Recombinant Saccharomyces cerevisiae Uncharacterized protein YMR134W (YMR134W)

What is YMR134W and why is it significant for research?

YMR134W is an essential gene in Saccharomyces cerevisiae that encodes a protein (Ymr134wp) of 237 amino acids. Its significance stems from its critical role in aerobic growth and its influence on several fundamental cellular processes. The protein has been identified as essential for viability, but remained uncharacterized until relatively recently. Research indicates it functions in ergosterol biosynthesis, which subsequently affects mitochondrial integrity, iron homeostasis, and oxidative stress resistance .

Methodologically, identifying the function of previously uncharacterized essential genes provides valuable insights into fundamental cellular processes. Researchers typically employ temperature-sensitive mutants, as complete deletion would be lethal, allowing for conditional expression studies under controlled conditions. This approach has been instrumental in uncovering YMR134W's role in cellular metabolism.

What are the known phenotypic effects of YMR134W mutation?

Temperature-sensitive mutations in YMR134W (YMR134W(ts)) result in several distinct phenotypic changes compared to wild-type cells, as summarized in the following table:

These phenotypic changes demonstrate that Ymr134wp impacts multiple cellular processes through its primary function in ergosterol biosynthesis, subsequently affecting membrane integrity and mitochondrial function .

How should I design experiments to study the function of YMR134W?

When designing experiments to study YMR134W, consider implementing a full factorial design that allows for examination of multiple factors simultaneously. Since YMR134W impacts multiple cellular processes, your experimental design should account for these diverse effects.

A recommended approach is a 2×3 factorial design examining:

• Strain factor: comparing wild-type and YMR134W(ts) strains

• Growth condition factor: analyzing cells under fermentative, respiratory, and stress conditions

This design would yield six experimental conditions, allowing comprehensive analysis of YMR134W function across different metabolic states. For each condition, measure multiple dependent variables including growth rate, ergosterol content, oxygen consumption, and iron levels .

Given that YMR134W is essential, use temperature-sensitive mutants rather than deletion strains. Schedule measurements at multiple time points after shifting to non-permissive temperature to capture both immediate and adaptive responses. Include biological replicates (minimum n=3) for each condition to ensure statistical robustness.

What control strains should be included when studying YMR134W function?

For rigorous investigation of YMR134W function, include the following control strains:

• Wild-type S. cerevisiae (essential baseline control)

• Temperature-sensitive strains of other genes in the ergosterol biosynthesis pathway (pathway-specific controls)

• Temperature-sensitive strains of unrelated essential genes (to distinguish general effects of compromised essential functions from YMR134W-specific effects)

• Wild-type strain treated with ergosterol biosynthesis inhibitors (to compare pharmacological versus genetic inhibition)

• YMR134W(ts) strain supplemented with exogenous ergosterol (for rescue experiments)

How can I investigate the molecular mechanism by which YMR134W affects iron homeostasis?

The relationship between YMR134W and iron homeostasis presents a complex research question requiring sophisticated methodological approaches. The observed phenotype—reduced mitochondrial iron despite increased cellular iron uptake—suggests disruption in iron trafficking rather than uptake mechanisms .

To investigate this relationship, implement the following methodological approach:

• Subcellular fractionation studies: Isolate different cellular compartments (cytosol, mitochondria, vacuoles) and quantify iron content using inductively coupled plasma mass spectrometry (ICP-MS) or colorimetric assays.

• Iron flux analysis: Use radioactive 55Fe to trace iron movement between compartments in wild-type versus YMR134W(ts) strains under different temperature conditions.

• Interactome analysis: Perform co-immunoprecipitation experiments with tagged Ymr134wp followed by mass spectrometry to identify protein interactions with iron transporters or regulators.

• Transcriptome analysis: Compare expression profiles of genes involved in iron regulation (e.g., Aft1/2-regulated genes) between wild-type and YMR134W(ts) strains.

• Membrane integrity assessment: Evaluate the impact of altered ergosterol content on the function of iron transporters using fluorescent probes for membrane fluidity.

This multi-faceted approach allows delineation of whether the iron homeostasis disruption is a direct effect of YMR134W function or a secondary consequence of altered membrane composition affecting iron transporter function and localization .

What techniques can be used to characterize the role of YMR134W in ergosterol biosynthesis?

To characterize YMR134W's role in ergosterol biosynthesis, employ the following advanced techniques:

• Metabolic profiling: Use gas chromatography-mass spectrometry (GC-MS) to quantify ergosterol and its precursors in wild-type versus YMR134W(ts) strains, helping identify at which step of the biosynthetic pathway YMR134W functions.

• Enzyme activity assays: Test the activities of various enzymes in the ergosterol biosynthesis pathway to determine if YMR134W directly affects a specific enzymatic step or has broader regulatory effects.

• Protein-protein interaction studies: Perform yeast two-hybrid or bimolecular fluorescence complementation assays to identify interactions between Ymr134wp and known ergosterol biosynthesis enzymes.

• Structural biology approaches: Use X-ray crystallography or cryo-electron microscopy to determine the three-dimensional structure of Ymr134wp, potentially revealing functional domains related to sterol metabolism.

• In vitro reconstitution: Purify recombinant Ymr134wp and test its activity with potential substrates from the ergosterol pathway in a defined system.

These approaches allow precise characterization of YMR134W's molecular function in ergosterol biosynthesis, distinguishing between enzymatic, regulatory, or structural roles .

How can I resolve the apparent contradiction between reduced mitochondrial iron and increased cellular iron in YMR134W mutants?

The observed phenomenon of reduced mitochondrial iron despite increased total cellular iron uptake in YMR134W(ts) cells presents an intriguing data interpretation challenge . This apparent contradiction can be approached methodologically through the following analytical framework:

• Compartmental analysis: Investigate whether iron accumulates in specific cellular compartments (e.g., vacuoles) by performing subcellular fractionation followed by quantitative iron measurements.

• Iron speciation studies: Determine if the chemical form of iron differs between wild-type and mutant cells using Mössbauer spectroscopy or X-ray absorption spectroscopy, as altered iron speciation could affect distribution.

• Membrane integrity assessment: Evaluate whether altered ergosterol content in mitochondrial membranes specifically impacts iron transporter function through membrane fluidity measurements and transporter activity assays.

• Regulatory pathway analysis: Investigate whether iron sensing and signaling pathways are disrupted, potentially leading to inappropriate upregulation of iron uptake despite adequate cellular iron levels.

• Time-course experiments: Track iron distribution immediately following temperature shift in YMR134W(ts) cells to distinguish primary from secondary effects.

These approaches help resolve whether the iron distribution anomaly is directly linked to YMR134W function or represents a cellular compensatory response to mitochondrial dysfunction resulting from ergosterol deficiency .

How should I interpret the increased oxidative stress sensitivity in YMR134W mutants?

The increased sensitivity to oxidative stress observed in YMR134W(ts) cells requires careful interpretation to distinguish between several possible mechanistic explanations . A methodological approach to this interpretation includes:

• Source identification: Determine whether increased reactive oxygen species (ROS) result from:

  • Mitochondrial dysfunction due to altered membrane composition

  • Disrupted iron homeostasis leading to increased free iron and subsequent Fenton reactions

  • Direct involvement of YMR134W in antioxidant functions

• Antioxidant system analysis: Measure the activities and expression levels of key antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) to determine if the baseline antioxidant capacity is compromised.

• Lipid peroxidation assessment: Quantify lipid peroxidation products to evaluate whether altered membrane composition increases vulnerability to oxidative damage.

• Gene expression profiling: Analyze transcriptional responses to oxidative stress in wild-type versus YMR134W(ts) cells to identify differences in stress adaptation.

• Genetic interaction studies: Perform epistasis experiments with mutants in known oxidative stress response pathways to position YMR134W in the cellular stress response network.

This systematic approach helps distinguish between direct involvement of YMR134W in oxidative stress resistance versus indirect effects stemming from its primary function in ergosterol biosynthesis and subsequent impacts on mitochondrial function and iron homeostasis .

What expression systems are suitable for producing recombinant YMR134W protein for in vitro studies?

For successful production of recombinant YMR134W protein, consider the following expression systems and methodological approaches:

• E. coli expression systems:

  • BL21(DE3) or Rosetta strains are suitable for His-tagged full-length YMR134W (1-237 amino acids) expression

  • Optimize expression conditions (temperature, IPTG concentration, induction time) to maximize soluble protein yield

  • Consider fusion tags (MBP, GST) if solubility is problematic with His-tag alone

• Yeast expression systems:

  • S. cerevisiae or Pichia pastoris systems may provide proper folding environment

  • Use strong inducible promoters (GAL1 for S. cerevisiae, AOX1 for P. pastoris)

  • Incorporate native secretion signals if membrane association is suspected

• Insect cell systems:

  • Baculovirus expression system for proteins requiring eukaryotic post-translational modifications

  • Particularly valuable if YMR134W undergoes regulatory modifications

• Purification strategy:

  • Two-step purification combining affinity chromatography and size exclusion

  • Include ergosterol or membrane mimetics during purification if membrane association is confirmed

  • Verify protein activity through binding assays with potential substrates from ergosterol pathway

Successful production of active recombinant YMR134W enables crucial biochemical characterization, including structural studies and in vitro activity assays to define its precise molecular function .

What are the key considerations for designing a thermosensitive YMR134W mutant strain?

Creating effective thermosensitive YMR134W mutant strains requires careful methodological planning. Consider the following approach:

• Mutation strategy selection:

  • Random mutagenesis coupled with temperature-sensitive phenotype screening

  • Site-directed mutagenesis targeting conserved domains or predicted structural elements

  • CRISPR-based saturation mutagenesis of specific protein regions

• Optimal temperature conditions:

  • Permissive temperature: 25-30°C (standard yeast growth temperature)

  • Non-permissive temperature: 37°C (high enough to inactivate the mutant protein without causing general heat stress)

  • Include temperature shift experiments to capture acute phenotypes

• Validation parameters:

  • Confirm protein expression levels at permissive temperature match wild-type

  • Verify protein destabilization or inactivation at non-permissive temperature

  • Assess reversibility of phenotypes upon return to permissive temperature

  • Quantify growth rates under various conditions to confirm conditional lethality

• Genetic background considerations:

  • Use strain backgrounds with well-characterized temperature responses

  • Consider creating multiple mutant alleles in different genetic backgrounds

  • Include wild-type controls from the same genetic background in all experiments

• Functional confirmation:

  • Measure ergosterol levels at permissive and non-permissive temperatures

  • Confirm respiratory capacity changes correlate with temperature shift

  • Validate iron distribution phenotypes are temperature-dependent

These guidelines ensure creation of reliable thermosensitive YMR134W strains that effectively mimic null phenotypes at non-permissive temperatures while maintaining normal function under permissive conditions .

How does YMR134W function interconnect with mitochondrial biogenesis and function?

The relationship between YMR134W and mitochondrial function represents a complex interaction between ergosterol biosynthesis, membrane integrity, and organelle function. The following methodological approach helps elucidate these interconnections:

• Mitochondrial morphology and dynamics:

  • Employ fluorescence microscopy with mitochondria-targeted fluorescent proteins to visualize changes in mitochondrial network morphology in YMR134W(ts) strains

  • Quantify fusion/fission events following temperature shift using time-lapse imaging

  • Measure changes in mitochondrial mass using flow cytometry with mitochondrial dyes

• Respiratory chain analysis:

  • Measure activities of individual respiratory chain complexes to identify specific defects

  • Perform oxygen consumption measurements using high-resolution respirometry

  • Assess membrane potential using potential-sensitive dyes like TMRM or JC-1

• Mitochondrial membrane composition:

  • Analyze lipid composition of isolated mitochondrial membranes from wild-type and YMR134W(ts) strains

  • Correlate ergosterol content with respiratory function and iron import capacity

  • Assess membrane fluidity using fluorescence anisotropy techniques

• Mitochondrial protein import:

  • Measure import efficiency of reporter proteins into mitochondria

  • Determine if iron-containing protein maturation is specifically affected

  • Evaluate the integrity of protein import machinery components

• Iron-sulfur cluster biogenesis:

  • Assess activities of iron-sulfur cluster-containing enzymes as indicators of cluster assembly

  • Measure expression of iron-sulfur cluster assembly machinery components

  • Perform in organello iron-sulfur cluster synthesis assays

This comprehensive approach reveals whether mitochondrial dysfunction in YMR134W mutants results directly from altered ergosterol content in mitochondrial membranes or indirectly through disrupted iron homeostasis affecting iron-dependent mitochondrial processes .

How can I design experiments to explore the relationship between YMR134W, ergosterol biosynthesis, and stress resistance?

To investigate the interconnection between YMR134W, ergosterol biosynthesis, and stress resistance, implement a multi-faceted experimental approach:

• Comparative stress response profiling:

  • Design a factorial experiment testing multiple stressors (oxidative, osmotic, temperature, cell wall)

  • Compare wild-type, YMR134W(ts), and strains with mutations in known ergosterol biosynthesis genes

  • Include combination treatments to identify stress-specific versus general vulnerability

  Strain	Control	Oxidative Stress	Osmotic Stress	Combined Stress
  Wild-type	Baseline growth	Response 1	Response 2	Response 3
  YMR134W(ts)	Baseline comparison	Specific sensitivity?	Specific sensitivity?	Synergistic effects?
  ERG mutants	Pathway control	Similar to YMR134W(ts)?	Similar to YMR134W(ts)?	Similar to YMR134W(ts)?

• Ergosterol supplementation experiments:

  • Supplement media with exogenous ergosterol under permissive and non-permissive conditions

  • Determine if ergosterol supplementation rescues specific stress sensitivities

  • Identify which phenotypes are directly linked to ergosterol deficiency

• Membrane integrity and stress response:

  • Measure membrane fluidity changes under different stress conditions

  • Assess localization and function of stress sensors that reside in membrane microdomains

  • Determine if altered membrane composition affects stress signaling pathway activation

• Transcriptional response analysis:

  • Perform RNA-seq comparing wild-type and YMR134W(ts) responses to different stressors

  • Identify differentially regulated stress response pathways

  • Determine whether stress response transcription factors depend on proper membrane composition

• Temporal analysis of adaptation:

  • Design time-course experiments tracking adaptation to chronic stress exposure

  • Compare immediate versus adaptive responses in wild-type and YMR134W(ts) strains

  • Determine if YMR134W mutants fail in initial response or in sustained adaptation

This comprehensive approach allows detailed characterization of how YMR134W-dependent ergosterol biosynthesis contributes to stress resistance mechanisms, distinguishing direct effects on stress signaling from indirect consequences of altered membrane composition .

What are promising approaches for identifying human homologs or functional counterparts of YMR134W?

Identifying human homologs or functional counterparts of YMR134W presents a significant challenge due to limited sequence conservation across species. A methodological framework for this investigation includes:

• Advanced bioinformatic approaches:

  • Profile-based searches using position-specific scoring matrices rather than simple sequence comparisons

  • Structure prediction-based searches focusing on conserved structural elements

  • Phylogenetic profiling to identify proteins with similar evolutionary patterns

  • Functional domain analysis identifying proteins with similar domain architecture

• Functional complementation studies:

  • Express candidate human genes in YMR134W(ts) yeast strains

  • Test for rescue of temperature sensitivity, ergosterol deficiency, and mitochondrial phenotypes

  • Create chimeric proteins combining domains from yeast and human candidates

• Comparative metabolic profiling:

  • Analyze changes in sterol metabolism in YMR134W(ts) yeast

  • Perform similar analyses in human cells with CRISPR-targeted disruption of candidate genes

  • Look for matching metabolic signatures indicating functional conservation

• Transcriptional response comparison:

  • Compare transcriptional changes in YMR134W mutant yeast with those in human cells following disruption of sterol metabolism

  • Identify conserved responsive gene networks indicating functional parallels

• Interactome comparative analysis:

  • Identify protein interaction partners of Ymr134wp in yeast

  • Screen for human proteins that interact with similar partners

  • Test whether these human proteins functionally complement YMR134W mutants

This systematic approach may identify human proteins that, despite limited sequence similarity, perform analogous functions in maintaining proper sterol metabolism, mitochondrial function, and iron homeostasis, potentially revealing new therapeutic targets for disorders of these pathways .

How should researchers design experiments to investigate YMR134W's potential role in antioxidant response?

To investigate YMR134W's potential role in antioxidant response, design experiments that distinguish direct involvement from indirect consequences using the following methodological framework:

• Comprehensive oxidative stress phenotyping:

  • Test multiple oxidants (hydrogen peroxide, menadione, paraquat) to distinguish between sensitivity to different types of reactive oxygen species

  • Determine dose-response relationships and calculate EC50 values for wild-type versus YMR134W(ts) strains

  • Design a full factorial experiment examining oxidative stress sensitivity under different carbon sources (fermentative versus respiratory)

• Antioxidant system analysis:

  • Measure baseline and stress-induced activities of key antioxidant enzymes:

    • Superoxide dismutases (SOD1, SOD2)

    • Catalase

    • Glutathione peroxidases

    • Thioredoxin system components

  • Compare antioxidant protein expression levels using western blotting

  • Analyze glutathione levels and redox state

• Redox proteomics approach:

  • Perform OxICAT or similar techniques to identify differentially oxidized proteins

  • Compare the redox proteomes of wild-type and YMR134W(ts) strains

  • Identify specific pathways affected by altered redox balance

• Genetic interaction studies:

  • Create double mutants combining YMR134W(ts) with deletions in key oxidative stress response genes

  • Test for synthetic interactions indicating parallel or compensatory pathways

  • Perform epistasis analysis to position YMR134W in the oxidative stress response network

• Transcriptional regulation analysis:

  • Analyze binding of oxidative stress response transcription factors (e.g., Yap1) to their targets

  • Determine if altered membrane composition affects transcription factor localization or activation

  • Measure stress-responsive gene expression dynamics using real-time reporters

This comprehensive approach allows determination of whether YMR134W directly participates in oxidative stress defense or if the observed oxidative stress sensitivity is a secondary consequence of altered ergosterol biosynthesis affecting membrane integrity and mitochondrial function .