Recombinant Saccharomyces cerevisiae Uncharacterized protein YGR168C (YGR168C)

What is the current functional classification of YGR168C protein?

YGR168C, previously designated as an uncharacterized protein, has been identified as a peroxisomal membrane protein and reclassified as PEX35. This protein functions as a regulator of peroxisome abundance in Saccharomyces cerevisiae. Its identification emerged from a high-content screen specifically designed to uncover mutants affecting peroxisome abundance. The protein exhibits a significant effect on peroxisome number and morphology, comparable to known peroxisomal biogenesis and inheritance proteins such as Pex12 and Vps1 .

How was YGR168C identified as a peroxisomal protein?

YGR168C was identified through a systematic, high-content screen in Saccharomyces cerevisiae designed to uncover mutants affecting peroxisome abundance. Researchers created a systematic library of gene mutants in yeast cells expressing fluorescent markers for peroxisomes, followed by automated microscopy and computational image analysis. The study revealed that deletion of YGR168C caused a significant decrease in peroxisome number, with effects comparable to mutations in known peroxisomal membrane proteins. When expressed with either a C- or N-terminal fluorescent tag under control of the medium-strength TEF1 promoter, YGR168C-tagged proteins localized to punctate structures that completely colocalized with the peroxisomal marker Pex3, confirming its peroxisomal localization .

What expression systems and purification methods are recommended for recombinant YGR168C protein?

For recombinant production of YGR168C protein, E. coli has been successfully used as an expression host. The recommended approach involves:

• Expression System: Use E. coli with the full-length YGR168C (1-376 amino acids) fused to an N-terminal His-tag.

• Purification Process: Purify using affinity chromatography, targeting the His-tag.

• Storage Recommendations: Store the purified protein at -20°C/-80°C upon receipt. For working aliquots, store at 4°C for up to one week. Avoid repeated freeze-thaw cycles.

• Reconstitution Protocol: Briefly centrifuge the vial prior to opening to bring contents to the bottom. Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Add 5-50% glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C.

The purified protein is typically greater than 90% pure as determined by SDS-PAGE and is stored in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

What fluorescent tagging strategies work best for visualizing YGR168C/PEX35 in vivo?

For effective visualization of YGR168C/PEX35 in vivo, researchers should consider:

• Promoter Selection: When expressed under its native promoter in glucose medium, YGR168C protein levels are very low and difficult to visualize above background autofluorescence. Using a medium-strength promoter such as TEF1 significantly improves visualization.

• Tag Positioning Options:

  • C-terminal fluorescent tags work effectively when expressed under stronger promoters

  • N-terminal fluorescent tags (such as GFP-YGR168C) also provide successful localization

• Compatible Markers for Colocalization: For confirmation of peroxisomal localization, use established peroxisomal markers such as Pex3-mCherry or Pex3-GFP, which show complete colocalization with tagged YGR168C.

• Alternative Visualization Method: For studying peroxisome morphology affected by YGR168C, transform strains with a plasmid expressing peroxisome-targeted GFP variant (EYFP-SKL) and analyze using fluorescence nanoscopy with nanobodies directed against GFP .

How can researchers effectively quantify changes in peroxisome abundance in YGR168C/PEX35 mutants?

To accurately quantify peroxisome abundance in YGR168C/PEX35 mutants:

• Multi-marker Validation: Use multiple peroxisomal markers (both matrix and membrane proteins) to verify phenotypes, as measurements can vary depending on the marker used.

• Automated Image Analysis Pipeline:

  • Acquire images using automated high-content microscopy

  • Use computational analysis to identify individual cells (using a fluorescent ER marker)

  • Measure both peroxisome size and number per cell

• Statistical Analysis Considerations:

  • Report peroxisome abundance values in arbitrary units

  • Only make direct comparisons between strains with the same marker

  • Use Z-scores to standardize comparisons across different experiments

• Super-resolution Microscopy: For detailed morphological analysis, use STED microscopy, which can reveal multi-lobular peroxisome morphology that may appear as enlarged peroxisomes under standard fluorescence microscopy .

What is the paradoxical effect of both deletion and overexpression of YGR168C on peroxisome abundance?

A particularly intriguing aspect of YGR168C/PEX35 function is that both deletion and overexpression cause a decrease in peroxisome number, albeit through different mechanisms:

This bidirectional effect suggests a finely balanced role for YGR168C in regulating peroxisome dynamics .

How does YGR168C/PEX35 expression change under different growth conditions?

YGR168C/PEX35 expression characteristics vary significantly under different conditions:

• Glucose Medium: Expression under native promoter is very low, with protein levels below detection threshold using standard fluorescence microscopy.

• Carbon Source Effects: The protein's effect on peroxisome morphology has been observed in both glucose and oleic acid media, suggesting it functions across different metabolic states. Overexpression leads to decreased peroxisomal fluorescent puncta in both conditions.

• Temporal Expression Patterns: Limited data exists on temporal regulation of YGR168C expression during cell cycle or stress conditions, representing a potential area for future research.

• Expression System Requirements: For experimental visualization, medium-strength promoters such as TEF1 are necessary to achieve detectable expression levels.

These expression characteristics are important considerations when designing experiments to study YGR168C function, as native expression levels may be insufficient for certain analytical techniques .

What experimental methods can determine the protein interactions of YGR168C/PEX35?

To investigate protein interactions of YGR168C/PEX35, researchers should consider:

• Formaldehyde Crosslinking:

  • Stabilize transient protein interactions

  • Can be applied at both native and overexpressed protein levels

  • Particularly valuable when working with membrane proteins

• Co-immunoprecipitation Strategies:

  • Use tagged versions (myc-YGR168C or GST-YGR168C) for pull-down experiments

  • Consider overexpression systems for stronger signals

  • Include appropriate controls to validate specific interactions

• Yeast Two-Hybrid Screening:

  • Can identify direct protein-protein interactions

  • May help establish the YGR168C interactome

• Proximity-based Labeling Methods:

  • BioID or APEX2 tagging can identify proteins in close proximity

  • Particularly useful for membrane proteins with difficult solubilization properties

• Proteomics Analysis:

  • Mass spectrometry following purification to identify interacting partners

  • Quantitative approaches can help distinguish specific from non-specific interactions

How can the SWAT-Tag strategy be applied to study YGR168C/PEX35?

The SWAp-Tag (SWAT) strategy offers a powerful approach for studying YGR168C/PEX35:

• SWAT Library Integration:

  • YGR168C can be tagged with an N-terminal SWAT acceptor module

  • This module contains a constitutive promoter and GFP tag

  • Integration allows visualization of protein localization

• Module Swapping Capabilities:

  • Once the initial SWAT acceptor module is integrated, it can be swapped with various donor modules

  • This enables rapid conversion between different tags (fluorescent proteins, affinity tags)

  • Tags can be exchanged without repeating the genomic integration process

• Experimental Applications:

  • Subcellular localization studies using different fluorescent tags

  • Protein interaction studies using affinity tags

  • Inducible expression studies by swapping in different promoters

• Technical Implementation:

  • The SWAT acceptor module contains a URA3 selection marker for integration

  • A truncated version of HygromycinB resistance cassette can be restored upon recombination

  • The I-SceI endonuclease under an inducible promoter facilitates the swap process

This approach is particularly valuable for proteins like YGR168C that require careful experimental design due to low native expression levels .

What growth phenotypes are associated with YGR168C/PEX35 deletion or overexpression?

The functional consequences of YGR168C/PEX35 manipulation include:

• Peroxisome-related Phenotypes:

  • Significant decrease in peroxisome number with deletion

  • Altered peroxisome morphology with overexpression (multi-lobular structures)

  • These phenotypes are comparable to defects in known peroxisomal biogenesis proteins

• Growth Characteristics:

  • Limited data exists on growth rate effects in standard media

  • Carbon source utilization phenotypes have not been fully characterized

  • Potential growth defects may be more pronounced under conditions requiring peroxisome function

• Metabolic Implications:

  • As a regulator of peroxisome abundance, YGR168C likely affects metabolic processes that depend on peroxisomes

  • This includes fatty acid β-oxidation, metabolism of reactive oxygen species, and other peroxisome-dependent pathways

• Experimental Analysis Methods:

  • Quantitative growth phenotyping in different carbon sources

  • Measurement of peroxisome-dependent metabolic activities

  • Genetic interaction profiling with other peroxisomal genes

How can inducible expression systems enhance studies of YGR168C/PEX35?

Inducible expression systems offer significant advantages for studying YGR168C/PEX35:

• YETI (Yeast Estradiol strains with Titratable Induction) System:

  • Enables transcriptional inducibility with single-gene precision at native loci

  • Contains Synthetic Genetic Array (SGA) screening markers and unique molecular barcodes

  • Allows high-throughput yeast genetics approaches

  • Can be characterized using quantitative growth phenotyping and pooled BAR-seq screens

• Z3EB42 Expression System:

  • Provides lower expression than Z3EV

  • Enables both conditional activation and repression

  • Particularly valuable for studying essential genes with low native expression

• Experimental Applications:

  • Titrate expression levels to identify dosage-dependent effects

  • Temporally control YGR168C expression to study acute versus chronic effects

  • Combine with fluorescent reporters to correlate expression levels with phenotypes

• Methodological Considerations:

  • For lowly expressed genes like YGR168C, choose systems that provide appropriate expression ranges

  • Approximately 40% of Z3pr-controlled essential genes have enough transcript to facilitate growth even without inducer

  • This characteristic should be considered when designing experiments

What are the technical challenges in purifying and working with recombinant YGR168C/PEX35?

Working with recombinant YGR168C/PEX35 presents several technical challenges:

• Membrane Protein Solubilization:

  • As a predicted five-transmembrane domain protein, YGR168C can be difficult to solubilize

  • Requires careful optimization of detergents and buffer conditions

• Expression System Selection:

  • E. coli has been successfully used as a heterologous expression system

  • Expression of the full-length protein (1-376 amino acids) with N-terminal His-tag has been achieved

  • Optimization may be required for high-yield production

• Stability Considerations:

  • Prone to aggregation and precipitation during purification and storage

  • Recommended storage in buffer with 6% Trehalose and 50% glycerol

  • Avoid repeated freeze-thaw cycles

• Reconstitution Protocols:

  • For functional studies, reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Addition of 5-50% glycerol helps maintain stability during storage

  • Working aliquots should be stored at 4°C and used within one week

• Quality Control Metrics:

  • Purity should be >90% as determined by SDS-PAGE

  • Functional validation through activity assays remains challenging due to limited knowledge of biochemical activities

What experimental approaches can determine the precise role of YGR168C/PEX35 in peroxisome fission?

To elucidate YGR168C/PEX35's role in peroxisome fission:

• Live Cell Imaging Techniques:

  • Time-lapse microscopy of fluorescently tagged peroxisomes in wild-type and mutant strains

  • Measure rates of peroxisome division events

  • Quantify dynamics of peroxisome elongation and constriction

• Genetic Interaction Analysis:

  • Create double mutants with known peroxisome fission factors (e.g., Dnm1, Vps1, Fis1)

  • Epistasis analysis to position YGR168C in the fission pathway

  • Synthetic genetic array screening to identify novel genetic interactions

• Biochemical Approaches:

  • In vitro reconstitution of peroxisome fission using purified components

  • Investigation of potential GTPase regulatory activities

  • Membrane tubulation or constriction assays

• Super-resolution Microscopy Studies:

  • Detailed visualization of YGR168C localization during fission events

  • Co-localization with other fission machinery components

  • Analysis of membrane curvature at fission sites

• Mathematical Modeling:

  • Develop models of peroxisome division incorporating YGR168C function

  • Simulate effects of YGR168C levels on peroxisome abundance and morphology

  • Test model predictions experimentally

How does YGR168C/PEX35 compare to peroxisomal membrane proteins in other organisms?

Comparative analysis of YGR168C/PEX35 across species:

• Homology Analysis:

  • Sequence homology searches to identify potential orthologs in other fungi, plants, and animals

  • Domain conservation analysis focusing on the five transmembrane domains and C-terminal region

  • Phylogenetic profiling to trace evolutionary relationships

• Functional Conservation Assessment:

  • Complementation studies using orthologs from other species

  • Investigation of whether human homologs can rescue yeast YGR168C deletion phenotypes

  • Analysis of functional domains conserved across species

• Systematic Comparison with Other PEX Proteins:

  • Compare structural features with other peroxisomal membrane proteins

  • Analyze expression patterns across conditions in different organisms

  • Investigate conservation of interaction partners

• Experimental Approach:

  • Heterologous expression of potential homologs in S. cerevisiae

  • CRISPR-based knockout or knockdown in mammalian cells

  • Microscopy-based analysis of peroxisome morphology across species

This comparative approach may reveal general principles of peroxisome regulation and identify YGR168C/PEX35 as a member of a conserved family of peroxisome regulators .

What high-throughput screening approaches can identify chemical modulators of YGR168C/PEX35 function?

To identify chemical modulators of YGR168C/PEX35:

• Reporter-based Screening Systems:

  • Develop fluorescent reporters that measure peroxisome abundance or morphology

  • Screen compound libraries for modulators that phenocopy YGR168C deletion or overexpression

  • Use automated microscopy and image analysis for quantification

• Growth-based Screens:

  • Utilize synthetic genetic interactions to create strains where growth depends on YGR168C function

  • Screen for compounds that suppress or enhance growth phenotypes

  • Employ pooled barcode-based screens for higher throughput

• Target-based Approaches:

  • Use purified recombinant YGR168C protein for biochemical screens

  • Develop binding assays using fluorescence polarization or thermal shift

  • Virtual screening based on predicted protein structure

• Methodology Considerations:

  • Primary screens with engineered reporter strains

  • Secondary validation in wild-type cells

  • Mechanism of action studies to confirm specificity

  • Structure-activity relationship analysis of identified hits

Chemical modulators would provide valuable tools for dissecting YGR168C function and potentially lead to broader applications in studying peroxisome biology .