Recombinant Saccharomyces cerevisiae Uncharacterized protein YER039C-A (YER039C-A)

What is currently known about the subcellular localization of YER039C-A?

YER039C-A has been identified as a multi-pass membrane protein according to the UniProt database . This classification indicates that the protein contains multiple membrane-spanning domains that integrate into cellular membranes. The specific membrane system (such as plasma membrane, endoplasmic reticulum, or mitochondrial membrane) has not been definitively established in the available data.

To determine the precise subcellular localization, researchers typically employ techniques such as:

• Fluorescent protein tagging and microscopy

• Subcellular fractionation followed by western blotting

• Immunolocalization using specific antibodies

The membrane localization suggests potential roles in membrane transport, signaling, or structural functions, but further experimental validation is required to confirm its exact location and function within the cell.

What are the optimal conditions for expression and purification of recombinant YER039C-A?

Based on the available data, recombinant YER039C-A has been successfully expressed in E. coli with an N-terminal His-tag . The following methodological approach is recommended:

Expression System:

• Host: E. coli (BL21 or Rosetta strains are commonly used for membrane proteins)

• Vector: pET series vectors with N-terminal His-tag

• Induction: IPTG concentration should be optimized (typically 0.1-1.0 mM)

• Temperature: Lower temperatures (16-25°C) often improve membrane protein folding

• Duration: Extended induction periods (16-24 hours) at lower temperatures

Purification Strategy:

• Cell lysis using detergent-containing buffers (e.g., n-dodecyl β-D-maltoside or CHAPS)

• Initial purification using Ni-NTA affinity chromatography

• Secondary purification using size exclusion chromatography

For maximum purity, the protocol should achieve >90% purity as determined by SDS-PAGE, consistent with the specifications mentioned in the product data .

How should researchers handle and store purified YER039C-A protein for optimal stability?

For optimal stability and activity maintenance of YER039C-A, the following handling and storage protocols are recommended:

Storage Conditions:

• Store at -20°C or preferably -80°C upon receipt

• Aliquoting is necessary to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

Buffer Composition:

• Tris/PBS-based buffer with pH 8.0

• Addition of 6% trehalose as a stabilizing agent

• Consider adding protease inhibitors for extended storage

Reconstitution Protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (recommended 50%) for long-term storage

Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of function, particularly for membrane proteins like YER039C-A.

What genetic interactions have been documented for YER039C-A and what do they suggest about its function?

The available data reveals that YER039C-A participates in numerous genetic interactions with various yeast proteins, suggesting potential functional roles. These interactions were documented in a study referenced by PubMed ID 27708008 .

Key Genetic Interaction Partners:

These genetic interactions span diverse cellular processes, with notable representation in membrane trafficking (ATC3, VAM7, BET3), suggesting YER039C-A may function in vesicular transport or membrane organization pathways. The interactions with cell cycle regulators (MET30) and protein quality control machinery (CDC48) further suggest roles in growth regulation or stress response mechanisms.

What experimental approaches are recommended to characterize the function of this uncharacterized protein?

To systematically characterize the function of YER039C-A, a multi-faceted experimental approach is recommended:

1. Phenotypic Profiling:

• Assess growth under various stress conditions (temperature, osmotic, pH, nutrient limitation)

• Screen for sensitivity to membrane-disrupting agents

• Analyze morphological changes in deletion or overexpression strains

2. Localization and Dynamics:

• Fluorescent protein tagging (C-terminal GFP fusion)

• Live-cell imaging to monitor subcellular distribution

• Colocalization studies with known compartment markers

3. Interaction Proteomics:

• Affinity purification coupled with mass spectrometry (AP-MS)

• Proximity-dependent labeling (BioID or APEX)

• Validation of key interactions using co-immunoprecipitation

4. Functional Genomics:

• CRISPR-Cas9 mediated gene editing to create functional mutants

• Synthetic genetic array (SGA) analysis to expand interaction network

• RNA-seq to identify transcriptional changes upon deletion/overexpression

5. Structural Analysis:

• Membrane topology mapping using cysteine accessibility methods

• Cryo-electron microscopy for structural determination

• Computational modeling and simulation of membrane integration

These approaches should be implemented in an iterative manner, with each experiment informing the design of subsequent investigations to gradually build a comprehensive functional profile of YER039C-A.

How can researchers investigate potential post-translational modifications of YER039C-A?

Although current databases indicate no documented post-translational modifications (PTMs) for YER039C-A , this likely reflects a lack of targeted studies rather than an absence of modifications. To investigate potential PTMs, researchers should employ the following methodological approach:

Mass Spectrometry-Based PTM Identification:

• Enrich the protein using immunoprecipitation or His-tag purification

• Perform proteolytic digestion with multiple enzymes (trypsin, chymotrypsin)

• Analyze peptides using high-resolution LC-MS/MS with fragmentation techniques optimized for PTM detection

• Search against PTM databases with variable modification parameters

Specific PTM Enrichment Strategies:

• Phosphorylation: TiO₂ or IMAC enrichment

• Glycosylation: Lectin affinity chromatography

• Ubiquitination: K-ε-GG antibody enrichment

In vivo PTM Detection:

• Metabolic labeling with PTM precursors

• Pharmacological inhibition of PTM-regulating enzymes

• Site-directed mutagenesis of predicted modification sites

Given YER039C-A's membrane localization, particular attention should be paid to modifications that regulate membrane protein trafficking and stability, such as phosphorylation, palmitoylation, and ubiquitination.

How should researchers address the lack of expression data for YER039C-A?

The Saccharomyces Genome Database indicates no expression data available for YER039C-A , presenting a significant research challenge. To address this gap, researchers should implement a comprehensive expression profiling strategy:

RNA-Level Expression Analysis:

• Design specific qRT-PCR primers spanning exon junctions

• Perform Northern blot analysis with strand-specific probes

• Implement RNA-seq with sufficient depth to capture low-abundance transcripts

• Analyze expression across diverse environmental conditions and stress responses

Protein-Level Expression Detection:

• Generate specific antibodies against unique peptide regions of YER039C-A

• Develop a targeted proteomics assay using selected reaction monitoring (SRM)

• Employ epitope tagging approaches (HA, FLAG, or GFP tags) for detection

• Utilize proximity labeling methods to capture transient expression

Regulatory Element Analysis:

• Characterize the promoter region using reporter constructs

• Identify transcription factor binding sites through ChIP-seq

• Investigate potential regulatory ncRNAs that might influence expression

• Analyze chromatin accessibility and histone modifications at the locus

The expression pattern of YER039C-A should be examined under various physiological and stress conditions, with particular attention to conditions that affect the phenotypes of interacting genes identified in genetic screens.

What is the recommended experimental design for investigating YER039C-A function in membrane dynamics?

Given YER039C-A's classification as a multi-pass membrane protein and its genetic interactions with membrane trafficking components, the following experimental design is recommended to investigate its role in membrane dynamics:

Step 1: Define Variables

• Independent variables: YER039C-A expression levels (wild-type, deletion, overexpression)

• Dependent variables: Membrane fluidity, lipid composition, vesicle trafficking rates

• Control variables: Temperature, growth phase, media composition

Step 2: Develop Specific Hypotheses

• H₁: YER039C-A influences membrane lipid organization

• H₂: YER039C-A regulates vesicular trafficking between specific compartments

• H₃: YER039C-A functions in membrane stress response pathways

Step 3: Experimental Treatments

• Generate strains with varying YER039C-A expression:

  • Wild-type control

  • YER039C-A deletion strain

  • Conditional expression strain (tetracycline-regulated promoter)

  • Point mutants affecting key domains

• Apply membrane-specific stressors:

  • Temperature shifts (heat shock/cold shock)

  • Membrane-disrupting agents (SDS, ethanol)

  • Osmotic stress conditions

Step 4: Measurement Techniques

• Membrane fluidity assessment:

  • Fluorescence anisotropy measurements

  • Electron paramagnetic resonance (EPR) spectroscopy

  • Laurdan generalized polarization

• Trafficking dynamics:

  • FM4-64 endocytic trafficking assays

  • Secretory pathway cargo transport rates

  • Localization of compartment-specific markers

• Lipidomic analysis:

  • Quantitative mass spectrometry of membrane lipids

  • Domain-specific lipid probes

  • Detergent resistance membrane fractionation

This systematic approach will provide insights into YER039C-A's specific role in membrane biology while controlling for confounding variables that might affect membrane dynamics .

How can researchers reconcile contradictory results regarding YER039C-A function?

When confronted with contradictory results regarding YER039C-A function, researchers should implement a systematic approach to reconciliation that incorporates the following methodological steps:

Standardize Experimental Conditions

• Establish consistent strain backgrounds

• Define precise growth conditions (media, temperature, growth phase)

• Standardize protein expression and purification protocols

• Develop validated detection and quantification methods

Implement Orthogonal Validation Approaches

• Validate findings using multiple independent techniques

• Combine in vivo, in vitro, and in silico approaches

• Verify results across different model systems where applicable

• Develop quantitative assays with appropriate statistical power

Design Factorial Experiments

• Systematically test combinations of variables that might explain discrepancies

• Include appropriate controls for each experimental condition

• Use statistical design of experiments (DOE) approaches

• Document and report all experimental parameters comprehensively

Collaborative Validation

• Establish multi-laboratory validation studies

• Implement blind experimental designs when possible

• Develop shared reagents and standardized protocols

• Pre-register experimental designs to minimize bias

Meta-Analysis of Results

• Develop a database of experimental conditions and outcomes

• Perform statistical meta-analysis of available data

• Identify patterns in contradictory results that might reveal condition-dependent functions

• Generate new testable hypotheses that might explain the observed discrepancies

This structured approach acknowledges that contradictory results may reflect context-dependent functions of YER039C-A rather than experimental errors, potentially revealing important insights about condition-specific roles of this uncharacterized protein.

What are the most promising future research directions for understanding YER039C-A function?

Based on the current knowledge and gaps identified in the available data, the following research directions hold significant promise for elucidating YER039C-A function:

• Integrative multi-omics approach: Combining transcriptomics, proteomics, and metabolomics analyses of YER039C-A mutants to identify affected pathways and processes.

• High-resolution structural studies: Determining the membrane topology and structural features using cryo-electron microscopy or NMR spectroscopy specialized for membrane proteins.

• Synthetic genetic interaction mapping: Expanding the genetic interaction network through systematic double mutant analysis to place YER039C-A in specific cellular pathways.

• Condition-specific functional characterization: Investigating YER039C-A function under diverse environmental conditions, particularly those that stress membrane systems.

• Comparative genomics approach: Analyzing potential homologs in other fungal species to identify evolutionarily conserved functions and structural features.