Recombinant Saccharomyces cerevisiae Uncharacterized protein YCR024C-B (YCR024C-B)

What is YCR024C-B and where is it found?

YCR024C-B is an uncharacterized protein found in the budding yeast Saccharomyces cerevisiae. It is encoded on chromosome III and is involved in bicistronic transcription with PMP1, another yeast gene. As an uncharacterized protein, its specific cellular function has not been fully elucidated, though it has been the subject of various research efforts to understand its role in yeast biology .

What is currently known about YCR024C-B's genomic context?

YCR024C-B exists in a bicistronic relationship with PMP1, where both genes are transcribed as part of the same mRNA transcript. This bicistronic arrangement appears to have functional significance, as YCR024C-B has a 3' untranslated region that may direct PMP1 subcellular localization. The gene is located on chromosome III near the MAT locus, a region that has been studied for its role in nuclear pore complex (NPC) assembly .

How has YCR024C-B been used in nuclear pore complex assembly studies?

YCR024C-B has been investigated in studies focusing on nuclear pore complex (NPC) assembly in Saccharomyces cerevisiae. In one significant study, researchers were attempting to identify genetic factors required for NPC assembly by testing whether YCR024C-B could complement the temperature-sensitive phenotype of an NPC assembly mutant strain (KRY141).

The experimental approach involved:

• Cloning YCR024C-B into a yeast expression plasmid

• Transforming both wild-type (YGS52) and NPC assembly mutant (KRY141) strains with this plasmid

• Testing growth at permissive (23°C) and non-permissive (34°C) temperatures

• Analyzing whether expression of wild-type YCR024C-B could rescue the temperature-sensitive phenotype

Results showed that while all strains grew at the permissive temperature, expressing YCR024C-B in KRY141 did not enable growth at the non-permissive temperature, indicating that YCR024C-B is not the gene mutated in KRY141 and is likely not directly involved in the specific NPC assembly pathway affected in this mutant .

What methodologies are used to express and purify recombinant YCR024C-B?

For researchers interested in studying YCR024C-B biochemically, recombinant expression and purification methods have been established:

• Expression system: E. coli bacterial expression system with an N-terminal His-tag

• Purification method: Likely affinity chromatography using the His-tag

• Final form: Lyophilized powder

• Storage buffer: Tris/PBS-based buffer containing 6% Trehalose, pH 8.0

• Reconstitution protocol:

  • Centrifuge vial briefly before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) for long-term storage

  • Aliquot and store at -20°C/-80°C

• Stability considerations: Avoid repeated freeze-thaw cycles; working aliquots can be stored at 4°C for up to one week

This methodology provides researchers with purified protein for functional, structural, or interaction studies that could help elucidate YCR024C-B's biological role.

How can researchers investigate the bicistronic relationship between YCR024C-B and PMP1?

To study the bicistronic relationship between YCR024C-B and PMP1, researchers can employ several methodological approaches:

• Transcriptomic analysis: RNA-seq or northern blot analysis to confirm and characterize the bicistronic transcript

• Ribosome profiling: To measure translation efficiency (TE) of both genes within the bicistronic transcript compared to monocistronic transcripts

• Reporter assays: Constructing reporter fusions to determine how the bicistronic arrangement affects expression of each gene

• Mutational analysis: Introducing mutations in potential regulatory elements within the bicistronic transcript to determine their effects

• Conservation analysis: Comparing the bicistronic arrangement across different yeast strains to assess evolutionary conservation

Research has shown that in bicistronic transcripts, the second gene (in this case, potentially PMP1) often shows lower translation efficiency than when expressed monocistronically. This suggests regulatory functions for the bicistronic arrangement .

What is the significance of bicistronic transcription in yeast, and how does it apply to YCR024C-B?

Bicistronic transcription in yeast represents an important regulatory mechanism that can affect gene expression. For YCR024C-B and PMP1:

• Regulatory potential: Research on bicistronic transcripts in yeast indicates they often serve regulatory functions. For example, in the YOR302W_CPA1 case, bicistronic transcription with translation of YOR302W can repress translation of CPA1 transcripts via ribosome stalling when arginine is present.

• Expression dynamics: Highly expressed bicistronic transcripts tend to be more conserved than lowly expressed ones, suggesting functional importance. Data shows that typically the first gene in bicistronic transcripts exhibits higher expression than the second gene.

• Translational efficiency: Studies comparing translation efficiency between genes in bicistronic transcripts revealed that the second gene often shows decreased translation efficiency compared to its monocistronic counterpart.

In the specific case of YCR024C-B and PMP1, the 3' untranslated region of YCR024C-B appears to direct PMP1 subcellular localization, suggesting an important functional relationship between these two genes mediated by their bicistronic arrangement .

What experimental design considerations are important when using YCR024C-B in genetic complementation studies?

When designing complementation studies involving YCR024C-B:

• Proper controls:

  • Positive control: Wild-type strain (e.g., YGS52) with empty vector and with YCR024C-B

  • Negative control: Mutant strain (e.g., KRY141) with empty vector

  • Test condition: Mutant strain with YCR024C-B expression vector

• Growth conditions:

  • Testing at both permissive temperature (23°C) and non-permissive temperature (34°C)

  • Using appropriate selective media to maintain plasmids

• Expression verification:

  • Confirming protein expression through western blotting or other techniques

  • Ensuring appropriate subcellular localization if relevant

• Phenotypic readouts:

  • Clear, quantifiable phenotypes (e.g., colony growth at restrictive temperature)

  • Secondary phenotypic assays to confirm results (e.g., microscopy for NPC assembly)

• Combinatorial testing:

  • Testing YCR024C-B in combination with its bicistronic partner PMP1

  • Previous research tested PMP1 and YCR024C-B together when investigating nuclear pore complex assembly mutants

How can researchers address contradictory data when investigating uncharacterized proteins like YCR024C-B?

When investigating uncharacterized proteins such as YCR024C-B, researchers often encounter conflicting or ambiguous data. Methodological approaches to address these issues include:

• Multiple experimental approaches:

  • Employing diverse techniques (genetic, biochemical, structural) to build converging lines of evidence

  • Example from research: "This workflow demonstrates the power of combining complementary techniques including in-cell crosslinking to discover high-confidence direct protein interactions without genetic modification, and to accurately predict and validate corresponding structural models."

• Validation with orthogonal methods:

  • Confirming findings using technically distinct approaches

  • Cross-referencing computational predictions with experimental data

• Comprehensive literature review:

  • Examining ALL available information about the protein and related systems

  • Placing findings in broader biological context

• Collaboration across disciplines:

  • Engaging researchers with different expertise (genetics, structural biology, systems biology)

  • Combining global approaches: "In this case, experimental data from global proteomic approaches, structure modeling, and in vivo validation converge to identify a novel protein-protein interaction and to demonstrate its biological function."

• Detailed documentation of experimental conditions:

  • Recording precise conditions that may influence results

  • For YCR024C-B, factors like temperature sensitivity are critical experimental variables

What computational approaches might help predict YCR024C-B function?

Modern computational approaches offer powerful ways to predict potential functions of uncharacterized proteins like YCR024C-B:

• AI-assisted structural prediction:

  • AlphaFold or similar tools can predict protein structure with high confidence

  • Structure prediction can reveal potential functional domains or interaction interfaces

  • "With this approach we identify the previously uncharacterized protein YneR, here renamed PdhI, as an inhibitor of the pyruvate dehydrogenase."

• Interaction network analysis:

  • Predicting protein-protein interactions based on sequence similarity or co-expression

  • Mapping YCR024C-B into the broader yeast interactome

• Evolutionary analysis:

  • Conservation patterns across species can indicate functional importance

  • "Highly expressed bicistronic transcripts are more conserved than lowly expressed bicistronic transcripts both within and between strains."

• Integrative multi-omics approaches:

  • Combining transcriptomic, proteomic, and phenomic data

  • Systems biology frameworks to place YCR024C-B in functional context

• Machine learning classification:

  • Training models on characterized proteins to predict features of uncharacterized ones

  • Feature extraction from sequence, structure, and genomic context

What specialized techniques could help determine YCR024C-B's role in cellular processes?

Several advanced experimental techniques could help elucidate YCR024C-B's cellular functions:

• Crosslinking mass spectrometry (XL-MS):

  • "Stabilizing the proteome prior to cell lysis is especially powerful for identifying interactions involving uncharacterized proteins."

  • Can capture transient or weak protein interactions that might be missed by traditional approaches

• Size exclusion chromatography with crosslinking:

  • "Crosslinking stabilized some members of complexes and aided their co-elution."

  • Helps identify stable protein complexes containing YCR024C-B

• Ribosome profiling for bicistronic transcripts:

  • "Here, we describe the genomic, transcriptomic, and ribosome profiling features of bicistronic transcripts in unicellular yeasts."

  • Can determine translation efficiency of YCR024C-B in its native context

• Fluorescence microscopy with tagged proteins:

  • "Fluorescence microscopy reveals the mislocalization of NPC proteins due to the failure to assemble NPCs."

  • Could determine subcellular localization and dynamics of YCR024C-B

• Systematic mutation analysis:

  • Creating targeted mutations to identify functional residues

  • Testing phenotypic effects under various conditions

These advanced techniques, when applied systematically, have the potential to reveal the function of YCR024C-B and its role in the bicistronic relationship with PMP1, ultimately contributing to our understanding of basic yeast biology and potentially conserved eukaryotic cellular processes.

How should researchers interpret temperature sensitivity data in YCR024C-B studies?

Temperature sensitivity assays are critical in YCR024C-B research, particularly in the context of NPC assembly studies. Researchers should consider:

• Growth comparison analysis:

  • Control strains at permissive (23°C) and non-permissive (34°C) temperatures

  • Careful quantification of colony size and growth rates

  • "It should be noted there is a difference between KRY141 and WT colony sizes indicating the difference in growth rates between the two strains even at the permissive temperature."

• Complete experimental matrix:

  Strain	Plasmid	Growth at 23°C	Growth at 34°C	Interpretation
  WT (YGS52)	Empty	Yes	Yes	Positive control
  WT (YGS52)	YCR024C-B	Yes	Yes	No negative effect
  KRY141	Empty	Yes	No	Negative control
  KRY141	YCR024C-B	Yes	No	No complementation
  KRY141	YCR024C-B+PMP1	Yes	No	No complementation

• Integration with other phenotypic data:

  • Correlating temperature sensitivity with other measurable phenotypes

  • Using microscopy or biochemical assays to confirm NPC assembly defects

• Statistical analysis:

  • Quantitative measurement of growth rates or colony sizes

  • Multiple biological and technical replicates to ensure reproducibility

What challenges exist in analyzing bicistronic transcript data for YCR024C-B and PMP1?

Analysis of bicistronic transcription presents several methodological challenges:

• Distinguishing transcriptional from translational effects:

  • "The second gene in the bicistronic transcripts is repressed in the bh and sh categories."

  • Need to separate effects on mRNA levels from effects on translation efficiency

• Quantifying relative expression:

  • Determining the relative expression levels of both genes in the bicistronic transcript

  • Comparing to monocistronic expression levels

• Identifying regulatory elements:

  • Locating specific sequence elements that control bicistronic expression

  • "YCR024C-B has a 3' untranslated region that directs PMP1 subcellular..."

• Categorizing expression levels:

  • "...genes in the lowly expressed category are not affected."

  • Proper categorization of expression levels to interpret results correctly

• Conservation analysis complexities:

  • "Highly expressed bicistronic transcripts are more conserved than lowly expressed bicistronic transcripts both within and between strains."

  • Analyzing evolutionary conservation patterns in context of expression levels

Researchers must employ a combination of genomic, transcriptomic, and translational analysis techniques to fully understand the bicistronic relationship between YCR024C-B and PMP1.

What are the most promising research directions for characterizing YCR024C-B?

Based on current knowledge, the most promising research avenues include:

• Detailed analysis of the bicistronic relationship with PMP1:

  • Investigating regulatory mechanisms between these genes

  • Determining how their co-expression affects cellular functions

• Comprehensive protein interaction studies:

  • Employing crosslinking techniques to stabilize interactions

  • "Stabilizing the proteome prior to cell lysis is especially powerful for identifying interactions involving uncharacterized proteins."

• Integration with systems biology approaches:

  • Placing YCR024C-B in broader cellular pathways

  • Using multi-omics data to establish functional context

• Structure-function analysis:

  • Determining the three-dimensional structure and relating it to potential functions

  • Identifying critical residues through targeted mutagenesis

• Evolutionary analysis across fungal species:

  • Examining conservation patterns to identify functionally important regions

  • Comparing bicistronic arrangements in related species

By pursuing these research directions with rigorous experimental designs and complementary methodologies, researchers can make significant progress in understanding this uncharacterized protein's biological role.

How can researchers best interpret negative results in YCR024C-B studies?

Negative results, such as the failure of YCR024C-B to complement the KRY141 mutation, provide valuable information when properly interpreted:

• Systematic elimination process:

  • "In this way, we have ruled out 4 potential genes: MAK32, PMP1, YCR024C-B, and YCR025C."

  • Negative results narrow down possibilities in a systematic research approach

• Experimental design validation:

  • Ensuring appropriate controls functioned as expected

  • Verifying experimental conditions were suitable for detecting complementation

• Alternative hypothesis generation:

  • Using negative results to formulate new hypotheses about protein function

  • Considering indirect roles or redundancy with other proteins

• Integration with positive findings:

  • Placing negative results in context with any positive findings about the protein

  • Building a comprehensive understanding that includes both what the protein does and does not do

• Publication of negative results:

  • Contributing to the scientific literature even when hypotheses are not confirmed

  • Preventing duplication of effort by other research groups