Recombinant Saccharomyces cerevisiae Putative uncharacterized membrane protein YBR113W (YBR113W)

What is YBR113W and how has it been identified?

YBR113W is a putative uncharacterized membrane protein in Saccharomyces cerevisiae with a full length of 160 amino acids . It was initially identified through genomic sequencing and ORF (Open Reading Frame) prediction. Experimental evidence for its transcription has been confirmed through RT-PCR analysis, where RNA products were first amplified by reverse-transcription, followed by PCR amplification of the resulting products . This demonstrated that YBR113W is actively transcribed in yeast cells, although its precise function remains to be fully characterized.

To study this protein, researchers typically employ the following methodology:

• Gene annotation and sequence analysis

• Transcriptional analysis (RT-PCR, RNA-seq)

• Protein expression verification

• Structural prediction of membrane topology

What expression systems are commonly used to produce recombinant YBR113W protein?

For yeast expression, the methodology typically involves:

• Cloning the YBR113W gene into appropriate yeast expression vectors

• Transformation into S. cerevisiae strains like EBY100 [MATa AGA1::GAL1-AGA1::URA3 ura3–52 trp1 leu2-delta200 his3-delta200 pep4::HIS3 prb11.6R can1 GAL]

• Selection on synthetic minimal media with appropriate auxotrophic markers

• Expression induction and verification

S. cerevisiae strain EBY100 is particularly useful as it can be exploited to express and secrete heterologous proteins and can be cultivated in either YPD Medium for maintenance or Synthetic Minimal Medium with Casamino Acids for selection .

What is known about the genomic context of YBR113W in S. cerevisiae?

YBR113W exists in a sense/antisense (S/AS) pairing with YBR112c, making it part of a notable genomic arrangement . Both ORFs in this pair are expressed, as demonstrated by RT-PCR using primers specific for each individual ORF. Importantly, when RT-PCR reactions were performed with primers encompassing both ORFs, no amplification was detected, ruling out the possibility that detection was due to a single, joint transcript .

This genomic context requires careful experimental design when studying YBR113W expression, particularly:

• Using strand-specific primers for accurate detection

• Controlling for potential read-through transcription

• Considering potential regulatory interactions between YBR113W and YBR112c

• Analyzing possible functional relationships between the paired ORFs

What methodological approaches are most suitable for characterizing the membrane topology of YBR113W?

As a putative membrane protein, determining the topology of YBR113W is crucial for understanding its function. Several complementary approaches should be considered:

• Computational prediction: Use membrane protein topology prediction algorithms to generate initial models based on hydrophobicity plots and transmembrane domain predictions.

• Protein tagging strategies:

  • N- and C-terminal tagging with reporter proteins to determine orientation

  • Internal epitope tagging at predicted loops

  • Comparison with known membrane protein structures

• Biochemical validation:

  • Protease protection assays to identify cytoplasmic vs. external domains

  • Glycosylation mapping for extracellular domains

  • Crosslinking studies to identify interaction interfaces

• Structural characterization:

  • Purification of recombinant protein in appropriate detergents

  • Circular dichroism to assess secondary structure content

  • Cryo-EM or X-ray crystallography for high-resolution structure determination

Since YBR113W is currently available as a His-tagged recombinant protein , researchers can leverage this resource for initial biochemical characterization before proceeding to more complex structural studies.

How can transcriptional analysis be optimized for studying YBR113W expression patterns?

Given the confirmed transcription of YBR113W and its genomic arrangement with YBR112c , several specialized approaches can enhance transcriptional analysis:

• RT-PCR optimization:

  • Design primers that differentiate between sense and antisense transcripts

  • Include controls without reverse transcription to confirm amplification from RNA

  • Sequence RT-PCR products to verify transcript identity

• RNA-seq considerations:

  • Use strand-specific library preparation methods

  • Apply sufficient sequencing depth to detect low-abundance transcripts

  • Analyze data with algorithms capable of resolving overlapping transcripts

• Temporal expression analysis:

  • Monitor expression under different growth conditions

  • Assess expression changes during different cell cycle phases

  • Compare expression patterns with genes of known function

• Single-cell approaches:

  • Apply single-cell RNA-seq to assess cell-to-cell variability in expression

  • Use RNA FISH (fluorescence in situ hybridization) to visualize transcript localization

These methodologies can help determine whether YBR113W expression is constitutive or condition-specific, providing clues to its biological role.

What gene manipulation strategies are most effective for functional characterization of YBR113W?

To elucidate the function of this uncharacterized protein, systematic genetic manipulation approaches are recommended:

• Gene deletion strategies:

  • Create precise YBR113W deletion strains using homologous recombination

  • Perform phenotypic profiling under diverse growth conditions

  • Consider the genomic context with YBR112c, potentially creating double knockouts

• Overexpression systems:

  • Develop controllable overexpression constructs using GAL1 promoter systems

  • Monitor phenotypic consequences of elevated YBR113W levels

  • Create fusion proteins with fluorescent tags for localization studies

• Complementation experiments:

  • Test whether YBR113W deletions can be complemented by homologs from related species

  • Evaluate specific protein domains through domain swapping experiments

  • Assess the impact of site-directed mutations on protein function

• Heterologous expression:

  • Express YBR113W in standard strain backgrounds like EBY100

  • Utilize secretion systems with α-factor secretion signal sequences

  • Apply selection on appropriate minimal media (SMD+CAA) with auxotrophic markers

A systematic application of these strategies can reveal phenotypes associated with YBR113W mutations, providing insights into its cellular role.

What protein purification strategies are most appropriate for recombinant YBR113W?

Purifying membrane proteins like YBR113W presents specific challenges. The following methodological approach is recommended:

• Expression optimization:

  • Test multiple expression systems (E. coli, yeast, insect cells)

  • Evaluate different fusion tags (His, GST, MBP) for improved solubility

  • Compare induction conditions (temperature, inducer concentration, duration)

• Membrane extraction:

  • Screen detergents for efficient solubilization (DDM, LMNG, digitonin)

  • Optimize detergent-to-protein ratios

  • Consider native nanodiscs or SMALPs for maintaining native-like environment

• Chromatography sequence:

  • Initial capture using affinity chromatography (Ni-NTA for His-tagged YBR113W)

  • Secondary purification via ion exchange or size exclusion chromatography

  • Quality assessment by SDS-PAGE, Western blotting, and mass spectrometry

• Stability assessment:

  • Monitor protein stability in various buffer conditions

  • Test additives like glycerol, specific lipids, or stabilizing ligands

  • Evaluate freeze-thaw stability for long-term storage

The commercially available His-tagged YBR113W protein (full length 1-160 amino acids) expressed in E. coli can serve as a reference standard for optimization of in-house purification protocols.

What interactome analysis methods would best identify YBR113W binding partners?

Understanding protein-protein interactions is crucial for characterizing uncharacterized proteins like YBR113W. A comprehensive interaction mapping strategy should include:

• Proximity-based methods:

  • BioID or TurboID fusion proteins to identify proximal proteins in vivo

  • APEX2 tagging for spatial proteomics in membrane compartments

  • Split-protein complementation assays for direct interaction verification

• Affinity-based approaches:

  • Co-immunoprecipitation using tagged YBR113W as bait

  • Tandem affinity purification for increased specificity

  • Crosslinking mass spectrometry to capture transient interactions

• Genetic interaction screening:

  • Synthetic genetic array (SGA) analysis with YBR113W deletion strains

  • Suppressor screens to identify genes that rescue YBR113W mutant phenotypes

  • Overexpression screens to identify genetic interactions

• Membrane-specific considerations:

  • Use detergent conditions that preserve native interactions

  • Consider membrane co-fractionation approaches

  • Apply split-ubiquitin yeast two-hybrid systems specific for membrane proteins

These methodologies should be applied with appropriate controls and validation to build a reliable YBR113W interactome map.

How can researchers effectively study the potential role of YBR113W in membrane-associated processes?

As a putative membrane protein, YBR113W may participate in various membrane-associated functions. The following research approaches can help elucidate its role:

• Subcellular localization:

  • Fluorescent protein tagging to determine precise membrane localization

  • Immunogold electron microscopy for high-resolution localization

  • Cell fractionation studies to biochemically confirm membrane association

• Membrane dynamics:

  • FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility

  • Single-particle tracking to monitor diffusion behavior

  • Lipidomic analysis to identify associated lipid species

• Functional assays:

  • Measure membrane integrity in YBR113W mutants

  • Assess transport activities across membranes

  • Evaluate stress responses related to membrane function

• Biophysical characterization:

  • Reconstitution in artificial membrane systems

  • Electrophysiological measurements if ion transport is suspected

  • Structural studies in lipid environments

These approaches should be prioritized based on preliminary data and computational predictions about YBR113W's potential membrane-associated functions.

What bioinformatic tools and resources are most valuable for YBR113W analysis?

Computational analysis provides essential context for experimental studies of YBR113W. The following resources and approaches are recommended:

• Sequence analysis tools:

  • Multiple sequence alignment to identify conserved regions

  • Homology modeling based on related proteins

  • Evolutionary analysis across fungal species

• Structural prediction:

  • Transmembrane domain prediction algorithms

  • Ab initio structural modeling

  • Protein-protein interaction interface prediction

• Functional annotation resources:

  • Gene Ontology enrichment for potential functional categories

  • Pathway analysis to identify possible biochemical roles

  • Domain and motif scanning for functional elements

• Expression data integration:

  • Analysis of existing transcriptomic datasets

  • Co-expression network analysis

  • Integration with proteomics databases

These computational approaches should be iteratively combined with experimental data to progressively refine hypotheses about YBR113W function.

What considerations should be made when designing RT-PCR experiments to verify YBR113W transcription?

Based on previous successful RT-PCR analysis of YBR113W , several key methodological considerations emerge:

• Primer design strategy:

  • Design primers specific to YBR113W sequence

  • Consider strand-specific primers to distinguish sense/antisense transcription

  • Include primers that span adjacent ORFs to test for joint transcripts

• Controls and validation:

  • Always include no-RT controls to rule out DNA contamination

  • Sequence RT-PCR products to confirm identity

  • Use positive controls with known expressed genes

• Experimental conditions:

  • Test multiple growth conditions to capture condition-specific expression

  • Consider time-course experiments to detect temporal regulation

  • Quantify expression levels using RT-qPCR

• Data interpretation:

  • Compare YBR113W expression to housekeeping genes

  • Analyze the relationship between YBR113W and YBR112c expression

  • Integrate results with existing transcriptomic datasets

These methodological details ensure robust validation of YBR113W transcription and provide a foundation for more detailed expression studies.

What are the key considerations for experimental design when studying uncharacterized proteins like YBR113W?

Investigating proteins with unknown function requires a systematic research strategy:

• Hypothesis development:

  • Generate initial hypotheses based on sequence features and genomic context

  • Consider evolutionary conservation patterns

  • Integrate predictions from multiple bioinformatic tools

• Experimental prioritization:

  • Begin with essential characterization (expression, localization, basic phenotypes)

  • Progress to more specialized analyses based on initial results

  • Design experiments with appropriate positive and negative controls

• Technology selection:

  • Choose methods that provide complementary lines of evidence

  • Consider both low-throughput targeted approaches and high-throughput screening

  • Select techniques appropriate for membrane proteins

• Data integration framework:

  • Establish systems for integrating diverse experimental results

  • Develop clear criteria for functional assignment

  • Plan for iterative refinement of hypotheses

This structured approach maximizes the chance of meaningful functional characterization while minimizing resource expenditure on less promising avenues.

What is currently known about the physiological role of YBR113W in S. cerevisiae?

Based on the available search results, YBR113W remains largely uncharacterized in terms of its physiological function. The current state of knowledge is limited to:

• Confirmed transcription: RT-PCR analysis has definitively established that YBR113W is transcribed, producing detectable mRNA that can be amplified and sequenced .

• Genomic context: YBR113W forms a sense/antisense pair with YBR112c, with both ORFs being independently expressed .

• Protein structure: YBR113W is classified as a putative membrane protein with 160 amino acids .

• Availability for research: Recombinant full-length YBR113W protein with His-tag is available for research purposes .

The lack of comprehensive pathway and functional annotation in the search results suggests that significant knowledge gaps remain regarding YBR113W's biological role.

What emerging technologies might accelerate functional characterization of YBR113W?

Several cutting-edge approaches could significantly advance understanding of YBR113W:

• CRISPR-based technologies:

  • CRISPRi for precise transcriptional repression

  • CRISPRa for targeted upregulation

  • Base editing for introducing specific mutations without double-strand breaks

• Single-cell approaches:

  • Single-cell proteomics to track YBR113W at the protein level

  • Spatial transcriptomics to analyze expression in specific cellular contexts

  • Live-cell imaging with advanced fluorescent tags

• Structural biology innovations:

  • Cryo-EM for membrane protein structures without crystallization

  • Hydrogen-deuterium exchange mass spectrometry for dynamic structural information

  • AlphaFold and related AI-based structure prediction tools

• Multi-omics integration:

  • Combined analysis of transcriptomics, proteomics, and metabolomics

  • Network-based approaches to place YBR113W in biological context

  • Machine learning methods to predict function from diverse data types

These technologies offer promising avenues to overcome the technical challenges that have limited functional characterization of membrane proteins like YBR113W.

How can a research methodology be designed to evaluate potential protein-protein interactions involving YBR113W?

A comprehensive methodology for studying YBR113W interactions should include:

For each identified interaction, validation experiments should be designed to confirm biological relevance and functional significance in the context of membrane biology.