Recombinant Saccharomyces cerevisiae Vacuolar membrane protein YNL058C (YNL058C)

What is YNL058C and what is known about its function?

YNL058C is a putative protein of unknown function found in Saccharomyces cerevisiae (baker's yeast). Green fluorescent protein (GFP)-fusion studies have demonstrated that the protein localizes to the vacuole, suggesting a role in vacuolar processes. Current evidence indicates that YNL058C is not an essential gene, as deletion mutants remain viable under standard laboratory conditions .

What genomic and proteomic resources are available for YNL058C research?

Multiple resources exist for researchers studying YNL058C:

• Saccharomyces Genome Database (SGD): Provides comprehensive genomic information including sequence data, functional annotations, and mutant phenotypes. Researchers can access this information at the YNL058C locus page .

• Protein databases: The UniProt accession number for YNL058C is P53947, which provides access to curated protein information .

• Recombinant protein: Commercially available recombinant YNL058C can be obtained for experimental studies, typically supplied in Tris-based buffer with 50% glycerol .

• Gene Ontology (GO) annotations: While limited due to the uncharacterized nature of the protein, GO annotations for YNL058C are available through SGD and provide computational predictions of function .

What are effective experimental approaches for characterizing YNL058C function?

When designing experiments to investigate YNL058C function, researchers should implement multiple complementary approaches:

• Reverse genetics: Generate deletion mutants (Δynl058c) and assess phenotypic consequences under various conditions. This requires clearly defined experimental variables and appropriate controls .

• Protein localization studies: While GFP-fusion studies have localized YNL058C to the vacuole, more detailed co-localization studies with known vacuolar markers can provide insights into specific subcompartments or domains.

• Protein-protein interaction studies:

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

  • Yeast two-hybrid screening

  • Proximity labeling approaches (BioID or APEX)

• Transcriptional profiling: Compare gene expression patterns between wild-type and Δynl058c strains to identify affected pathways.

• Metabolomic analysis: Characterize changes in vacuolar metabolites in Δynl058c strains.

How should researchers design controls for YNL058C functional studies?

Proper experimental controls are essential for YNL058C research:

• Genetic controls:

  • Wild-type (WT) strain with identical genetic background

  • Complementation strain (Δynl058c expressing YNL058C from a plasmid)

  • Strains with deletions in genes of known vacuolar function for comparison

• Technical controls:

  • Empty vector controls for overexpression studies

  • Isogenic controls for growth conditions

  • Negative and positive controls for protein-protein interaction studies

• Validation controls:

  • Use multiple methods to confirm findings

  • Include known vacuolar membrane proteins as reference points

  • Implement rescue experiments to confirm phenotype specificity

The experimental design should carefully account for control variables (e.g., temperature, media composition) and potential confounding variables (e.g., secondary mutations, strain background effects) to ensure reproducible results .

What approaches can be used to express and purify recombinant YNL058C for biochemical studies?

Recombinant expression and purification of membrane proteins like YNL058C presents specific challenges. The following methodological approach is recommended:

• Expression system selection:

  • Heterologous expression in E. coli (using specialized strains for membrane proteins)

  • Homologous expression in S. cerevisiae (preferred for maintaining native folding)

  • Alternative eukaryotic systems (P. pastoris, insect cells) for higher yields

• Construct design:

  • Include affinity tags (His6, FLAG, etc.) for purification

  • Consider fusion partners to increase solubility (MBP, SUMO, etc.)

  • Design constructs with and without predicted transmembrane domains

• Purification strategy:

  • Membrane isolation through differential centrifugation

  • Solubilization screening with different detergents (DDM, LMNG, CHAPS)

  • Affinity chromatography followed by size exclusion chromatography

  • Consider amphipol or nanodisc reconstitution for stabilization

• Quality control:

  • SDS-PAGE and western blotting for purity assessment

  • Mass spectrometry for identity confirmation

  • Circular dichroism for secondary structure analysis

  • Thermal stability assays to optimize buffer conditions

Commercially available recombinant YNL058C is supplied in Tris-based buffer with 50% glycerol, which has been optimized for this protein . Researchers developing their own purification protocols may use these buffer conditions as a starting point.

How can researchers investigate the potential role of YNL058C in cellular processes?

To understand YNL058C's role in cellular processes, researchers should consider multifaceted approaches:

• Phenotypic profiling under stress conditions:

  • Osmotic stress (high salt, sorbitol)

  • pH stress (acidic and alkaline conditions)

  • Nutrient limitation (nitrogen, carbon, phosphate)

  • Oxidative stress (H₂O₂, menadione)

  • Cell wall/membrane stress (Calcofluor White, SDS)

• Vacuolar function assays:

  • Vacuolar pH measurement using pH-sensitive fluorescent proteins

  • Vacuolar inheritance during cell division

  • Vacuolar fragmentation/fusion dynamics

  • Protein sorting to the vacuole

  • Vacuolar enzyme activities (e.g., carboxypeptidase Y)

• Genetic interaction mapping:

  • Synthetic genetic array (SGA) analysis with Δynl058c

  • Targeted genetic interactions with known vacuolar proteins

  • Suppressor screens to identify functional relationships

• Phosphorylation analysis:
  Based on the sequence analysis, YNL058C contains numerous potential phosphorylation sites. The protein has been identified as containing DDK/CDK consensus sites, suggesting it may be regulated by cell-cycle dependent kinases .

How might researchers investigate potential regulatory relationships involving YNL058C?

Understanding the regulatory network surrounding YNL058C requires investigation at multiple levels:

• Transcriptional regulation:

  • Promoter analysis for transcription factor binding sites

  • ChIP-seq to identify transcription factors binding the YNL058C promoter

  • Reporter gene assays to map regulatory elements

• Post-translational modifications:

  • Phosphorylation mapping by mass spectrometry

  • Investigation of ubiquitination and other modifications

  • Mutagenesis of predicted modification sites to assess functional consequences

• Protein turnover and stability:

  • Cycloheximide chase assays to measure protein half-life

  • Proteasome inhibition studies

  • Analysis of protein levels across growth phases and stress conditions

• Regulatory network inference:

  • Integration of multiple data types to build comprehensive models

  • Network analysis to identify regulatory hubs connected to YNL058C

  • Analysis of transcriptional responses related to YNL058C modulation

Research suggests that bidirectional terminators in S. cerevisiae play a role in preventing spurious transcription from invading neighboring genes, which could be relevant for understanding YNL058C expression regulation .

What strategies can help resolve contradictory findings about YNL058C?

When faced with conflicting results regarding YNL058C function or characteristics, researchers should:

• Systematic parameter investigation:

  • Thoroughly document and compare experimental conditions

  • Test strain background effects (BY4741 vs. W303 vs. S288C)

  • Validate key reagents (antibodies, constructs) across laboratories

  • Establish standardized protocols for core assays

• Multi-laboratory validation:

  • Implement blind testing procedures

  • Share strains, reagents, and detailed protocols

  • Perform parallel experiments with standardized controls

• Data integration approaches:

  • Develop computational models that accommodate apparently contradictory data

  • Employ Bayesian analysis to update confidence in specific hypotheses

  • Consider context-dependent mechanisms that might explain divergent results

• Publication of negative and contradictory results:

  • Document experimental variations that lead to different outcomes

  • Transparently report all experimental attempts, not just successful ones

  • Consider preprint platforms for rapid dissemination of contradictory findings

What are common challenges in studying YNL058C and how can they be addressed?

Researchers working with YNL058C may encounter several technical challenges:

• Protein detection issues:

  • Low endogenous expression levels

  • Limited antibody availability or specificity

  • Solution: Use epitope tagging strategies (HA, FLAG, V5) or fluorescent protein fusions

• Functional redundancy:

  • Potential overlapping functions with other vacuolar membrane proteins

  • Solution: Generate multiple gene deletions or employ conditional alleles

• Membrane protein solubility:

  • Challenges in extracting and maintaining native conformation

  • Solution: Screen multiple detergent conditions; consider nanodiscs or amphipols

• Phenotypic subtlety:

  • Mild or condition-specific phenotypes that are difficult to detect

  • Solution: Employ sensitive high-throughput methods; test diverse environmental conditions

• Experimental variation:

  • Day-to-day variability in results due to subtle environmental factors

  • Solution: Implement robust statistical design with sufficient replication and appropriate controls

What statistical considerations are important when analyzing data from YNL058C experiments?

When analyzing experimental data related to YNL058C:

What emerging technologies might enhance YNL058C characterization?

Several cutting-edge approaches could advance our understanding of YNL058C:

• CRISPR-based technologies:

  • CRISPRi for tunable gene repression

  • Base editing for introducing specific mutations

  • CRISPR activation for overexpression studies

  • CRISPR screens for genetic interaction mapping

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed localization

  • FRET/FLIM for protein-protein interaction studies

  • Live-cell imaging with minimal photobleaching

  • Correlative light and electron microscopy for ultrastructural context

• Structural biology approaches:

  • Cryo-electron microscopy for membrane protein structures

  • Integrative structural biology combining multiple data types

  • Molecular dynamics simulations to model membrane interactions

• Single-cell technologies:

  • Single-cell transcriptomics to identify cell-to-cell variation

  • Single-cell proteomics for protein abundance quantification

  • Microfluidic approaches for precise environmental control

How might systems biology approaches enhance understanding of YNL058C function?

Systems biology offers powerful frameworks for integrating diverse data types to understand YNL058C in a broader cellular context:

• Multi-omics integration:

  • Combined analysis of transcriptomic, proteomic, and metabolomic data

  • Network inference from multiple data types

  • Identification of emergent properties not visible in single datasets

• Mathematical modeling:

  • Kinetic models of vacuolar processes

  • Flux balance analysis incorporating vacuolar functions

  • Stochastic modeling of protein dynamics

• Evolutionary approaches:

  • Comparative genomics across fungal species

  • Analysis of selection pressures on YNL058C

  • Investigation of protein family relationships

• Automated experimental design:

  • Machine learning approaches to prioritize experiments

  • Iterative cycles of prediction and validation

  • Optimal experimental design for parameter estimation in models

The integration of these approaches within a systems biology framework will likely yield the most comprehensive understanding of YNL058C function, moving beyond isolated studies to place this protein within its broader cellular and evolutionary context.