Recombinant Saccharomyces cerevisiae Uncharacterized protein YHL026C (YHL026C)

What experimental approaches should I use to begin characterizing the uncharacterized protein YHL026C in S. cerevisiae?

To begin characterizing YHL026C, implement a systematic approach combining multiple methodologies:

• Sequence analysis: Conduct bioinformatic analysis to identify conserved domains, potential functions, and homology to characterized proteins.

• Gene expression profiling: Determine under which conditions YHL026C is expressed using RNA-seq or microarray analysis.

• Protein localization: Create a YHL026C-GFP fusion construct to visualize subcellular localization, similar to methods used for localizing Yll056cp to the cytoplasm .

• Phenotypic characterization of deletion mutants: Generate a YHL026C knockout strain and assess growth under various conditions to identify potential phenotypes.

• Overexpression studies: Utilize vectors like pYC130 (low-copy) or pAD4 (high-copy with ADH1 promoter) to observe the effects of overexpressing YHL026C .

This multi-faceted approach will provide initial insights into YHL026C's potential function within the cellular context.

What are the optimal conditions for expressing recombinant YHL026C protein in S. cerevisiae?

For optimal expression of recombinant YHL026C in S. cerevisiae:

• Vector selection: Choose between centromere-based low-copy-number plasmids like pYC130 or 2μ-based high-copy-number plasmids like pAD4 containing the ADH1 promoter . The choice depends on whether native-like expression or overexpression is desired.

• Growth media: Standard YPD (2% glucose, 1% yeast extract, 2% peptone) is suitable for general protein expression, while synthetic defined media can be used for more controlled conditions .

• Expression timing: Monitor growth curves to determine optimal harvest time, typically early to mid-log phase for most recombinant proteins.

• Temperature optimization: Express at lower temperatures (20-25°C rather than 30°C) to improve folding of complex proteins.

• Consider strain background: Wild-type laboratory strains may not be optimal; strains with reduced proteolytic activity or enhanced protein folding capacity may yield better results .

Remember that expression conditions may need to be empirically determined for YHL026C specifically, as uncharacterized proteins often require customized protocols.

How can I determine the subcellular localization of YHL026C?

To determine the subcellular localization of YHL026C:

• Fluorescent protein tagging: Create a C- or N-terminal fusion with GFP using PCR-based integration techniques. Similar approaches were used for Yll056cp, which was successfully localized to the cytoplasm .

• Microscopy analysis: Utilize fluorescence microscopy to visualize the tagged protein in live cells. Include appropriate organelle markers (e.g., DAPI for nucleus, MitoTracker for mitochondria) for co-localization studies.

• Biochemical fractionation: Perform cellular fractionation followed by Western blotting to detect the protein in different cellular compartments.

• Alternative tagging strategies: If GFP fusion affects protein function, consider smaller epitope tags (HA, Myc, FLAG) followed by immunofluorescence.

• Temporal analysis: Examine localization under different growth conditions and cell cycle stages, as some proteins relocalize in response to environmental changes.

This comprehensive approach will provide reliable information about where YHL026C functions within the cell, offering insights into its potential roles.

What systems biology approaches can help decipher the function of YHL026C?

Systems biology offers powerful approaches for understanding uncharacterized proteins like YHL026C:

• Transcriptional response analysis: Identify conditions that significantly alter YHL026C expression. Similar to Yll056c, which showed upregulation under high furfural or 5-(hydroxymethyl)-2-furaldehyde concentrations .

• Interactome mapping:

  • Perform affinity purification-mass spectrometry (AP-MS) to identify protein interaction partners

  • Conduct yeast two-hybrid screens to detect binary interactions

  • Analyze genetic interaction networks through synthetic genetic array (SGA) analysis

• Metabolomic profiling: Compare metabolite profiles between wild-type and YHL026C deletion strains to identify metabolic pathways affected.

• Integration with existing datasets: Cross-reference findings with global phenotypic and genetic interaction datasets from large-scale studies.

• Comparative genomics: Analyze conservation patterns and co-evolution with functionally related genes across fungal species.

These approaches can reveal biological processes in which YHL026C participates, even without initial functional information.

How can I optimize purification of recombinant YHL026C for structural studies?

Optimizing purification of YHL026C for structural studies requires careful consideration:

• Expression construct design:

  • Include affinity tags (His6, GST, MBP) to facilitate purification

  • Consider fusion partners that enhance solubility

  • Design constructs with flexible linkers and protease cleavage sites

• Expression optimization:

  • Test expression in different yeast strains, including those with enhanced protein folding capacity

  • Explore reduced cultivation temperatures (20-25°C) to improve folding

  • Investigate the unfolded protein response (UPR) status during expression, as reduced translational activity has been associated with higher yields of functional protein

• Purification strategy:

  • Implement multiple purification steps, typically beginning with affinity chromatography

  • Include size exclusion chromatography to ensure homogeneity

  • Consider stability buffers with additives that maintain protein integrity

• Sample quality assessment:

  • Verify protein folding using circular dichroism or thermal shift assays

  • Assess protein homogeneity using dynamic light scattering

  • Perform pilot crystallization screens to evaluate sample quality

• Protein engineering:

  • If initial constructs prove problematic, design truncated versions based on domain prediction

  • Consider surface entropy reduction for crystallization

These methodological considerations will increase the likelihood of obtaining pure, homogeneous YHL026C suitable for structural studies.

What approaches can be used to identify potential enzymatic activities of YHL026C?

To identify potential enzymatic activities of YHL026C:

• Structure-based prediction:

  • Perform detailed bioinformatic analysis to identify conserved catalytic motifs

  • Use structural modeling to predict active sites and substrate binding pockets

  • Compare with enzymes of known function that share structural features

• Substrate screening:

  • Test activity against substrate libraries based on structural predictions

  • Perform untargeted metabolomic analysis comparing wild-type and YHL026C deletion strains

  • Use cell extracts with recombinant YHL026C to identify altered metabolite profiles

• Enzyme assay development:

  • Establish appropriate buffer conditions, temperature, pH, and cofactor requirements

  • Determine optimal enzyme concentration and reaction time

  • Develop sensitive detection methods for potential products

• Kinetic analysis:

  • If activity is identified, characterize kinetic parameters (Km, Vmax, kcat)

  • Similar to how Yll056cp was characterized with different aldehydes, showing varying Vmax, Kcat, and Km values for different substrates

• Inhibitor studies:

  • Test effects of metal ions, salts, and chemical additives on enzyme activity

  • Evaluate temperature and pH stability under different conditions

This systematic approach led to the characterization of Yll056cp as an NADH-dependent aldehyde reductase capable of reducing at least seven aldehyde compounds, despite being previously uncharacterized .

What are the most effective strategies for generating YHL026C deletion and point mutation strains?

For generating YHL026C deletion and point mutation strains, consider these strategies:

• CRISPR-Cas9 system:

  • Design guide RNAs targeting YHL026C

  • Provide repair templates for precise deletions or point mutations

  • Select transformants on appropriate media and confirm edits by sequencing

• Traditional homologous recombination:

  • Create PCR products with 40-60 bp homology arms flanking a selection marker

  • Transform into S. cerevisiae and select on appropriate media

  • Confirm integration by PCR and sequencing

• Two-step gene replacement:

  • Use URA3 counterselection with 5-FOA for markerless mutations

  • Particularly useful for introducing subtle point mutations

• Random mutagenesis:

  • Treat cells with chemical mutagens like EMS (ethyl methanesulfonate)

  • Screen for phenotypes of interest, similar to the approach used to generate TFL-resistant mutants

  • Sequence the gene to identify mutations

• Plasmid shuffle technique:

  • Useful if YHL026C is essential

  • Maintain a wild-type copy on a URA3 plasmid while introducing mutations

  • Select for mutant function on 5-FOA media

Each approach has advantages depending on the specific research question, with CRISPR offering the highest precision for targeted modifications.

How can I design experiments to identify genetic interactions with YHL026C?

To identify genetic interactions with YHL026C:

• Synthetic Genetic Array (SGA) analysis:

  • Cross a YHL026C deletion strain with the yeast deletion collection

  • Select double mutants using appropriate markers

  • Analyze growth phenotypes to identify synthetic lethal/sick interactions

  • Quantify colony sizes to identify both negative and positive genetic interactions

• Targeted gene deletion analysis:

  • Create double deletions with genes in pathways of interest

  • Perform phenotypic assays under various stress conditions

  • Quantify growth rates in liquid culture for precise interaction measurements

• Overexpression screening:

  • Transform a YHL026C deletion strain with an overexpression library

  • Screen for suppression of deletion phenotypes

  • Identify dosage suppressors that may function in the same pathway

• Chemical-genetic profiling:

  • Test sensitivity of YHL026C deletion to a library of compounds

  • Compare profiles with other deletion strains to identify shared functions

  • Cluster results to place YHL026C within functional categories

• Transcriptional profiling:

  • Compare gene expression changes in YHL026C mutants versus wild-type

  • Identify transcription factors potentially regulating these responses, similar to how transcription factors Yap1p, Hsf1p, Pdr1/3p, Yrr1p, and Stb5p were identified as likely controllers of YLL056C expression

These methods will help place YHL026C within cellular networks and identify functional relationships with other genes.

What methods should I use to identify post-translational modifications of YHL026C?

To identify post-translational modifications (PTMs) of YHL026C:

• Mass spectrometry-based approaches:

  • Purify tagged YHL026C from yeast cells

  • Perform tryptic digestion followed by LC-MS/MS analysis

  • Use multiple fragmentation methods (CID, ETD, HCD) for comprehensive PTM detection

  • Implement enrichment strategies for specific modifications (TiO2 for phosphorylation, lectin affinity for glycosylation)

• Site-directed mutagenesis validation:

  • Mutate identified modification sites to non-modifiable residues

  • Assess functional consequences through phenotypic assays

  • Compare protein stability and localization between wild-type and mutant proteins

• Specific PTM detection methods:

  • Phosphorylation: Phos-tag SDS-PAGE, phospho-specific antibodies

  • Ubiquitination: Immunoprecipitation under denaturing conditions

  • Glycosylation: Mobility shift assays with glycosidases

  • Acetylation: Western blotting with anti-acetyl-lysine antibodies

• Temporal PTM dynamics:

  • Analyze modifications under different growth conditions and stresses

  • Implement pulse-chase labeling to determine modification turnover rates

• PTM-dependent interactions:

  • Use modified and unmodified peptides as baits in pull-down assays

  • Identify proteins that interact specifically with modified forms

These approaches will provide insights into how YHL026C activity, stability, and interactions might be regulated through post-translational modifications.

How can I investigate protein-protein interactions involving YHL026C?

To investigate protein-protein interactions involving YHL026C:

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

  • Express epitope-tagged YHL026C in S. cerevisiae

  • Perform gentle cell lysis to preserve protein complexes

  • Purify using antibody-conjugated beads and identify interacting proteins by MS

  • Include appropriate controls to filter out non-specific interactions

• Yeast two-hybrid (Y2H) screening:

  • Use YHL026C as bait against a prey library or targeted candidates

  • Implement membrane-based Y2H systems if YHL026C is membrane-associated

  • Validate positive interactions using alternative methods

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse YHL026C and potential interaction partners with complementary fragments of a fluorescent protein

  • Visualize interactions in vivo through reconstituted fluorescence

  • Assess spatial distribution of interactions within the cell

• Proximity-dependent labeling:

  • Fuse YHL026C with BioID or APEX2 enzymes

  • Identify proximal proteins through biotinylation and streptavidin pulldown

  • Particularly useful for identifying weak or transient interactions

• Co-immunoprecipitation validation:

  • Express differentially tagged proteins in the same cells

  • Perform reciprocal co-immunoprecipitation experiments

  • Verify interactions under different cellular conditions

These complementary approaches will generate a comprehensive interaction network for YHL026C, providing functional insights through guilt-by-association principles.

How is YHL026C expression regulated in response to environmental conditions?

To investigate YHL026C expression regulation:

• Transcriptional profiling across conditions:

  • Perform RT-qPCR or RNA-seq under various stress conditions

  • Analyze expression during different growth phases and nutrient limitations

  • Compare with datasets of known stress-responsive genes

• Promoter analysis:

  • Perform in silico analysis to identify potential transcription factor binding sites

  • Create reporter constructs with the YHL026C promoter driving fluorescent protein expression

  • Conduct promoter deletion/mutation studies to identify regulatory elements

• Transcription factor identification:

  • Use chromatin immunoprecipitation (ChIP) to identify factors binding the YHL026C promoter

  • Screen transcription factor deletion strains for altered YHL026C expression

  • This approach could reveal regulators similar to Yap1p, Hsf1p, Pdr1/3p, Yrr1p, and Stb5p that were identified for YLL056C

• Post-transcriptional regulation:

  • Analyze mRNA stability and half-life under different conditions

  • Investigate the role of RNA-binding proteins in regulating YHL026C mRNA

  • Examine potential microRNA-mediated regulation

• Epigenetic regulation:

  • Assess chromatin modifications at the YHL026C locus using ChIP-seq

  • Test the effects of histone modification inhibitors on expression

Understanding the regulation of YHL026C will provide insights into its physiological roles and the conditions under which it functions.

What approaches can determine if YHL026C is involved in RNA decay pathways similar to other characterized S. cerevisiae proteins?

To determine if YHL026C functions in RNA decay pathways:

• Comparative protein sequence analysis:

  • Analyze YHL026C for domains characteristic of RNA decay factors

  • Compare with known components of RNA decay pathways in S. cerevisiae

  • Look for similarity to factors involved in the 18S nonfunctional ribosomal RNA decay pathway

• RNA stability assays:

  • Compare decay rates of reporter mRNAs in wild-type and YHL026C deletion strains

  • Analyze stability of natural transcripts using transcription inhibition and RNA-seq

  • Examine specific RNA decay intermediates by Northern blot

• Protein localization studies:

  • Determine if YHL026C co-localizes with known RNA decay factors like P-bodies or stress granules

  • Perform immunofluorescence under stress conditions that induce RNA decay

• Biochemical RNA binding assays:

  • Test direct RNA binding using electrophoretic mobility shift assays (EMSA)

  • Perform RNA immunoprecipitation to identify bound transcripts in vivo

  • Use PAR-CLIP or CLIP-seq to map binding sites transcriptome-wide

• Genetic interaction analysis:

  • Test for synthetic phenotypes with deletions of known RNA decay factors

  • Examine if YHL026C deletion affects nonsense-mediated decay, no-go decay, or non-functional 18S rRNA decay pathways

These approaches will reveal whether YHL026C functions in established RNA surveillance pathways or represents a novel factor in RNA metabolism.

Table 1: Comparison of Expression Systems for Studying Uncharacterized Yeast Proteins