration of nutrient sensing, stress response, and longevity pathways in eukaryotic cells.