Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YMR158W-B (YMR158W-B)

What experimental methods are most appropriate for confirming the subcellular localization of YMR158W-B?

Determining the subcellular localization of YMR158W-B requires a multi-faceted approach combining both genetic tagging and microscopy techniques. Based on current research methodologies, the most reliable approach involves creating an S-peptide tagged version of the protein. This technique has proven successful with the related YMR158W protein, which was found to localize to the small subunit of the mitoribosome. When studying YMR158W, researchers found that using a plasmid-based expression system with an S-peptide tag allowed for effective tracking of the protein through cellular fractionation and sucrose density gradient centrifugation .

For YMR158W-B specifically, the experimental protocol should include:

• Cloning the YMR158W-B gene into a plasmid vector (such as pSHLeu) to create an S-peptide fusion

• Transforming the construct into a yeast strain with the chromosomal copy deleted

• Isolating mitochondria through differential centrifugation

• Separating mitoribosomal subunits via sucrose density gradient centrifugation

• Analyzing fractions through SDS-PAGE and Western blotting to detect the tagged protein

Note that when implementing this protocol for YMR158W, researchers observed that the S-tagged protein appeared approximately 10 kDa larger than expected on SDS-PAGE, likely due to the nature of the S-tag itself . This should be anticipated when designing detection methods for YMR158W-B as well.

How does YMR158W-B contribute to mitochondrial function in Saccharomyces cerevisiae?

YMR158W-B's contribution to mitochondrial function can be assessed through a systematic phenotypic analysis of mutant strains. Based on studies of the related YMR158W protein, which demonstrated that cells carrying only tagged versions grew poorly in non-fermentable media (YPGE), indicating an essential role in mitochondrial function , similar approaches can be applied to YMR158W-B.

The methodological approach should include:

• Generation of YMR158W-B deletion strains (ymr158w-b::KAN)

• Complementation assays with wild-type and mutant versions of YMR158W-B

• Growth assessments on fermentable (glucose) vs. non-fermentable (glycerol, ethanol) carbon sources

• Mitochondrial respiration measurements using oxygen consumption assays

• Analysis of mitochondrial translation using 35S-methionine labeling in the presence of cycloheximide

When conducting these experiments, it's crucial to maintain proper controls, including wild-type strains and strains with deletions in known mitoribosomal proteins. This allows for comparative analysis of phenotypic severity and functional relationships.

What is the relationship between YMR158W and YMR158W-B in terms of functional redundancy?

Investigating functional redundancy between YMR158W and YMR158W-B requires a genetic approach combined with biochemical characterization. While the available research indicates that YMR158W localizes to the small subunit of mitoribosome and is essential for mitochondrial function , the relationship with YMR158W-B needs to be systematically characterized.

The experimental approach should include:

• Creation of single deletion strains (Δymr158w and Δymr158w-b)

• Construction of double deletion strains (if viable)

• Cross-complementation studies (expressing YMR158W in Δymr158w-b strains and vice versa)

• Mitoribosome assembly analysis in each genetic background

• Comparative mitochondrial translation efficiency measurements

Table 1: Hypothetical Growth Phenotypes to Assess Functional Redundancy

This type of systematic analysis would provide clear insights into whether these proteins can functionally substitute for each other, or if they have distinct non-overlapping roles in mitochondrial function.

What are the recommended protocols for purifying recombinant YMR158W-B protein for in vitro studies?

Purification of recombinant YMR158W-B for in vitro studies requires careful consideration of its biochemical properties and potential association with mitoribosomal components. Based on experimental approaches used for similar mitoribosomal proteins, the following protocol is recommended:

• Clone the YMR158W-B coding sequence into a bacterial expression vector with a 6xHis or GST tag

• Express in E. coli BL21(DE3) cells at reduced temperature (18°C) to enhance proper folding

• Lyse cells in buffer containing appropriate detergents (0.5% Triton X-100) and protease inhibitors

• Purify using affinity chromatography (Ni-NTA for His-tagged proteins)

• Apply size exclusion chromatography to remove aggregates and obtain homogeneous protein

When designing the expression construct, consider that mitoribosomal proteins often have mitochondrial targeting sequences that may affect solubility when expressed in bacterial systems. It may be necessary to remove these sequences or optimize expression conditions to improve yield.

For functional studies, the purified protein should be assessed for RNA binding capability and interaction with other mitoribosomal components through techniques such as electrophoretic mobility shift assays (EMSA) and in vitro reconstitution experiments.

What approaches can be used to determine the specific RNA binding sites of YMR158W-B in mitochondrial ribosomal RNA?

Characterizing the RNA binding properties of YMR158W-B requires sophisticated techniques that can map protein-RNA interactions with high resolution. Given that YMR158W has been localized to the small subunit of the mitoribosome , similar methodologies can be applied to study YMR158W-B's RNA interactions.

The recommended methodological approach includes:

• UV Crosslinking and Immunoprecipitation (CLIP): This technique involves in vivo crosslinking of protein-RNA complexes, followed by immunoprecipitation of YMR158W-B (using tagged versions) and sequencing of the bound RNA fragments. This provides a transcriptome-wide view of binding sites.

• RNA Footprinting Assays: Using chemical probing reagents such as dimethyl sulfate (DMS) or selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to identify regions of mitochondrial rRNA protected by YMR158W-B binding.

• Cryo-EM Structural Analysis: High-resolution structural determination of mitoribosome containing YMR158W-B can reveal the precise molecular contacts between the protein and rRNA components.

• Mutational Analysis: Systematic mutagenesis of YMR158W-B followed by RNA binding studies to identify critical residues involved in RNA recognition.

These approaches should be performed in parallel with studies on wild-type and mutant versions of the protein to establish structure-function relationships. When analyzing the data, pay particular attention to conserved RNA structural elements that may represent functionally important interaction sites.

How can researchers design experiments to resolve conflicting data regarding YMR158W-B's role in mitoribosome assembly?

When faced with conflicting data about YMR158W-B's role in mitoribosome assembly, a systematic experimental design approach is essential. Based on the experimental design principles outlined in scientific inquiry methodologies , researchers should implement the following strategy:

• Clear Hypothesis Formulation: Define specific, testable hypotheses regarding YMR158W-B's role, based on existing conflicting data.

• Variable Identification and Control: Carefully identify all variables that might influence experimental outcomes, including:

  • Independent Variable: YMR158W-B presence/absence or mutation type

  • Dependent Variable: Mitoribosome assembly status or function (with quantifiable metrics)

  • Controlled Variables: Growth conditions, genetic background, expression levels

• Time-Course Assembly Analysis: Implement pulse-chase experiments to track mitoribosome assembly over time, comparing wild-type and YMR158W-B mutant strains.

• Quantitative Proteomics: Use SILAC (Stable Isotope Labeling with Amino acids in Cell culture) approaches to quantitatively compare mitoribosomal protein composition between wild-type and mutant strains.

• Genetic Interaction Mapping: Conduct synthetic genetic array (SGA) analysis with YMR158W-B against known mitoribosomal assembly factors to identify functional relationships.

Table 2: Experimental Design Matrix for Resolving Conflicting Data

Each experiment should include at least three independent biological replicates to ensure statistical validity, and data analysis should employ appropriate statistical methods to determine significance levels .

What are the most effective methods for analyzing YMR158W-B post-translational modifications and their impact on protein function?

Investigating post-translational modifications (PTMs) of YMR158W-B requires a combination of mass spectrometry-based proteomics and functional assays. The methodological approach should include:

• Sample Preparation Optimization:

  • Purify YMR158W-B under native conditions to preserve PTMs

  • Implement parallel purifications from different growth conditions (fermentative vs. respiratory growth)

  • Use phosphatase inhibitors and deacetylase inhibitors during purification to prevent PTM loss

• Mass Spectrometry Analysis:

  • Employ both bottom-up (peptide-level) and top-down (intact protein) proteomics

  • Use multiple fragmentation methods (CID, ETD, HCD) to maximize PTM detection

  • Implement targeted MS/MS approaches for suspected modification sites

• Functional Correlation:

  • Generate site-specific mutants that either prevent modification (e.g., S→A for phosphorylation sites) or mimic constitutive modification (e.g., S→D for phosphorylation)

  • Assess the impact of these mutations on YMR158W-B function in vivo

  • Determine if modifications change in response to cellular stress or metabolic shifts

• Temporal Dynamics:

  • Analyze modification patterns across the cell cycle and during mitochondrial biogenesis

  • Identify the enzymes responsible for adding/removing modifications through genetic screens

Table 3: Common Post-Translational Modifications to Investigate in YMR158W-B

This comprehensive approach will reveal not only which modifications exist on YMR158W-B but also how they regulate its function in mitochondrial ribosomes under different cellular conditions.

How can researchers effectively design experiments to study the evolutionary conservation of YMR158W-B across fungal species?

Studying the evolutionary conservation of YMR158W-B requires a comparative genomics approach combined with functional characterization. The methodological workflow should include:

• Sequence-Based Analysis:

  • Perform BLAST and HMM-based searches to identify orthologs across fungal lineages

  • Conduct multiple sequence alignments to identify conserved domains and residues

  • Apply phylogenetic analysis to reconstruct the evolutionary history of the gene family

• Structural Conservation Assessment:

  • Use protein structure prediction tools (AlphaFold, RoseTTAFold) to model YMR158W-B and its orthologs

  • Compare predicted structures to identify conserved structural elements beyond sequence conservation

  • Identify potential functional sites based on structural conservation

• Functional Complementation Experiments:

  • Clone orthologs from diverse fungal species into S. cerevisiae expression vectors

  • Transform these constructs into YMR158W-B deletion strains

  • Assess the ability of each ortholog to rescue the deletion phenotype

• Domain Swap Experiments:

  • Create chimeric proteins containing domains from different species' orthologs

  • Test these chimeras for functional complementation

  • Identify which regions confer species-specific functions

Table 4: Cross-Species Functional Complementation Analysis Framework

When implementing this framework, it's important to maintain consistent experimental conditions across all species being tested. Additionally, consider the impact of codon usage differences and optimize coding sequences for expression in S. cerevisiae when necessary.

What are the critical parameters to control when designing experiments to study YMR158W-B's interaction with other mitoribosomal proteins?

When investigating protein-protein interactions involving YMR158W-B, experimental design must carefully control several critical parameters to ensure reliable and reproducible results. Based on established experimental design principles , the following methodological considerations are essential:

• Tag Selection and Positioning:

  • Compare N-terminal vs. C-terminal tags to determine which minimally impacts function

  • Validate that tagged versions complement deletion phenotypes before proceeding

  • Consider using multiple tag types (FLAG, HA, GFP) to confirm results across methods

• Interaction Detection Methods:

  • Implement at least two independent methods (e.g., co-immunoprecipitation and proximity labeling)

  • Include appropriate negative controls (non-specific IgG, unrelated mitochondrial proteins)

  • Control for interaction specificity using mutant versions with altered binding properties

• Experimental Conditions:

  • Define precise buffer compositions that maintain native interactions

  • Test interactions under different metabolic conditions (fermentative vs. respiratory growth)

  • Evaluate the impact of detergent type and concentration on interaction stability

• Quantification Approaches:

  • Use quantitative proteomics (SILAC or TMT labeling) to measure interaction strengths

  • Apply appropriate statistical analyses to differentiate specific from non-specific interactions

  • Perform competition assays to assess binding hierarchies within multiprotein complexes

When applying these methods, researchers should prioritize experimental designs with at least three independent biological replicates and include statistical analysis to determine confidence intervals for each detected interaction . Additionally, validation of key interactions using orthogonal methods is essential for establishing biological significance.

How can researchers design experiments to determine if YMR158W-B is involved in mitochondrial translation regulation rather than structural ribosome assembly?

Distinguishing between structural and regulatory roles for YMR158W-B requires careful experimental design focusing on both ribosome assembly and translation dynamics. The methodological approach should include:

• Conditional Depletion System:

  • Create an auxin-inducible degron (AID) tagged YMR158W-B strain

  • Monitor both ribosome assembly and translation activity at different time points after depletion

  • Determine whether translation defects precede or follow assembly defects

• Translation Efficiency Measurements:

  • Implement ribosome profiling to measure translation efficiency genome-wide

  • Compare the impact on mitochondrial vs. cytoplasmic translation

  • Analyze ribosome occupancy patterns on specific mitochondrial mRNAs

• Resolution of Structure vs. Function:

  • Use cryo-EM to determine if YMR158W-B remains associated with functionally active ribosomes

  • Compare structures of initiating, elongating, and terminating ribosomes

  • Identify if YMR158W-B changes position during the translation cycle

• mRNA-Specific Effects:

  • Perform RNA immunoprecipitation to identify if YMR158W-B binds specific mitochondrial mRNAs

  • Measure translation rates of individual mitochondrial genes using reporter constructs

  • Test if YMR158W-B preferentially affects certain classes of mitochondrial genes

Table 5: Experimental Readouts to Distinguish Structural vs. Regulatory Roles

This comprehensive approach allows researchers to differentiate between a primarily structural role (like other small subunit mitoribosomal proteins) and a regulatory role in translation, which would be indicated by mRNA-specific effects without major assembly defects.